Objective To improve the chemically-activated luciferase expression (CALUX)bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms ofDLCs. Method A recombinant vector was construc...Objective To improve the chemically-activated luciferase expression (CALUX)bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms ofDLCs. Method A recombinant vector was constructed and used to transfect humanhepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor(AhR)-meditated luciferase gene expression. The reliability of luciferase induction in thiscell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection timewas examined and a comparison was made by using the commonly used ethoxyresoufin-O-deethylase (EROD) activity induction assay. Result The results suggested that theluciferase activity in recombinant cells was peaked at about 4 h and then decreased to astable activity by 14 h after TCDD treatment. The detection limit of this cell line was0.11pmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%.Conclusion The luciferase induction is 30-fold more sensitive than EROD induction, thedetection time is 68 h shorter and the detection procedure is also simpler.展开更多
Objective:To construct a PSA luciferase report plasmid and monitor the growth and metastasis of prostate cancer after emasculation in SCID mice.Methods:PSA promoter sequence and luciferase gene were amplified by PCR a...Objective:To construct a PSA luciferase report plasmid and monitor the growth and metastasis of prostate cancer after emasculation in SCID mice.Methods:PSA promoter sequence and luciferase gene were amplified by PCR and subsequently inserted into pZsCreen1-1 vector to construct pPSA-FL-Luc vector.LNCaP cells that were stably transfected with pPSA-FL-Luc were used to establish a SCID mouse xenograft model.Then,the growth and metastasis of prostate cancer were monitored via living imaging.Results:We successfully constructed a PSA luciferase piasmid,pPSA-FL-Luc.DHT enhanced lucifcrase activity in a concentration-dependent manner in 293 T cells with pPSA-FL-Luc transfection.Prostate cancer SCID mouse model was established with pPSA-FL-Luc transfected LNCaP cells.In tumor bearing mice with or without emasculation,pPSA-FL-Lue piasmid was applied to monitored tumor growth and metastasis based on bioluminescence imaging.Conclusions:We construct a pPSA-FL-Luc piasmid,which stably expresses luciferase and can be applied to monitor tumor development in a prostate SCID mouse model.展开更多
BACKGROUND Previously,we have successfully constructed replication-competent hepatitis B virus(HBV)vectors by uncoupling the P open reading frame(ORF)from the preC/C ORF to carefully design the transgene insertion sit...BACKGROUND Previously,we have successfully constructed replication-competent hepatitis B virus(HBV)vectors by uncoupling the P open reading frame(ORF)from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence.Consequently,the replication-competent HBV vectors carrying foreign genes,including pCH-BsdR,carrying blasticidin resistance gene(399 bp),and pCH-hrGFP,carrying humanized renilla green fluorescent protein gene(720 bp),were successfully obtained.However,the replication efficiency of the former is higher but it is tedious to use,while that of the latter is poor and cannot be quantified.Hence,we need to search for a new reporter gene that is convenient and quantifiable for further research.AIM To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies.METHODS We utilized the replication-competent HBV viral vectors constructed by our laboratory,combined with the secreted luciferase reporter gene,to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase(SecNluc).HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene.RESULTS The replication-competent HBV vector carrying the SecNluc reporter gene pCHsNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression.HBV replication intermediates could be produced from this vector.Via transfection with pTRE-sNLuc and selection by hygromycin,we obtained isolated cell clones,named HBV-NLuc-35 cells,which could secrete secNLuc recombinant viruses,and were sensitive to existing anti-HBV drugs.Using differentiated HepaRG cells,it was verified that recombinant HBV possessed infectivity.CONCLUSION Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability,and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene.More importantly,the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.展开更多
A simple and rapid expression and purification method of recombinant firefly luciferase was developed for bacteria detection. A modified luciferase gene from North American firefly Photinus pyralis was cloned into pET...A simple and rapid expression and purification method of recombinant firefly luciferase was developed for bacteria detection. A modified luciferase gene from North American firefly Photinus pyralis was cloned into pET28a expression vector and the recombinant protein was produced in Escherichia coli BL21. The recombinant luciferase,equipped with a polyhistidine affinity tag,was purified by immobilized metal ion affinity chromatography (IMAC). The approach generated an abundant expression and an efficient purification of a recombinant luciferase with final yield 1.995mg/L of cell culture. Experiments on the recombinant luciferase also showed that the relative light units (RUL) of the enzyme were 5.8×108,and the specific activity was 2.9×1010 RLU/mg. By applying adenosine triphosphate (ATP) bioluminescence to detection of the coin bacteria using the recombinant protein,the ATP content of bacteria was 9.48×10-16mol/mL,and was identical to the bacteria counts (4500CFU/mL) in order of magnitude. Taken together,our results provided a simple and efficacious method of the preparation of recombinant luciferase,which could be applied in the determination of bacteria via ATP bioluminescence.展开更多
To analyze miR-139 target sites in 3' UTR of GHR gene in dairy cow mammary gland, a GHR 3' UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary g...To analyze miR-139 target sites in 3' UTR of GHR gene in dairy cow mammary gland, a GHR 3' UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary gland epithelial cells (DCMECs). The miR-139 targeting GHR 3' UTR was predicted by Target Scan 5.1 software, 3' UTR fragment of GHR was amplified by PCR from RNA of DCMECs. PCR products were cloned into Spe Ⅰ/Hind Ⅱ modified pMIR-Report vector. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into DCMECs using lipofectamine 2000 transfection reagent. The dualluciferase reporter assay system was used to quantitiate the reporter activity. The results showed that a 107 bp 3' UTR fragment of GHR gene was successfully cloned into the pMIR-Report vector, which authenticated by Spe Ⅰ/Hind Ⅲ digestion and DNA sequencing. The luciferase activity of reporter construction treated with miR-139 decreased 20.87% compared with the control group. It was concluded that the GHR3' UTR-luciferase reporter vector had been successfully constructed. The luciferase activity of the reporter could be suppressed by miR- 139.展开更多
