Cloning of BjNAC102 Promoter and Construction of Expression Vector in Brassica juncea

Transcription factor NAC102 plays an important role in the abiotic stress responses of plants.In this study,the promoter sequence of 3000 bp located in the upstream of the BjNAC102 gene was cloned from Brassica juncea‘Sichuan Yellow Seed’by using the homologous cloning method.The expression vector of the GUS gene driven by the BjNAC102 promoter was constructed by seamless cloning technology.The results showed that the sequence of the promoter of the BjNAC102 gene contained many cis-acting elements involved in light responsiveness,gibberellinresponsive element,and auxin-responsive element.It was speculated that BjNAC102 played an important role in the abiotic stress response in Brassica juncea.The expression vector of the promoter of the BjNAC102 gene was constructed,which layed a foundation for further studies of the expression pattern of the BjNAC102 gene in Brassica juncea.

Intramammary expression and therapeutic effect of a human lysozyme-expressing vector for treating bovine mastitis

To develop a gene therapy strategy for treating bovine mastitis, a new mammary-specific vector containing human lysozyme (hLYZ) cDNA and kanamycin resistance gene was constructed for intramammary expression and clinical studies. After one time acupuncture or intracisternal infusion of healthy cows with 400 μg of the p215C3LYZ vector, over 2.0 μg/ml of rhLYZ could be detected by enzymatic assay for about 3 weeks in the milk samples. Western blotting showed that rhLYZ secreted into milk samples from the vector-injected cows had molecular weight similar to that of the natural hLYZ in human colostrums. Twenty days after the primary injection, the quarters were re-injected with the same vector by quarter acupuncture and even higher concentrations of rhLYZ could be detected. Indirect competitive ELISA of milk samples showed that the vector injection did not induce detectable humoral immune response against hLYZ. Clinical studies showed that twice acupuncture of quarters with the p215C3LYZ vector had overt therapeutic effect on clinical and subclinical mastitis previously treated with antibiotics, including disappearance of clinical symptoms and relatively high microbiological cure rates. These data provide a solid rationale for using the vector to develop gene therapy for treating bovine mastitis.

Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B

BACKGROUND: Melanoma differentiation associated gene-7 (mda-7) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways. In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma (HCC) in vitro. METHODS: Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modified Eagle's medium without serum (control). The others were transfected with adenovirus expressing the mda-7 gene (Ad.mda-7) or adenovirus vector serving as negative control (Ad.vec). The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was confirmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and flow cytometry were used to assess tumor cell proliferation and the cell cycle. Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells. The expression of MDA-7, Bcl-2 and Bax proteins were detected by Western blotting. RESULT S: The mda-7 gene was expressed in Hep3B and L-02 cells. The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml, respectively. mda-7 induced Hep3B growth suppression and apoptosis, compared with Ad.mda-7 and control (P<0.01). In addition, cell block in G2/M was identified by exposure of HCC cells to secreted MDA-7 protein, but this was not found in L-02. The gene expression of Bcl-2 was markedly decreased in Hep3B but not in L-02. CONCLUSIONS: mda-7 selectively induces growth inhibition and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the antiapoptosis protein Bcl-2. It could be an ideal gene for gene therapy in HCC.

Construction of a novel Shigella live-vector strain co-expressing CS3 and LTB/STm of enterotoxigenic E.coli

AIM: To construct and evaluate a polyvalent recombinant vaccine strain Shigella flexneri2a T32 against enterotoxigenic E.coli/(ETEC). METHODS: By using a host-plasmid balanced lethal system based on asd gene, a polyvalent recombinant strain was constructed to highly express CS3 and regularly express fusion enterotoxin of LIB subunit and mutant ST (LTB/STm) in a vaccine strain Shigella flexneri 2a T32 with specific deletion of asd gene. Fimbria CS3 was observed by immunofluorescence and electron microscopy assay. The security of LTB/STm was examined by ileal loop assay and suckling mouse assay. To evaluate this new candidate vaccine, it was compared with a previous vaccine strain in plasmid and protein level, growth assay and immunogenicity in Balb/c mice. RESULTS: The newly constructed vaccine expressed CS3 and grew better than the previously constructed vaccine except for the lower expression of LTB/STm. Serum IgG and mucosal IgA against CS3, LTB, ST, and host lipopolysaccharide (LPS) were produced after immunization of Balb/c mice by oral route with the new strain. The titers were not significantly different from the Balb/c mice with the previous strain. CONCLUSION: This novel candidate diarrheal vaccine can effectively induce serum and mucosal antibody responses against ETEC and Shigella.

