According to the published gene sequence of the major surface antigen (P30) of Toxoplasma gondii, a pair of primers were designed and synthesized. Using polymerase chain reaction (PCR), the coding sequences of P30 gen...According to the published gene sequence of the major surface antigen (P30) of Toxoplasma gondii, a pair of primers were designed and synthesized. Using polymerase chain reaction (PCR), the coding sequences of P30 gene were amplified from a Chinese strain of T. gondii, The amplified gene fragment and plasmid pB220 were digested with EcoRI and BamHI and then ligated. The inserted gene fragment was sequenced by the chain termination method, the reading reveals that nucleotide sequence determined was the same as the P30 sequecne of RH strain pubilished by Burg (1988), except that one base was changed. The recombinant plasmid containing P30 gene was transformed to E. coli DH5α.After temperature inducing culture, the total cellular proteins were analysed by SDS-PAGE and Western blot. The results show that the p30 gene cloned into the plasmid could express in E. coli, and the expression product had immunogenicity.展开更多
To explore a novel strategy for antisense gene therapy of cancer,the coding sequence of hum an proliferating cell nuclear antigen(PCNA) c DNA was reversely inserted into the eukaryotic vector p L XSN by molecular cl...To explore a novel strategy for antisense gene therapy of cancer,the coding sequence of hum an proliferating cell nuclear antigen(PCNA) c DNA was reversely inserted into the eukaryotic vector p L XSN by molecular cloning techniques and transferred into bladder cancer EJcells with li- posome. The PCNA expression in transferred cells was dynamically detected by immunofluo- rescence and RT- PCR techniques. Changes of proliferation activities of cancer cells were assayed by MTT colorim etric and cloning formation m ethods.In the experiment,the antisense eukaryotic vector was successfully constructed and nam ed as p L APSN.After transfection with it for1- 7 days,PCNA protein and m RNA levels in cancer cells were blocked by16 .74 % - 84 .2 1% (P< 0 .0 5 ) and2 3.2 7% - 86 .15 % (P<0 .0 5 ) respectively.The proliferation activities of transferred cells were inhibited by 2 7.91% - 6 2 .0 7% (P<0 .0 1) ,with cloning formation abilities being de- creased by 5 0 .81% (P<0 .0 1) . Itwas concluded that the in vitro proliferation activities of cancer cells could be effectively inhibited by blocking PCNA expression with antisense technique,which could serve as an ideal strategy for gene therapy of bladder cancer.展开更多
Objective: To clone core gene cDNA of Chinese hepatitis C virus (HCV) into eukaryotic expression vector pTM3 and to express HCV core antigen in HepG2 cells. Methods: Core gene cDNA of HCV was introduced into eukaryoti...Objective: To clone core gene cDNA of Chinese hepatitis C virus (HCV) into eukaryotic expression vector pTM3 and to express HCV core antigen in HepG2 cells. Methods: Core gene cDNA of HCV was introduced into eukaryotic expression vector pTM3. Using vaccinia virus/bacteriophage T7 hybrid expression system, HepG2cells were trans feeted with the recombinant plasmid pTM3-Q534 by lipofectin. Results: From the transfected bacteria Top10F, 2pTM3-Q534 clones containing the recombinant plasmid were identified from randomly selected 10ampicillin-resistant colonies. By indirect immunofluorescence technique HCV core protein was identified in HepG2cells trans feeted with the recombinant plasmid. Conclusion: The construction of a recombinant plasmid and the expression of core gene cDNA of HCV in HepG2 were successful.展开更多
[ Objective ] This study aimed to clone and express formyltransferase (Wbkc) gene from Brucella abortus in E. coli, purify the expressed protein and analyze its immunogenicity. [Method] A gene encoding 27 -35 ku for...[ Objective ] This study aimed to clone and express formyltransferase (Wbkc) gene from Brucella abortus in E. coli, purify the expressed protein and analyze its immunogenicity. [Method] A gene encoding 27 -35 ku formyltransferase (Wbkc) was amplified from the genomic DNA of BruceUa abortus by PCR. The amplified fragments were digested with BamH I and Sal I, and inserted into pET28a vector. The constructed recombinant plasmid pET 28a-Wbkc was trans- formed into E. coli BL21 and was induced to express the fusion protein. Subsequently, the protein was purified by histidine-binding resin column chromatography, and the immunogenicity was detected by Western blot assay. The recombinant plasmid was identified by colony PCR, double digestion and sequencing analysis. [ Result] Wbkc was successfully cloned and expressed in E. coli. A specific protein band of 29 ku was detected by SDS-PAGE. Western blot showed specific im- munoreactivity of the purified fusion protein. [ Conclusion] This study provided a solid foundation for further investigating diagnostic proteins for brucellosis and developing Brucella gene-deletion vaccines.展开更多