The Aryl hydrocarbon receptor(AhR)ligands exhibiting modulating activity represents a new class of anticancer agents that can be directed towards several tumors.We have examined AhR expression in human colon cancer an...The Aryl hydrocarbon receptor(AhR)ligands exhibiting modulating activity represents a new class of anticancer agents that can be directed towards several tumors.We have examined AhR expression in human colon cancer and adjacent non-tumor tissue.AhR expression level was about 2-7 times higher in tumor tissue samples than in the adjacent non-tumor samples(in 82%of all the samples).We were unable to find any increase of ABCG2 expression on the level of the transcription,while the expression of MDR2 was increased in half of the tumors compared to the levels of expression in normal adjacent tissue.We have used FICZ as a potent high affinity ligand of the AhR to calibrate the reporter cell line HEK293TAhR-luc as a potent high affinity ligand of the AhR.The concentration of xenobiotic response element(XRE)ligands is higher,than in the blood of healthy people in 86%of the patients.The proposed test system will allow the use of the AhR ligand level as an additional diagnostic marker in the treatment of colon cancer.展开更多
Coxsackievirus belongs to the Picornaviridae family and is one of the major pathogens that cause hand,foot and mouth disease(HFMD)in infants and children with potential serious complications and even deaths.The pathog...Coxsackievirus belongs to the Picornaviridae family and is one of the major pathogens that cause hand,foot and mouth disease(HFMD)in infants and children with potential serious complications and even deaths.The pathogenesis of this virus is not fully elucidated and no vaccine or antiviral drug has been approved.In this study,a full-length infectious cDNA clone of coxsackievirus B5 virus was assembled and the recombinant virus displayed similar growth kinetics and ability to cause cytopathic effects as the parental virus.Luciferase reporter was then incorporated to generate both full-length and subgenomic replicon(SGR)reporter viruses.The full-length reporter virus is suitable for high-throughput antiviral screening,while the SGR is a useful tool to study viral-host interactions.More importantly,the full-length reporter virus has also been shown to infect the suckling mouse model and the reporter gene could be detected using an in vivo imaging system,thus providing a powerful tool to track viruses in vivo.In summary,we have generated coxsackievirus B5 reporter viruses and provided unique tools for studying virus-host interactions in vitro and in vivo as well as for high-throughput screenings(HTS)to identify novel antivirals.展开更多
Background:To study the effects of the main diterpene esters in Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)on the transcriptional activity and protein expression of liver X receptor(LXR).Methods:The effe...Background:To study the effects of the main diterpene esters in Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)on the transcriptional activity and protein expression of liver X receptor(LXR).Methods:The effect of the main diterpene ester components in Semen Euphorbiae on the viability of HEK293 cells were studied by MTT assay.The LXR-Luc plasmid vector was transfected into HEK293 cells and treated with Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)for 24 h.The effect of the main diterpene ester components of Semen Euphorbiae on LXR-Luc luciferase activity was investigated by dual luciferase reporter gene system,and the expression of LXRαprotein was detected by Western Blot.Results:Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)could significantly reduce the relative luciferase activity(RLU)of LXRα,and the expression level of LXRαprotein was significantly down-regulated.Conclusion:Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)can inhibit the expression of LXR protein level,which may be achieved by inhibiting the transcriptional activity of LXR.展开更多
Background Osteosarcoma (OS) is the most common primary malignant tumor of bone. Mouse models of human OS can invariably provide greater insight into the complex mechanisms that underlie the development and pathogen...Background Osteosarcoma (OS) is the most common primary malignant tumor of bone. Mouse models of human OS can invariably provide greater insight into the complex mechanisms that underlie the development and pathogenesis of this aggressive tumor. Bioluminescence technology favored tracing cancer cells in vivo. In this study, an OS model was described and evaluated using human OS cell line, Saos2, labeled with luciferase (Saos2-1uc). Methods Saos2 cells were infected by lentivirus loading a firefly luciferase gene. Luciferase expression of Saos2-1uc cells was characterized both in vitro and in vivo. Specific biologic and oncologic features of Saos2-1uc cells were analyzed. The OS was established as orthotopic xenografts in nude mice. Both orthotopic tumors and spontaneous lung metastasis were analyzed. Results Tumorigenesis and spontaneous lung metastasis in nude mice could be monitored in vivo through in vivo imaging system. The enhancement in proliferation, migration and invasion abilities and the attenuation in adhesion ability were observed in Saos2-1uc cells compared with Saos2 cells. Furthermore, there were the up-regulation of Osteocalcin, CCRIO, CXCR1 and ID1 and the down-regulation of ALP, collagen I, CCR1, CCR3, CXCR3, NID and N-cadherin in Saos2-1uc cells compare to Saos2 cells. The rate of spontaneous lung metastasis in Saos2-1uc cells was higher than that in Saos2 cells, although without significant difference. Conclusions Lentivirus transfection may cause alteration of gene expression profiles and further biological functions. This model can be used in the elucidation of molecular mechanisms of tumorigenesis and the screening of new therapeutic agents.展开更多