CONSTRUCTION AND IDENTIFICATION OF RETROVIRAL VECTOR EXPRESSING HUMAN INTERLEUKIN-17 GENE

Human interleukin 17(IL17) is a newly found cytokine secreted by activated T lymphocytes. Many of its characteristics remain unknown. To provide a way for understanding its role in immunology and hematology further, we constructed a recombinant retroviral vector pL17SN containing the human IL17 gene about 1.3kb with the coding region. The constructed retroviral vector packaged in CRIP cells could infect human primary fibroblasts, and could stimulate the primary fibroblasts secreting human GMCSF and IL6. The retroviral vector containing the human IL17 gene we constructed may present an efficient way to understand the biological functions of human IL17 and to investigate applications of IL17 in cancer gene therapy.

CONSTRUCTION OF THE DICISTRONIC ADENOVIRUS VECTOR EXPRESSING BIOACTIVE HUMAN INTERLEUKIN-12

The full-length cDNA encoding the subunits p40 and p35 of human interleukin12(hIL12) were cloned separately by RTPCR, linked together by internal ribosomal entry site (IRES) of encephalomyocarditis virus which initiates capindependent translation to form a dicistronic gene fragment. The dicistronic fragment was placed between the cytomegalovirus (CMV) promoter and SV40 polyA signal to form a dicistronic expression cassette. Subsequently, the dicistronic expression cassette was inserted into E1 region of Ad5 genome in cosmid vector pAx1cw of E1substitution type. By homologous recombination with EcoT22Idigested Ad5 DNATPC in 293 cells, the replicationdeficient recombinant adenoviruses of hIL12 were generated efficiently. After infected with hIL12 recombinant adenoviruses in vitro, 293 cells, human hepatocellular carcinoma cells HepG2, and primary human skin fibroblasts expressed and secreted hIL12 at comparable levels (30～60ng/ 106cells/24hr), which could stimulate the proliferation and IFNγ production of human lymphoblasts. These suggest that the dicistronic adenovirus vector of hIL12 could effectively mediate the expression of bioactive hIL12 and might be used in cancer gene therapy.

Construction and Identification of Human Tissue Kallikrein Gene Eukaryotic Expressing Vector

To clone and sequence the human tissue kallikrein gene of Chinese, and to construct eukaryotic expression recombinant of KK, total RNA was extracted from human pancreas and human tissue kallikrein gene cDNA was amplified by PCR after reverse-transcription by using Oligo（dT） primer. The original kallikrein cDNA was recovered and filled with Klenow enzyme and inserted into KS plasmid. After restriction endonuclease digestion, KK cDNA was sequenced by ABI 377 analyzer. Then the KK gene was amplified from pBluescript KSKK and inserted into pcDNA3. A sequence comparison showed that the cloned kallikrein gene was only one nucleotide different from that reported in the Genbank. The coding amino acid was Asp in the Genbank gene, while the coding amino acid of Chinese kallikrein gene was Asn. The KK cDNA fragment was inserted into the eukaryotic expression vector pcDNA3. The cloned kallikrein gene and the pcDNA3KK can be used for further study in gene therapy.

Construction and identification of a vector expressing TRAM siRNA in mammalian cells

Objective:To construct and identify a vector expressing TRAM siRNA in mammalian cells.Methods:It was constructed that the vector named R-pSUPER-EGFP used to transcribe functional TRAM siRNA. Two of pair 64 nt TRAM gene specific target sequences were inserted into the downstream of the H1 promoter. The recombinants were transformed into E. coli JM109, and finally their veracity was confirmed by double cutting with the enzymes and sequencing. R-pSUPER-EGFP was transfected into RAW264.7 cell by using Lipofectamine TM2000, and the expression of TRAM was detected by Western blotting. Results:Two different recombinant plasmids containing corresponding TRAM gene specific target sequences were constructed, transfected into RAW264.7 cell line successively, which can specifically restrain expression of TRAM protein. Conclusion:The optimizing method in constructing the recombinant vector serves other plasmid-based RNA interference research. Therefore, the recombinant vectors establish the basis for research on the function of TRAM gene.