AIM: To investigate the immunogenidty of a novel DNA vacoine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS: A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plas...AIM: To investigate the immunogenidty of a novel DNA vacoine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS: A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes (CTLs) of mice was quantitatively measured by lactate dehydrogenase release assay. RESULTS: HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1:97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector:target ratio of 20:1 in mice that received pSW3891/HBc DNA vaccine. CONCLUSION: pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.展开更多
AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vecto...AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method.RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control.Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively.CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).展开更多
INTRODUCTIONGastric epithelial dysplasia (GED) hypothetically is a straight-forward concept: dysplastic epithelium replacing the normal gastric epithelium of the stomach [1].In the stomach ,like any other segment of t...INTRODUCTIONGastric epithelial dysplasia (GED) hypothetically is a straight-forward concept: dysplastic epithelium replacing the normal gastric epithelium of the stomach [1].In the stomach ,like any other segment of the gut ,it is defined as an unequivocal non-invasive epithelial change[2,3].The observation of gastric dysplasia as a cancerous lesion was recognized over a century ago ,but it is only after the advent of gastroscopy that its clinical significance has been stressed[4-7].展开更多
Objective To investigate the expression of the preferentially expressed antigen of melanoma (PRAME) gene in acute leukemia and its clinical significance. Methods The level of expressed PRAME mRNA in bone marrow mono...Objective To investigate the expression of the preferentially expressed antigen of melanoma (PRAME) gene in acute leukemia and its clinical significance. Methods The level of expressed PRAME mRNA in bone marrow mononuclear cells from 34 patients with acute leukemia (AL) and in 12 bone marrow samples from healthy volunteers was measured via RT-PCR. Correlation analyses between PRAME gene expression and the clinical characteristics (gender, age, white blood count, immunophenotype of leukemia, percentage of blast cells, and karyotype) of the patients were performed. Results The PRAME gene was expressed in 38.2% of all 34 patients, in 40.7% of the patients with acute myelogenous leukemia (AML, n=27), and in 28.6% of the patients with acute lymphoblastic leukemia (ALL, n=7), but was not expressed in the healthy volunteers. The difference in the expression levels between AML and ALL patients was statistically significant. The rate of gene expression was 80% in M~, 33.3% in M2, and 28.6% in M~. Gene expression was also found to be correlated with CDl5 and CD33 expression and abnormal karyotype, but not with age, gender; white blood count or percentage of blast cells. Conclusions The PRAME gene is highly expressed in acute leukemia and could be a useful marker to monitor minimal residual disease. This gene is also a candidate target for the immunotherapy of acute leukemia.展开更多
To obtain the recombinant core domain of porcine zone pellucida 3β (cZP3β) for the further research on its functions Methods The nucleotide sequence region from 44 to 306 codons of pZP3β entire eDNA was obtained ...To obtain the recombinant core domain of porcine zone pellucida 3β (cZP3β) for the further research on its functions Methods The nucleotide sequence region from 44 to 306 codons of pZP3β entire eDNA was obtained by PCR and then was cloned into pET-3c vector. After being identified, recon was transformed into E.coli BL21 (DE3) pLysS and then induced by IPTG. Results The recombinant cZP3β was expressed in E. coli up to 15% of total cellular proteins, and was made sure by Western blot analysis. Conclusion The research on expression of core domain of pZP3β could benefit to further investigation of its immunogenicity and the development of antigen preparation.展开更多