SARS-CoV-2 main protease(M^(pro))is one of the most extensively exploited drug targets for COVID-19.Structurally disparate compounds have been reported as M^(pro) inhibitors,raising the question of their target specif...SARS-CoV-2 main protease(M^(pro))is one of the most extensively exploited drug targets for COVID-19.Structurally disparate compounds have been reported as M^(pro) inhibitors,raising the question of their target specificity.To elucidate the target specificity and the cellular target engagement of the claimed M^(pro) inhibitors,we systematically characterize their mechanism of action using the cell-free FRET assay,the thermal shift-binding assay,the cell lysate Protease-Glo luciferase assay,and the cell-based FlipGFP assay.Collectively,our results have shown that majority of the M^(pro) inhibitors identified from drug repurposing including ebselen,carmofur,disulfiram,and shikonin are promiscuous cysteine inhibitors that are not specific to M^(pro),while chloroquine,oxytetracycline,montelukast,candesartan,and dipyridamole do not inhibit M^(pro) in any of the assays tested.Overall,our study highlights the need of stringent hit validation at the early stage of drug discovery.展开更多
Background Developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1) remains a grand challenge after more than two decades of intensive effort. It is partially due to the lack of suitable a...Background Developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1) remains a grand challenge after more than two decades of intensive effort. It is partially due to the lack of suitable animal models for screening and prioritizing vaccine candidates. In this study, we aim to develop a mice model to test HIV-1 vaccine efficacy. Methods We constructed a recombinant vaccinia expressing firefly luciferase and HIV-1 Gag fusion protein based on Tiantan strain, an attenuated but replication-competent poxvirus (rTTV-lucgag). By quantifying the luciferase activity as its read out, we defined the biodistribution of Tiantan strain poxvirus in mice inoculated intraperitoneally and attempted to apply this model to evaluate the HIV-1 vaccine efficacy. Results Our data demonstrated that the rTTV-lucgag was able to express high level of luciferase (〈106 relative luciferase units (RLU)/mg protein) and HIV-1 Gag (〉3 folds increase comparing to the control). After intraperitoneal inoculation, this virus had dominant replication in the ovary, uterus, and cervix of mice and the luciferase activities in those organs are significantly correlated with viral titers (r^2=0.71, P 〈0.01). Pre-immunization with an HIV gag DNA vaccine reduced the luciferase activity in ovary from (6006±3141) RLU/mg protein in control group to (1538±463) RLU/mg protein in vaccine group (P=0.1969). Conclusions The luciferase activity in ovary could represent viral replication in vivo; this rTTV-lucgag/mice model may be suitable to assess the protective efficacy of cytotoxic T-cell responses to HIV Gag with less tedious work and high through-put.展开更多
Severe fever with thrombocytopenia syndrome(SFTS),caused by SFTS virus(SFTSV)infection,was first reported in 2010 in China with an initial fatality of up to 30%.The laboratory confirmation of SFTSV infection in terms ...Severe fever with thrombocytopenia syndrome(SFTS),caused by SFTS virus(SFTSV)infection,was first reported in 2010 in China with an initial fatality of up to 30%.The laboratory confirmation of SFTSV infection in terms of detection of viral RNA or antibody levels is critical for SFTS diagnosis and therapy.In this study,a new luciferase immunoprecipitation system(LIPS)assay based on p REN2 plasmid expressing SFTSV NP gene and tagged with Renilla luciferase(Rluc),was established and used to investigate the levels of antibody responses to SFTSV.Totally 464 serum samples from febrile patients were collected in the hospital of Shaoxing City in Zhejiang Province in 2019.The results showed that 82 of the 464 patients(17.7%)had antibody response to SFTSV,which were further supported by immunofluorescence assays(IFAs).Further,q RT-PCR and microneutralization tests showed that among the 82 positive cases,15 patients had viremia,10 patients had neutralizing antibody,and one had both(totally 26 patient).However,none of these patients were diagnosed as SFTS in the hospital probably because of their mild symptoms or subclinical manifestations.All the results indicated that at least the 26 patients having viremia or neutralizing antibody were the missed diagnosis of SFTS cases.The findings suggested the occurrence of SFTS and the SFTS incidence were higher than the reported level in Shaoxing in 2019,and that LIPS may provide an alternative strategy to confirm SFTSV infection in the laboratory.展开更多
The Middle East respiratory syndrome(MERS)is a lethal zoonosis caused by MERS coronavirus(MERS-CoV)and poses a significant threat to public health worldwide.Therefore,a rapid,sensitive,and specific serologic test for ...The Middle East respiratory syndrome(MERS)is a lethal zoonosis caused by MERS coronavirus(MERS-CoV)and poses a significant threat to public health worldwide.Therefore,a rapid,sensitive,and specific serologic test for detecting anti-MERS-CoV antibodies in both humans and animals is urgently needed for the successful management of this illness.Here,we evaluated various novel luciferase immunosorbent assays(LISA)based on nucleocapsid protein(NP)as well as fragments derived from spike protein(S)including subunit 1(S1),N terminal domain(NTD),receptorbinding domain(RBD)and subunit 2(S2)of S for the detection of MERS-CoV-specific IgG.Fusion proteins,including nanoluciferase(NLuc)and various fragments derived from the NP or S protein of MERS-CoV,were expressed in human embryonic kidney 293 T cells.LISAs that detected anti-MERS-CoV IgG were further developed using cell lysates expressing various fusion proteins.Panels of human or animal samples infected with MERS-CoV were used to analyze the sensitivity and specificity of various LISAs in reference to a MERS-CoV RT-PCR,commercial S1-based ELISA,and pseudovirus particle neutralization test(ppNT).Our results showed that the S1-,RBD-,and NP-LISAs were more sensitive than the NTD-and S2-LISAs for the detection of anti-MERS-CoV IgG.Furthermore,the S1-,RBD-,and NP-LISAs were more sensitive(by at least 16-fold)than the commercially available S1-ELISA.Moreover,the S1-,RBD-,and NPLISA specifically recognized anti-MERS-CoV IgG and did not cross-react with samples derived from other human CoV(OC43,229E,HKU1,NL63)-infected patients.More importantly,these LISAs proved their applicability and reliability for detecting anti-MERS-CoV IgG in samples from camels,monkeys,and mice,among which the RBD-LISA exhibited excellent performance.The results of this study suggest that the novel MERS-CoV RBD-and S1-LISAs are highly effective platforms for the rapid and sensitive detection of anti-MERS-CoV IgG in human and animal samples.These assays have the potential to be used as serologic tests for the management and control of MERS-CoV infection.展开更多