Recombinant adenoviral vector expressing the tumor necrosis factor-related apoptosis-inducing ligand gene suppresses human pancreatic cancer growth

Objective: To investigate the antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene transfection mediated by adenovirus into human pancreatic carcinoma cell line Panc-1, and the mechanisms involved in this effect. Methods: TRAIL gene was transfected into pancreatic cancer cell line Panc-1 by an adenovirus vector (Ad-TRAIL). Level of TRAIL mRNA expression was determined using RT-PCR, and TRAIL protein synthesis was evaluated with Western blot. Cell-growth activities were determined by MTT assay. The bystander effect was observed by co-culturing the Panc-1 cells with the transfected TRAIL gene at different ratios. Apoptosis in pancreatic cancer cells was detected by flow cytometry. Procaspase-8 and procaspase-3 were determined by Western blot. Results: The stable overexpression of TRAIL was de-tected in Panc-1 cells transfected by Ad-TRAIL. Ad-TRAIL significantly inhibited of cell viability of Panc-1 cells. Furthermore, co-culture of cancer cells transfected with TRAIL with that nontransfected resulted in the cell death of both cells by bystander effect. Moreover, the percentage of apoptotic cells was significantly higher in the Ad-TRAIL-treatment group compared to the control groups (P < 0.01). And there was a diminished amount of procaspase-8 and procaspase-3 after infection with Ad-TRAIL. Conclusion: The overexpression of TRAIL gene in Panc-1 cells by Ad-TRAIL exerts its antitumor effects, and the mechanisms involved in this effect may be proapoptosis and bystander effect.

The construction and application of the adenovirus vector coexpressing the bioactlve heterodimers of human IL-12

Interleukin-12, a heterodimer produced mainlyby antigen presenting cells (APCs), plays vital rolesin the induction of Thl-mediated immune responsesand has the potential to treat cancer and infectiousdiseases. Systemic administration of IL-12 wasobserved to be associated with severe toxicity. To takefull advantage of its immunoregulatory activities andminimize its toxicities, local administration of IL-12may be the ideal approach. Intratumoral injection

Glutamatergic CYLD deletion leads to aberrant excitatory activity in the basolateral amygdala:association with enhanced cued fear expression

Neuronal activity,synaptic transmission,and molecular changes in the basolateral amygdala play critical roles in fear memory.Cylindromatosis(CYLD)is a deubiquitinase that negatively regulates the nuclear factor kappa-B pathway.CYLD is well studied in non-neuronal cells,yet underinvestigated in the brain,where it is highly expressed.Emerging studies have shown involvement of CYLD in the remodeling of glutamatergic synapses,neuroinflammation,fear memory,and anxiety-and autism-like behaviors.However,the precise role of CYLD in glutamatergic neurons is largely unknown.Here,we first proposed involvement of CYLD in cued fear expression.We next constructed transgenic model mice with specific deletion of Cyld from glutamatergic neurons.Our results show that glutamatergic CYLD deficiency exaggerated the expression of cued fear in only male mice.Further,loss of CYLD in glutamatergic neurons resulted in enhanced neuronal activation,impaired excitatory synaptic transmission,and altered levels of glutamate receptors accompanied by over-activation of microglia in the basolateral amygdala of male mice.Altogether,our study suggests a critical role of glutamatergic CYLD in maintaining normal neuronal,synaptic,and microglial activation.This may contribute,at least in part,to cued fear expression.

Experimental study on siRNA expressing vector-based RNA interference targeting c-Myc in human hepatocellular carcinoma cell line BEL-7402

Objective: To explore the inhibition of the expression of c-Myc in human hepatocellular carcinoma via vector-based RNA interference (RNAi). Methods: Two RNA interference DNA templates targeting c-Myc oncogene were designed via online software and synthesized. By ligation, the fragments were inserted into pSilencer 1.0-U6 to construct the recombinant siRNA expressing plasmids. The identified recombinants were introduced into BEL-7402 cells with Lipofactamine. The inhibition of c-Myc expression, together with the expression of CDK4, hTERT and Gadd45β in c-Myc down-regulated BEL-7402 cells, were analyzed by semi-quantitative RT-PCR. Results: Two recombinant plasmids pSic-myc-1 and pSic-myc-2, which direct the yields of siRNAs targeting c-Myc in cells, were constructed. Among which, pSic-myc-2 was shown to trigger a RNAi-mediated inhibition of expression of c-Myc in BEL-7402 by up to 90%. In c-Myc knockdown BEL-7402 cells, the expression of CDK4 and hTERT were down-regulated with a ratio of 85% and 57%, respectively, while the expression of Gadd45β was up-regulated by up to 110%. Conclusion: The expression of c-Myc in BEL-7402 could be suppressed by vector-based RNA interference successfully. The knockdown of c-Myc in turn resulted in the changes of expression of genes related to cell proliferation and apoptosis. Thus, our study provided a preliminary data in searching of a c-Myc-targeted RNAi therapy of human hepatocellular carcinoma.