[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the ...[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the GenBank,primers were designed for amplification of MPT-64 gene,and the recombinant plasmid pET-32a-MPT-64 was constructed.The recombinant plasmid was expressed in prokaryotic expression vector to obtain fusion protein.[Result]Protective antigen gene MPT-64 was successfully cloned.The recombinant plasmid pET-32a-MPT-64 was obtained.MPT-64 fusion protein was successfully expressed.[Conclusion]This study laid solid foundation for the prevention,diagnosis,treatment of tuberculosis and the development of tuberculosis vaccines.展开更多
Objective:To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods:MAGE...Objective:To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods:MAGE-A9 cDNA was cloned from human hepatocellular carcinoma tissue by using RT-PCR, and then subcloned into the plasmid pMD18-T. After sequencing, the MAGE-A9 was cloned into the prokaryotic expression vector pBAD/gⅢ to construct the recombinant expression vector pBAD/gⅢ - MAGE-A9, and was transformed into E. coli TOP10. The recombinant MAGE-A9 protein was expressed under induction of L-Arabinose, and was purified through Hitrap column. The anti-MAGE-A9 monoclonal antibody was generated. The expression of MAGE-A9 in hepatocellular carcinoma specimens was examined through ABC assay. Results:The cDNA sequence of the cloned MAGE-A9 gene was consistent with the reported sequence. By affinity column and SDS-PAGE, the purified MAGE-A9 fusion protein displayed a band of Mr 35,000, and subsequently the anti-MAGE-A9 monoclonal antibody was obtained. We found that MAGE-A9 expressed in the cytoplast of positive cells and MAGE-A9 antigen was detected in 8 cases out of 39 (21%) hepatocellular carcinoma specimens. Conclusion:MAGE-A9 antigen was expressed in a fair proportion of hepatocellular carcinoma specimens, these patients might be suitable candidates for immune involving antigen, encoded by the MAGE-A9 gene.展开更多
The Epstein-Barr virus membrane antigen was constructed and inserted into vaccinia virus, Tian-tan strain in order to study the effect of this virus on EB infection and tumorogenesis. The EBV-derived membrane antigen ...The Epstein-Barr virus membrane antigen was constructed and inserted into vaccinia virus, Tian-tan strain in order to study the effect of this virus on EB infection and tumorogenesis. The EBV-derived membrane antigen was expressed under the control of a 7.5 K promoter of vaccinia virus. The antibody against the membrane antigen of EB virus was produced on rabbits vaccinated with recombinant vaccinia virus.展开更多
The gene encoding the core and NS3 proteins of hepatitis C virus was amplified by PCR, respectirely. Two genes were fused and formed the recombinant plamid pHCV CN (Core+NS3) and pHCV NC (NS3+Core). The fused plasmid ...The gene encoding the core and NS3 proteins of hepatitis C virus was amplified by PCR, respectirely. Two genes were fused and formed the recombinant plamid pHCV CN (Core+NS3) and pHCV NC (NS3+Core). The fused plasmid were expressed in E.coli 06. The pHC CN plasmid expressed fussion protein was shown by a major band with a expected molecular weight about 68 kD on SDS PAGE. However, the pHCV NC plasmid expressed fussion protein was 66 kD in molecular weight. The results indicate that the pHCV NC fussion protein differs from the pHCV CN fussion protein in mulecular weight. It suggests that the fusion way is important for the structure of recombinant proteins.展开更多