Conformational dynamics contribute importantly to enzyme catalysis,such that targeted conformational constraint may affect catalysis.Firefly luciferases undergo extensive structural change during catalysis;key residue...Conformational dynamics contribute importantly to enzyme catalysis,such that targeted conformational constraint may affect catalysis.Firefly luciferases undergo extensive structural change during catalysis;key residues form a hydrophobic pocket,excluding water and enabling maximally energetic light production.Point mutants almost always luminesce at longer wavelengths(lower energy)than the wild type.Conformational constraint,using dipeptide analogue 3 at a position critical for optimized excited state structure,produced luciferase emission at a shorter wavelength by∼10 nm.Incomparison,introduction of conformationally constrained analogues 4,5,or 7 afforded luciferases emitting at longer wavelengths,while a related unconstrained luciferase(analogue 6)exhibited wild-type emission.The constrained luciferases tested were more stable than the wild type.Protein modeling demonstrated that the“inside”or“outside”orientation of the conformationally constrained dipeptide led to the shorter or longer emission wavelength,respectively.More broadly,these results suggest that local conformational constraint can control specific elements of enzyme behavior,both in vitro and in vivo.This represents the first example of studying enzyme function by introducing conformationally constrained dipeptides at a specific protein position.The principles discovered here in luciferase modification will enable studies to control the wavelength emission and photophysical properties of modified luciferases.展开更多
Firefly luciferase (EC1. 13. 12. 7) from Photinus pyralis catalyzes oxydecarboxylation of D-luciferin in the presence of MgATP accompanied by visible light emission with the maximum wavelength of 562 nm. The total rea...Firefly luciferase (EC1. 13. 12. 7) from Photinus pyralis catalyzes oxydecarboxylation of D-luciferin in the presence of MgATP accompanied by visible light emission with the maximum wavelength of 562 nm. The total reaction is as follows:展开更多
Zika virus(ZIKV) causes rash, moderate fever, conjunctivitis, and arthralgia, and has serious connection with neurological complications;therefore, it is a major threat to public health. A rapid and supersensitive met...Zika virus(ZIKV) causes rash, moderate fever, conjunctivitis, and arthralgia, and has serious connection with neurological complications;therefore, it is a major threat to public health. A rapid and supersensitive method for detecting anti-ZIKV antibodies in humans and animals is thus urgently required. Here, we report an NS1-based luciferase immunosorbent assay(LISA), developed to detect ZIKV-specific IgG. Fusion proteins including a reporter Nano-luciferase(NLuc) and various fragments of ZIKV NS1 protein were expressed in 293 T cells. LISA was performed using the above cell lysates containing the expressed fusion proteins. Sample panels of humans and animals infected with ZIKV were examined for sensitivity of LISA, relative to those of ZIKV RT-PCR, commercial NS1-based ELISA, and micro-neutralization(MN) assays.Specificity and potential cross-reactivity were also evaluated using various convalescent serum samples derived from patients infected with dengue virus(DENV), Japanese encephalitis virus(JEV), and hepatitis C virus(HCV). Results indicated the optimal antigenic domain for anti-ZIKV IgG detection was located within 172–352 amino acids(aa) of ZIKV NS1 protein. NS1-based LISA performs better than commercial ELISA in anti-ZIKV Ig G detection. LISA was shown to be at least fourfold more sensitive than commercial ELISA, and could detect anti-ZIKV Ig G in various animal hosts without the need of species-specific labeled antibody. This novel assay is potentially useful for the rapid and sensitive detection of anti-ZIKV IgG in human and animal samples.展开更多
The 6-His tagged firefly luciferase was highly expressed in E. coli and purified to homogeneity by affinity chromatography and gel filtration. After immunizing Balb/c mice with the antigen, 6 hybridomas clones were fo...The 6-His tagged firefly luciferase was highly expressed in E. coli and purified to homogeneity by affinity chromatography and gel filtration. After immunizing Balb/c mice with the antigen, 6 hybridomas clones were found to secrete monoelonal antibodies (mAbs) and the mAbs were also purified separately. The competitive binding experiments show that 2 mAbs can bind heat-denatured antigen or its proteolytic fragments but not the native lueiferase, suggesting that their epitopes might be accommodated in the internal segments of the protein. On the other hand, the other 4 mAbs are capable of binding both native and denatured antigens. It infers that their epitopes locate in the segments on the protein surface. The results also suggest that the six mAbs are all sequence-specific.展开更多
文摘Objective To improve the chemically-activated luciferase expression (CALUX)bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms ofDLCs. Method A recombinant vector was constructed and used to transfect humanhepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor(AhR)-meditated luciferase gene expression. The reliability of luciferase induction in thiscell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection timewas examined and a comparison was made by using the commonly used ethoxyresoufin-O-deethylase (EROD) activity induction assay. Result The results suggested that theluciferase activity in recombinant cells was peaked at about 4 h and then decreased to astable activity by 14 h after TCDD treatment. The detection limit of this cell line was0.11pmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%.Conclusion The luciferase induction is 30-fold more sensitive than EROD induction, thedetection time is 68 h shorter and the detection procedure is also simpler.