Construction of a lentiviral vector over - expressing BDNF and its transfection into adipose derived stem cell lines in vitro

Objective:To construct a recombinant lentiviral vector that co-express ZsGreen and BDNF and establish an Adipose derived stem cell (ADSC) line with stable BDNF expression down- regulation.Methods: BDNF were designed and inserted into the lentiviral vector p HBLV-CMVIE-Zs Green-Puro. After identification by DNA sequencing, the lentiviral vectors carrying BDNF were packaged in 293 cells. The transfection efficiency was observed under fluorescence microscope;the lentiviral particles were collected to infect mouse ADSCs, and BDNF level were assessed using real-time PCR and Western blotting.Results: DNA sequencing demonstrated successful construction of the BDNF lentivirus vectors. Real- time PCR and Western blotting showed a high level BDNF mRNA and protein.Conclusion: We have successfully established an ADSC model with stable BDNF expression which establish the basis for the potential application of ADSCs-BDNF in the treatment of AD.

A Preliminary Analysis on the Construction and Expression of Eukaryotic Expression Vectors of Mytilin and Myticin from Mytilus coruscus

[Objective] The aim was to study the construction and expression of eukaryotic expression vectors of antibacterial peptides （mytilin and myticin） from Mytilus coruscus.[Method] By the screening of antibacterial peptide genes of mytilin and myticin of Mytilus coruscus,five antibacterial peptide genes were selected.Then,the relative eukaryotic expressing vectors were constructed by the use of PCR technique and DNA recombinant technology.Subsequently,they were transferred in to S78 Saccharomyces cerevisia by using LiAC transformation method,and then preliminary expressing analysis was carried out.[Result] Five eukaryotic expressing vectors of antibacterial peptides from Mytilus coruscus were successfully constructed,and the results of mRNA detection revealed that the five antibacterial peptides from Mytilus coruscus were successfully transcribed.[Conclusion] The results provide a basis for using genetic engineering to express antibacterial peptides of mytilin and myticin from Mytilus coruscus,and for developing the further study of antibacterial peptides from Mytilus coruscus based on this.

The Construction and Identification of Eukaryotic Expression Vector pEGFP-N1-hTERT

[Objective] The aim of this study is to construct eukaryotic expression vector pEGFP-N1-hTERT and observe its expression in eukaryotic cells.[Method]The eukaryotic expression vector pEGFP-N1-hTERT was constructed with pC1-neo-hTERT and pEGFP-N1 plasmids,and the accuracy of human telomerase reverse transcriptase(hTERT)gene fragment was confirmed by double enzyme digestion and DNA sequencing analysis.After transfecting pEGFP-N1-hTERT into rat fetal neural stem cells(NSCs),the protein localization of human telomerase reverse transcriptase were indirectly observed through green fluorescent protein in the cells,and the correctness of constructed pEGFP-N1-hTERT was certificated by RT-PCR and Western Blot analysis.[Result]The eukaryotic expression vector pEGFP-N1-hTERT had correct structure and could express in eukaryotic cells.[Conclusion]This study laid a foundation for the establishment of immortalized NSCs line in rats.

Construction and Identification of siRNA Expression Vector Targeting Nucleocapsid Protein N gene of PRRSV

[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucleocapsid protein N gone sequence of PRRSV were designed or synthesized, and then inserted into CMV promoter downstream to clone into pSilencer 4,1 -CMV eukaryotic expression vector. The recombinant expression vector was identified by enzyme digestion and DNA sequencing. [ Result] The results showed that the siRNA interference recombinant plasmid vector pSilencer-N targeting nucleocapsid protein gone expression had been successfully constructed. [ Conclusion] This study lays a foundation for studies on the controlling PRRSV by RNA interference technique .

Construction of Double Cross-over Expression Vector for Chloroplast Multicistron in Brassica napus L.