AIM: To evaluate the relationship between the expression of lipopolysaccharides (LPS) binding protein (LBP) and CD14 mRNA and the severity of liver injury in alcohol-fed rats. METHODS: Twenty Wistar rats were divided ...AIM: To evaluate the relationship between the expression of lipopolysaccharides (LPS) binding protein (LBP) and CD14 mRNA and the severity of liver injury in alcohol-fed rats. METHODS: Twenty Wistar rats were divided into two groups:ethanol-fed group (group E) and control group (group C). Group E was fed with ethanol(5-12 g x kg(-1) x d(-1)) and group C received dextrose instead of ethanol. Rats of the two groups were sacrificed at 4 weeks and 8 weeks. Levels of endotoxin and alanine transaminase (ALT) in blood were measured, and liver pathology was observed under light and electronic microscopy. Expressions of LBP and CD14 mRNA in liver tissues were determined by RT-PCR analysis. RESULTS: Plasma endotoxin levels were increased more significantly in group E(129+/-21) ng x L(-1) and (187+/-35) ng x L(-1) at 4 and 8 wk than in control rats(48+/-9) ng x L(-1) and (53+/-11) ng x L(-1), respectively (P【0.05). Mean values of plasma ALT levels were (1867+/-250) nkat x L(-1) and (2450+/-367) nkat x L(-1) in Group E. The values were increased more dramatically in ethanol-fed rats than in Group C after 4 and 8 weeks. In liver section from ethanol-fed rats, there were marked pathological changes (steatosis, cell infiltration and necrosis). In ethanol-fed rats, ethanol administration led to a significant increase in LBP and CD14 mRNA levels compared with the control group (P【0.05). CONCLUSION: Ethanol administration led to a significant increase in endotoxin levels in serum and LBP and CD14 mRNA expressions in liver tissues. The increase of LBP and CD14 mRNA expression might wake the liver more sensitive to endotoxin and liver injury.展开更多
INTRODUCTIONFas/ FasL system has been identified as a keymediator of apoptosis in tumor cells[1-4]. Theoccurrence and development of neoplasm are closelyrelated to apoptosis[5-7] Most chemotherapeuticdrugs kill cancer...INTRODUCTIONFas/ FasL system has been identified as a keymediator of apoptosis in tumor cells[1-4]. Theoccurrence and development of neoplasm are closelyrelated to apoptosis[5-7] Most chemotherapeuticdrugs kill cancer cells mainly by inducingapoptosis[8-14].'展开更多
AIM:To evaluate how widely Helicobacter pylori (H. pylori ) HopE and HopV porins are expressed among Chilean isolates and how seroprevalent they are among infected patients in Chile.METHODS: H. pylori hopE and hopV ge...AIM:To evaluate how widely Helicobacter pylori (H. pylori ) HopE and HopV porins are expressed among Chilean isolates and how seroprevalent they are among infected patients in Chile.METHODS: H. pylori hopE and hopV genes derived from strain CHCTX-1 were cloned by polymerase chain reaction (PCR), sequenced and expressed in Escherichia coli AD494 (DE3). Gel-purified porins were used to prepare polyclonal antibodies. The presence of both genes was tested by PCR in a collection of H. pylori clinical isolates and their expression was detected in lysates by immunoblotting. Immune responses against HopE, HopV and other H. pylori antigens in sera from infected and non-infected patients were tested by Western blotting using these sera as f irst antibody on recombinant H. pylori antigens.RESULTS: PCR and Western blotting assays revealed that 60 and 82 out of 130 Chilean isolates carried hopE and hopV genes, respectively, but only 16 and 9, respectively, expressed these porins. IgG serum immunoreactivity evaluation of 69 H. pylori-infected patients revealed that HopE and HopV were infrequently recognized (8.7% and 10.1% respectively) compared to H. pylori VacA (68.1%) and CagA (59.5%) antigens. Similar values were detected for IgA serum immunoreactivity against HopE (11.6%) and HopV (10.5%) although lower values for VacA (42%) and CagA (17.4%) were obtained when compared to the IgG response.CONCLUSION: A scarce expression of HopE and HopV among Chilean isolates was found, in agreement with the infrequent seroconversion against these antigens when tested in infected Chilean patients.展开更多
BACKGROUND The relationship between hepatitis B surface antigen(HBsAg)-positive carrier status and liver cancer has been extensively studied.However,the epigenetic changes that occur during progression from HBsAg-posi...BACKGROUND The relationship between hepatitis B surface antigen(HBsAg)-positive carrier status and liver cancer has been extensively studied.However,the epigenetic changes that occur during progression from HBsAg-positive carrier status or cirrhosis to liver cancer are unknown.The epigenetic modification of DNA hydroxymethylation is critical in tumor development.Further,5-hydroxymethylcytosine(5hmC)is an important base for DNA demethylation and epigenetic regulation.It is also involved in the assembly of