基金supported by grant-from the National Natural Science Foundation of China(81101717)Animal Plarform Project of Department of Science and Technology of Zhejiang Province(2012C37081)+1 种基金Doctoral Fund of Ministry of Education of China(20110101120111)Zhejiang Medical and Health Science and Technology Plan(2013KYB086)
文摘Objective:To construct a PSA luciferase report plasmid and monitor the growth and metastasis of prostate cancer after emasculation in SCID mice.Methods:PSA promoter sequence and luciferase gene were amplified by PCR and subsequently inserted into pZsCreen1-1 vector to construct pPSA-FL-Luc vector.LNCaP cells that were stably transfected with pPSA-FL-Luc were used to establish a SCID mouse xenograft model.Then,the growth and metastasis of prostate cancer were monitored via living imaging.Results:We successfully constructed a PSA luciferase piasmid,pPSA-FL-Luc.DHT enhanced lucifcrase activity in a concentration-dependent manner in 293 T cells with pPSA-FL-Luc transfection.Prostate cancer SCID mouse model was established with pPSA-FL-Luc transfected LNCaP cells.In tumor bearing mice with or without emasculation,pPSA-FL-Lue piasmid was applied to monitored tumor growth and metastasis based on bioluminescence imaging.Conclusions:We construct a pPSA-FL-Luc piasmid,which stably expresses luciferase and can be applied to monitor tumor development in a prostate SCID mouse model.
基金Supported by the National Natural Science Foundation of China,No.81672041the National Major Science and Technology Special Project for Infectious Diseases of China,No.2012ZX10004503-012
文摘BACKGROUND Previously,we have successfully constructed replication-competent hepatitis B virus(HBV)vectors by uncoupling the P open reading frame(ORF)from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence.Consequently,the replication-competent HBV vectors carrying foreign genes,including pCH-BsdR,carrying blasticidin resistance gene(399 bp),and pCH-hrGFP,carrying humanized renilla green fluorescent protein gene(720 bp),were successfully obtained.However,the replication efficiency of the former is higher but it is tedious to use,while that of the latter is poor and cannot be quantified.Hence,we need to search for a new reporter gene that is convenient and quantifiable for further research.AIM To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies.METHODS We utilized the replication-competent HBV viral vectors constructed by our laboratory,combined with the secreted luciferase reporter gene,to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase(SecNluc).HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene.RESULTS The replication-competent HBV vector carrying the SecNluc reporter gene pCHsNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression.HBV replication intermediates could be produced from this vector.Via transfection with pTRE-sNLuc and selection by hygromycin,we obtained isolated cell clones,named HBV-NLuc-35 cells,which could secrete secNLuc recombinant viruses,and were sensitive to existing anti-HBV drugs.Using differentiated HepaRG cells,it was verified that recombinant HBV possessed infectivity.CONCLUSION Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability,and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene.More importantly,the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.
基金supported by the National Key Technology R & D Program (2006BAK02A13)the National Basic Research Program of China (973Program, No.2007CB714507)
文摘A simple and rapid expression and purification method of recombinant firefly luciferase was developed for bacteria detection. A modified luciferase gene from North American firefly Photinus pyralis was cloned into pET28a expression vector and the recombinant protein was produced in Escherichia coli BL21. The recombinant luciferase,equipped with a polyhistidine affinity tag,was purified by immobilized metal ion affinity chromatography (IMAC). The approach generated an abundant expression and an efficient purification of a recombinant luciferase with final yield 1.995mg/L of cell culture. Experiments on the recombinant luciferase also showed that the relative light units (RUL) of the enzyme were 5.8×108,and the specific activity was 2.9×1010 RLU/mg. By applying adenosine triphosphate (ATP) bioluminescence to detection of the coin bacteria using the recombinant protein,the ATP content of bacteria was 9.48×10-16mol/mL,and was identical to the bacteria counts (4500CFU/mL) in order of magnitude. Taken together,our results provided a simple and efficacious method of the preparation of recombinant luciferase,which could be applied in the determination of bacteria via ATP bioluminescence.
基金Supported by the National Key Basic Research and Development (973) Plan (2011CB100804)Northeast Agricultural University Innovation Team Project (CXT005-1-1/CXT005-1-2)
文摘To analyze miR-139 target sites in 3' UTR of GHR gene in dairy cow mammary gland, a GHR 3' UTR- luciferase reporter vector was constructed and the effect of miRNA on its activity was evaluated in dairy cow mammary gland epithelial cells (DCMECs). The miR-139 targeting GHR 3' UTR was predicted by Target Scan 5.1 software, 3' UTR fragment of GHR was amplified by PCR from RNA of DCMECs. PCR products were cloned into Spe Ⅰ/Hind Ⅱ modified pMIR-Report vector. The luciferase reporter vector and miRNA eukaryotic expression vector were transferred into DCMECs using lipofectamine 2000 transfection reagent. The dualluciferase reporter assay system was used to quantitiate the reporter activity. The results showed that a 107 bp 3' UTR fragment of GHR gene was successfully cloned into the pMIR-Report vector, which authenticated by Spe Ⅰ/Hind Ⅲ digestion and DNA sequencing. The luciferase activity of reporter construction treated with miR-139 decreased 20.87% compared with the control group. It was concluded that the GHR3' UTR-luciferase reporter vector had been successfully constructed. The luciferase activity of the reporter could be suppressed by miR- 139.