[Objective] This study aimed to construct Brassica napus chloroplast multi- cistron double cross-over expression vector, to lay the foundation for the genetic engi- neering research of Brassica napus chloroplast. [Method] Two primers were designed based on the known Brassica napus chloroplast DNA sequences AF267640 and Z50868 in GenBank. By using PCR method, two Brassica napus L. chloroplast DNA fragments were obtained, which were named RbcL and ACCD. The two Brassica na- pus chloroplast DNA homologous fragments were then cloned into plasmid pMD18-T to obtain recombinant plasmid pHBM715. Tandem expression cassette harboring spectinomycin-resistant gene aadA, mannanase gene man and green fluorescent pro- tein gene gfp was cloned into the plasmid pHBM715, thereby constructing Brassica napus chloroplast multicistron double cross-over expression vector pHBM716, which was transformed into Escherichia coil for expression and identification. [Result] Plate qualitative analysis was conducted for the functional identification of expression cas- sette in the constructed Brassica napus chloroplast multicistron double cross-over ex- pression vector, results showed that the three genes of the same multicistron were all expressed in E. coil [Conclusion] This study successfully constructed Brassica napus chloroplast multicistron double cross-over expression vector, which laid the foundation for the genetic engineering of Brassica napus chloroplast.

Construction of Prokaryotic Expression Vectors of EBP1 Gene from Nervilia Fordii (Hance) Schltr.

[Objective] To construct prokaryotic expression vectors encoding gene Erb3binding protein （EBP1）, which plays important roles in regulating plant organ size from Nervilia fordii （Hance） Schltr. [Methods] PCR products of NfEBP1 with particular restriction sites and expression vectors, pET-28 and pET-16b were digested. Ligation, transformation and selection were performed to construct the recombinant plasmids pET-28-NfEBP1 and pET-16-NfEBP1. The recombinant plasmids were transformed into E. coli BL21 using heat -shock transformation. [Results] Recombinant plasmids pET-28-NfEBP1-1188 and pET-16-NfEBP1-1188 were constructed and transformed into expressional host cells, E. coli BL21, and validated by colony PCR, sequencing and double digestion. [Conclusion] Prokaryotic expression vectors of EBP1 gene from N. fordii were successfully constructed, which laid the foundation for characterization of the gene function.

Construction of Recombinant Retroviral Vector Containing HIV-1 Tat Gene and Functional Detection of Expressed Tat in Target Cells

Objective: To construct recombinant retroviral vector containing HIV-1 Tatgene and evaluate the junction of the expressed Tat in target cells. Methods: HIV-1 Tat_(101) genewas recovered from pEV plasmid by Hind Ⅲ digestion and cloned into expression plasmid LZESpBMN-Z toconstruct recombinant retroviral expression plasmid named LZRS-Tat_(101). Using the method ofcalcium phosphate, the construct of LZRS-Tat_(101) was then transfected into packaging cell linesPhoenix (ΦNX) which contained env and gal genes encoding structural proteins and pol gene codingfor 3 enzymes ( reverse transcriptase, protease and integrate) essential for retroviral integrationand replication . The stable transfected cell lines was obtained using puromycin to screen for morethan 3 days. Then, immunohistochemical (IHC ) staining was carried out to detect the expressionlevel of Tat_(101) protein in both transiently and stably trancfected ΦNX, respectively. Thesupematants containing recombinant virus collected from transient and stable transfected cells wereemployed to infect 293 cells, respectively, and the expressed Tat in 293 cells was tested by Westernblot. Meantime, the supematants of infected 293 cells was further added to HL3T1 cells which wereHela cell lines containing an HIV-1-LTR/CAT reporter construct to establish a co-culture system.After co-culture for 72 hours, the protein was extracted from HL3T1 cells and used for CAT activityassay. Results: After LZRS- Tat_(101) was transfected into ΦNX, the amount of expressed Tat intransient transfection cells was significantly higher than that in stable transfection cells; Tatcould be detected not only in 293 cells but also in the supematants from 293 cells culture, and Tatin the supematants could activate HIV-1 LTR promoter in HL3T1, resulting in high 'expression of CATlocated at the downstream of LTR. Conclusion: The construct of recombinant retrovirus LZRS-Tat_(101) could express Tat protein in target cells and the expressed Tat was functionally activeand can really exhibit the ability to activate transcription.