chromosomes and the regulation of gene expression.However,the mechanism of action of 5hmC in HBsAgpositive carriers or patients with cirrhosis who develop liver cancer has not been fully elucidated.AIM To investigate the possible epigenetic mechanism of HBsAg-positive carriers and hepatocellular carcinoma(HCC)progression from cirrhosis.METHODS Forty HBsAg-positive carriers,forty patients with liver cirrhosis,and forty patients with liver cancer admitted to the First People's Hospital of Yongkang between March 2020 and November 2021 were selected as participants.Free DNA was extracted using a cf-DNA kit.cfDNA was extracted by 5hmC DNA sequencing for principal component analysis,the expression profiles of the three groups of samples were detected,and the differentially expressed genes(DEGs)modified by hydroxymethylation were screened.Bioinformatic analysis was used to enrich DEGs,such as in biological pathways.RESULTS A total of 16455 hydroxymethylated genes were identified.Sequencing results showed that 32 genes had significant 5hmC modification differences between HBsAg carriers and liver cancer patients,of which 30 were upregulated and 2 downregulated in patients with HCC compared with HBsAg-positive carriers.Significant 5hmC modification differences between liver cirrhosis and liver cancer patients were identified in 20 genes,of which 17 were upregulated and 3 were downregulated in patients with HCC compared with those with cirrhosis.These genes may have potential loci that are undiscovered or unelucidated,which contribute to the development and progression of liver cancer.Analysis of gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes showed that the major signaling pathways involved in the differential genes were biliary secretion and insulin secretion.The analysis of protein interactions showed that the important genes in the protein-protein interaction network were phosphoenolpyruvate carboxykinase and solute carrier family 2.CONCLUSION The occurrence and development of liver cancer involves multiple genes and pathways,which may be potential targets for preventing hepatitis B carriers from developing liver cancer.展开更多
文摘According to the published gene sequence of the major surface antigen (P30) of Toxoplasma gondii, a pair of primers were designed and synthesized. Using polymerase chain reaction (PCR), the coding sequences of P30 gene were amplified from a Chinese strain of T. gondii, The amplified gene fragment and plasmid pB220 were digested with EcoRI and BamHI and then ligated. The inserted gene fragment was sequenced by the chain termination method, the reading reveals that nucleotide sequence determined was the same as the P30 sequecne of RH strain pubilished by Burg (1988), except that one base was changed. The recombinant plasmid containing P30 gene was transformed to E. coli DH5α.After temperature inducing culture, the total cellular proteins were analysed by SDS-PAGE and Western blot. The results show that the p30 gene cloned into the plasmid could express in E. coli, and the expression product had immunogenicity.
基金This studywassupported by a grant from the NationalNatural Sciences Foundation ofChina(No. 39770 739)
文摘To explore a novel strategy for antisense gene therapy of cancer,the coding sequence of hum an proliferating cell nuclear antigen(PCNA) c DNA was reversely inserted into the eukaryotic vector p L XSN by molecular cloning techniques and transferred into bladder cancer EJcells with li- posome. The PCNA expression in transferred cells was dynamically detected by immunofluo- rescence and RT- PCR techniques. Changes of proliferation activities of cancer cells were assayed by MTT colorim etric and cloning formation m ethods.In the experiment,the antisense eukaryotic vector was successfully constructed and nam ed as p L APSN.After transfection with it for1- 7 days,PCNA protein and m RNA levels in cancer cells were blocked by16 .74 % - 84 .2 1% (P< 0 .0 5 ) and2 3.2 7% - 86 .15 % (P<0 .0 5 ) respectively.The proliferation activities of transferred cells were inhibited by 2 7.91% - 6 2 .0 7% (P<0 .0 1) ,with cloning formation abilities being de- creased by 5 0 .81% (P<0 .0 1) . Itwas concluded that the in vitro proliferation activities of cancer cells could be effectively inhibited by blocking PCNA expression with antisense technique,which could serve as an ideal strategy for gene therapy of bladder cancer.