基金the grants from Russian Science Foundation(№14-50-00068).
文摘The Aryl hydrocarbon receptor(AhR)ligands exhibiting modulating activity represents a new class of anticancer agents that can be directed towards several tumors.We have examined AhR expression in human colon cancer and adjacent non-tumor tissue.AhR expression level was about 2-7 times higher in tumor tissue samples than in the adjacent non-tumor samples(in 82%of all the samples).We were unable to find any increase of ABCG2 expression on the level of the transcription,while the expression of MDR2 was increased in half of the tumors compared to the levels of expression in normal adjacent tissue.We have used FICZ as a potent high affinity ligand of the AhR to calibrate the reporter cell line HEK293TAhR-luc as a potent high affinity ligand of the AhR.The concentration of xenobiotic response element(XRE)ligands is higher,than in the blood of healthy people in 86%of the patients.The proposed test system will allow the use of the AhR ligand level as an additional diagnostic marker in the treatment of colon cancer.
基金supported by the National Natural Science Foundation of China (82272309).
文摘Coxsackievirus belongs to the Picornaviridae family and is one of the major pathogens that cause hand,foot and mouth disease(HFMD)in infants and children with potential serious complications and even deaths.The pathogenesis of this virus is not fully elucidated and no vaccine or antiviral drug has been approved.In this study,a full-length infectious cDNA clone of coxsackievirus B5 virus was assembled and the recombinant virus displayed similar growth kinetics and ability to cause cytopathic effects as the parental virus.Luciferase reporter was then incorporated to generate both full-length and subgenomic replicon(SGR)reporter viruses.The full-length reporter virus is suitable for high-throughput antiviral screening,while the SGR is a useful tool to study viral-host interactions.More importantly,the full-length reporter virus has also been shown to infect the suckling mouse model and the reporter gene could be detected using an in vivo imaging system,thus providing a powerful tool to track viruses in vivo.In summary,we have generated coxsackievirus B5 reporter viruses and provided unique tools for studying virus-host interactions in vitro and in vivo as well as for high-throughput screenings(HTS)to identify novel antivirals.
基金supported by National Natural Science Foundation of China(Grant No.82074021).
文摘Background:To study the effects of the main diterpene esters in Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)on the transcriptional activity and protein expression of liver X receptor(LXR).Methods:The effect of the main diterpene ester components in Semen Euphorbiae on the viability of HEK293 cells were studied by MTT assay.The LXR-Luc plasmid vector was transfected into HEK293 cells and treated with Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)for 24 h.The effect of the main diterpene ester components of Semen Euphorbiae on LXR-Luc luciferase activity was investigated by dual luciferase reporter gene system,and the expression of LXRαprotein was detected by Western Blot.Results:Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)could significantly reduce the relative luciferase activity(RLU)of LXRα,and the expression level of LXRαprotein was significantly down-regulated.Conclusion:Euphorbia factor L_(1),L_(2),L_(3),L_(7a),L_(7b)and L_(8)can inhibit the expression of LXR protein level,which may be achieved by inhibiting the transcriptional activity of LXR.
基金the grants from the National Natural Science Foundation of China,the Shanghai Science and Technology Development Fund,the Program of Key Disciplines of Shanghai Municipal Education Commission,the Doctoral Innovation Foundation of Shanghai Jiaotong University
文摘Background Osteosarcoma (OS) is the most common primary malignant tumor of bone. Mouse models of human OS can invariably provide greater insight into the complex mechanisms that underlie the development and pathogenesis of this aggressive tumor. Bioluminescence technology favored tracing cancer cells in vivo. In this study, an OS model was described and evaluated using human OS cell line, Saos2, labeled with luciferase (Saos2-1uc). Methods Saos2 cells were infected by lentivirus loading a firefly luciferase gene. Luciferase expression of Saos2-1uc cells was characterized both in vitro and in vivo. Specific biologic and oncologic features of Saos2-1uc cells were analyzed. The OS was established as orthotopic xenografts in nude mice. Both orthotopic tumors and spontaneous lung metastasis were analyzed. Results Tumorigenesis and spontaneous lung metastasis in nude mice could be monitored in vivo through in vivo imaging system. The enhancement in proliferation, migration and invasion abilities and the attenuation in adhesion ability were observed in Saos2-1uc cells compared with Saos2 cells. Furthermore, there were the up-regulation of Osteocalcin, CCRIO, CXCR1 and ID1 and the down-regulation of ALP, collagen I, CCR1, CCR3, CXCR3, NID and N-cadherin in Saos2-1uc cells compare to Saos2 cells. The rate of spontaneous lung metastasis in Saos2-1uc cells was higher than that in Saos2 cells, although without significant difference. Conclusions Lentivirus transfection may cause alteration of gene expression profiles and further biological functions. This model can be used in the elucidation of molecular mechanisms of tumorigenesis and the screening of new therapeutic agents.
基金This research was supported by the National Institute of Allergy and Infectious Diseasess at the National Instiute of Health(NIH,USA,grants AI147325,AI157046,and AI158775)the Arizona Biomedical Research Centre Young Investigator grant(ADHS18-198859,USA)to Jun Wang.
文摘SARS-CoV-2 main protease(M^(pro))is one of the most extensively exploited drug targets for COVID-19.Structurally disparate compounds have been reported as M^(pro) inhibitors,raising the question of their target specificity.To elucidate the target specificity and the cellular target engagement of the claimed M^(pro) inhibitors,we systematically characterize their mechanism of action using the cell-free FRET assay,the thermal shift-binding assay,the cell lysate Protease-Glo luciferase assay,and the cell-based FlipGFP assay.Collectively,our results have shown that majority of the M^(pro) inhibitors identified from drug repurposing including ebselen,carmofur,disulfiram,and shikonin are promiscuous cysteine inhibitors that are not specific to M^(pro),while chloroquine,oxytetracycline,montelukast,candesartan,and dipyridamole do not inhibit M^(pro) in any of the assays tested.Overall,our study highlights the need of stringent hit validation at the early stage of drug discovery.
基金This research was supported by the National Natural Science Foundation of China (No. 30771915), the National Grand Program on Key Infectious Disease (No. 2008ZX10001-02 Project 2 and 2008ZX10001-012) from the China Ministry of Health & Ministry of Sciences and Technology, China.
文摘Background Developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1) remains a grand challenge after more than two decades of intensive effort. It is partially due to the lack of suitable animal models for screening and prioritizing vaccine candidates. In this study, we aim to develop a mice model to test HIV-1 vaccine efficacy. Methods We constructed a recombinant vaccinia expressing firefly luciferase and HIV-1 Gag fusion protein based on Tiantan strain, an attenuated but replication-competent poxvirus (rTTV-lucgag). By quantifying the luciferase activity as its read out, we defined the biodistribution of Tiantan strain poxvirus in mice inoculated intraperitoneally and attempted to apply this model to evaluate the HIV-1 vaccine efficacy. Results Our data demonstrated that the rTTV-lucgag was able to express high level of luciferase (〈106 relative luciferase units (RLU)/mg protein) and HIV-1 Gag (〉3 folds increase comparing to the control). After intraperitoneal inoculation, this virus had dominant replication in the ovary, uterus, and cervix of mice and the luciferase activities in those organs are significantly correlated with viral titers (r^2=0.71, P 〈0.01). Pre-immunization with an HIV gag DNA vaccine reduced the luciferase activity in ovary from (6006±3141) RLU/mg protein in control group to (1538±463) RLU/mg protein in vaccine group (P=0.1969). Conclusions The luciferase activity in ovary could represent viral replication in vivo; this rTTV-lucgag/mice model may be suitable to assess the protective efficacy of cytotoxic T-cell responses to HIV Gag with less tedious work and high through-put.
基金supported by the National Program on Key Research Project of China(2018YFE0200400,2019YFC1200700)the National Natural Science Foundation of China(U20A20135)+1 种基金the Strategic Biological Resources Capacity Building Project of Chinese Academy of Sciences(KFJ-BRP-017-06)the Key deployment projects of Chinese Academy of Sciences(KJZD-SW-L11)
文摘Severe fever with thrombocytopenia syndrome(SFTS),caused by SFTS virus(SFTSV)infection,was first reported in 2010 in China with an initial fatality of up to 30%.The laboratory confirmation of SFTSV infection in terms of detection of viral RNA or antibody levels is critical for SFTS diagnosis and therapy.In this study,a new luciferase immunoprecipitation system(LIPS)assay based on p REN2 plasmid expressing SFTSV NP gene and tagged with Renilla luciferase(Rluc),was established and used to investigate the levels of antibody responses to SFTSV.Totally 464 serum samples from febrile patients were collected in the hospital of Shaoxing City in Zhejiang Province in 2019.The results showed that 82 of the 464 patients(17.7%)had antibody response to SFTSV,which were further supported by immunofluorescence assays(IFAs).Further,q RT-PCR and microneutralization tests showed that among the 82 positive cases,15 patients had viremia,10 patients had neutralizing antibody,and one had both(totally 26 patient).However,none of these patients were diagnosed as SFTS in the hospital probably because of their mild symptoms or subclinical manifestations.All the results indicated that at least the 26 patients having viremia or neutralizing antibody were the missed diagnosis of SFTS cases.The findings suggested the occurrence of SFTS and the SFTS incidence were higher than the reported level in Shaoxing in 2019,and that LIPS may provide an alternative strategy to confirm SFTSV infection in the laboratory.
基金This work was supported by the following grants:the National Major Project for Control and Prevention of Infectious Disease in China(No.2018ZX10101002 and 2018ZX10732401)the National Key Research and Development Program of China(No.2016YFD0500301 and 2017YFC1200503)。
文摘The Middle East respiratory syndrome(MERS)is a lethal zoonosis caused by MERS coronavirus(MERS-CoV)and poses a significant threat to public health worldwide.Therefore,a rapid,sensitive,and specific serologic test for detecting anti-MERS-CoV antibodies in both humans and animals is urgently needed for the successful management of this illness.Here,we evaluated various novel luciferase immunosorbent assays(LISA)based on nucleocapsid protein(NP)as well as fragments derived from spike protein(S)including subunit 1(S1),N terminal domain(NTD),receptorbinding domain(RBD)and subunit 2(S2)of S for the detection of MERS-CoV-specific IgG.Fusion proteins,including nanoluciferase(NLuc)and various fragments derived from the NP or S protein of MERS-CoV,were expressed in human embryonic kidney 293 T cells.LISAs that detected anti-MERS-CoV IgG were further developed using cell lysates expressing various fusion proteins.Panels of human or animal samples infected with MERS-CoV were used to analyze the sensitivity and specificity of various LISAs in reference to a MERS-CoV RT-PCR,commercial S1-based ELISA,and pseudovirus particle neutralization test(ppNT).Our results showed that the S1-,RBD-,and NP-LISAs were more sensitive than the NTD-and S2-LISAs for the detection of anti-MERS-CoV IgG.Furthermore,the S1-,RBD-,and NP-LISAs were more sensitive(by at least 16-fold)than the commercially available S1-ELISA.Moreover,the S1-,RBD-,and NPLISA specifically recognized anti-MERS-CoV IgG and did not cross-react with samples derived from other human CoV(OC43,229E,HKU1,NL63)-infected patients.More importantly,these LISAs proved their applicability and reliability for detecting anti-MERS-CoV IgG in samples from camels,monkeys,and mice,among which the RBD-LISA exhibited excellent performance.The results of this study suggest that the novel MERS-CoV RBD-and S1-LISAs are highly effective platforms for the rapid and sensitive detection of anti-MERS-CoV IgG in human and animal samples.These assays have the potential to be used as serologic tests for the management and control of MERS-CoV infection.
基金supported by research grants R01GM12367 and R35GM140819 from the National Institutes of General Medical Sciences,NIH.
文摘Conformational dynamics contribute importantly to enzyme catalysis,such that targeted conformational constraint may affect catalysis.Firefly luciferases undergo extensive structural change during catalysis;key residues form a hydrophobic pocket,excluding water and enabling maximally energetic light production.Point mutants almost always luminesce at longer wavelengths(lower energy)than the wild type.Conformational constraint,using dipeptide analogue 3 at a position critical for optimized excited state structure,produced luciferase emission at a shorter wavelength by∼10 nm.Incomparison,introduction of conformationally constrained analogues 4,5,or 7 afforded luciferases emitting at longer wavelengths,while a related unconstrained luciferase(analogue 6)exhibited wild-type emission.The constrained luciferases tested were more stable than the wild type.Protein modeling demonstrated that the“inside”or“outside”orientation of the conformationally constrained dipeptide led to the shorter or longer emission wavelength,respectively.More broadly,these results suggest that local conformational constraint can control specific elements of enzyme behavior,both in vitro and in vivo.This represents the first example of studying enzyme function by introducing conformationally constrained dipeptides at a specific protein position.The principles discovered here in luciferase modification will enable studies to control the wavelength emission and photophysical properties of modified luciferases.
文摘Firefly luciferase (EC1. 13. 12. 7) from Photinus pyralis catalyzes oxydecarboxylation of D-luciferin in the presence of MgATP accompanied by visible light emission with the maximum wavelength of 562 nm. The total reaction is as follows:
基金the National Key Research and Development Program of China(2016YFD0500301)the National Major Project for Control and Prevention of Infectious Disease in China(2018ZX10101002 and 2017YFC1200503)+1 种基金the Bureau of Science and Information Technology of Guangzhou Municipality,China(201604020011,201704020219)National Key R&D Program of China(2018ZX10732401).
文摘Zika virus(ZIKV) causes rash, moderate fever, conjunctivitis, and arthralgia, and has serious connection with neurological complications;therefore, it is a major threat to public health. A rapid and supersensitive method for detecting anti-ZIKV antibodies in humans and animals is thus urgently required. Here, we report an NS1-based luciferase immunosorbent assay(LISA), developed to detect ZIKV-specific IgG. Fusion proteins including a reporter Nano-luciferase(NLuc) and various fragments of ZIKV NS1 protein were expressed in 293 T cells. LISA was performed using the above cell lysates containing the expressed fusion proteins. Sample panels of humans and animals infected with ZIKV were examined for sensitivity of LISA, relative to those of ZIKV RT-PCR, commercial NS1-based ELISA, and micro-neutralization(MN) assays.Specificity and potential cross-reactivity were also evaluated using various convalescent serum samples derived from patients infected with dengue virus(DENV), Japanese encephalitis virus(JEV), and hepatitis C virus(HCV). Results indicated the optimal antigenic domain for anti-ZIKV IgG detection was located within 172–352 amino acids(aa) of ZIKV NS1 protein. NS1-based LISA performs better than commercial ELISA in anti-ZIKV Ig G detection. LISA was shown to be at least fourfold more sensitive than commercial ELISA, and could detect anti-ZIKV Ig G in various animal hosts without the need of species-specific labeled antibody. This novel assay is potentially useful for the rapid and sensitive detection of anti-ZIKV IgG in human and animal samples.
基金Project supported by the National Climbing Project of China.
文摘The 6-His tagged firefly luciferase was highly expressed in E. coli and purified to homogeneity by affinity chromatography and gel filtration. After immunizing Balb/c mice with the antigen, 6 hybridomas clones were found to secrete monoelonal antibodies (mAbs) and the mAbs were also purified separately. The competitive binding experiments show that 2 mAbs can bind heat-denatured antigen or its proteolytic fragments but not the native lueiferase, suggesting that their epitopes might be accommodated in the internal segments of the protein. On the other hand, the other 4 mAbs are capable of binding both native and denatured antigens. It infers that their epitopes locate in the segments on the protein surface. The results also suggest that the six mAbs are all sequence-specific.