文摘Objective: To clone core gene cDNA of Chinese hepatitis C virus (HCV) into eukaryotic expression vector pTM3 and to express HCV core antigen in HepG2 cells. Methods: Core gene cDNA of HCV was introduced into eukaryotic expression vector pTM3. Using vaccinia virus/bacteriophage T7 hybrid expression system, HepG2cells were trans feeted with the recombinant plasmid pTM3-Q534 by lipofectin. Results: From the transfected bacteria Top10F, 2pTM3-Q534 clones containing the recombinant plasmid were identified from randomly selected 10ampicillin-resistant colonies. By indirect immunofluorescence technique HCV core protein was identified in HepG2cells trans feeted with the recombinant plasmid. Conclusion: The construction of a recombinant plasmid and the expression of core gene cDNA of HCV in HepG2 were successful.
基金Supported by National Natural Science Foundation of China(31260608)Key Science and Technology Project for Colleges and Universities in Inner Mongolia Autonomous Region(NJZZ12117)Scientific and Technological Cooperation Project between Tongliao City and Universities in Inner Mongolia Autonomous Region(SXZD2012131)
文摘[ Objective ] This study aimed to clone and express formyltransferase (Wbkc) gene from Brucella abortus in E. coli, purify the expressed protein and analyze its immunogenicity. [Method] A gene encoding 27 -35 ku formyltransferase (Wbkc) was amplified from the genomic DNA of BruceUa abortus by PCR. The amplified fragments were digested with BamH I and Sal I, and inserted into pET28a vector. The constructed recombinant plasmid pET 28a-Wbkc was trans- formed into E. coli BL21 and was induced to express the fusion protein. Subsequently, the protein was purified by histidine-binding resin column chromatography, and the immunogenicity was detected by Western blot assay. The recombinant plasmid was identified by colony PCR, double digestion and sequencing analysis. [ Result] Wbkc was successfully cloned and expressed in E. coli. A specific protein band of 29 ku was detected by SDS-PAGE. Western blot showed specific im- munoreactivity of the purified fusion protein. [ Conclusion] This study provided a solid foundation for further investigating diagnostic proteins for brucellosis and developing Brucella gene-deletion vaccines.
基金Supported by the 135 Project of Jiangsu Province, No. 044
文摘AIM: To investigate the immunogenidty of a novel DNA vacoine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS: A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes (CTLs) of mice was quantitatively measured by lactate dehydrogenase release assay. RESULTS: HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1:97 200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector:target ratio of 20:1 in mice that received pSW3891/HBc DNA vaccine. CONCLUSION: pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.
基金Supported by the National Natural Science Foundation of China, No. 30371270 the Major Program of Department of Science and Technology of Zhejiang Province, No. 2003C13015
文摘AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method.RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control.Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively.CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).
基金Supported by the Science Fund of Health Department,No.95A2141.and the Science Fund of Health Bureau of Shanghai.No.982019
文摘INTRODUCTIONGastric epithelial dysplasia (GED) hypothetically is a straight-forward concept: dysplastic epithelium replacing the normal gastric epithelium of the stomach [1].In the stomach ,like any other segment of the gut ,it is defined as an unequivocal non-invasive epithelial change[2,3].The observation of gastric dysplasia as a cancerous lesion was recognized over a century ago ,but it is only after the advent of gastroscopy that its clinical significance has been stressed[4-7].
文摘Objective To investigate the expression of the preferentially expressed antigen of melanoma (PRAME) gene in acute leukemia and its clinical significance. Methods The level of expressed PRAME mRNA in bone marrow mononuclear cells from 34 patients with acute leukemia (AL) and in 12 bone marrow samples from healthy volunteers was measured via RT-PCR. Correlation analyses between PRAME gene expression and the clinical characteristics (gender, age, white blood count, immunophenotype of leukemia, percentage of blast cells, and karyotype) of the patients were performed. Results The PRAME gene was expressed in 38.2% of all 34 patients, in 40.7% of the patients with acute myelogenous leukemia (AML, n=27), and in 28.6% of the patients with acute lymphoblastic leukemia (ALL, n=7), but was not expressed in the healthy volunteers. The difference in the expression levels between AML and ALL patients was statistically significant. The rate of gene expression was 80% in M~, 33.3% in M2, and 28.6% in M~. Gene expression was also found to be correlated with CDl5 and CD33 expression and abnormal karyotype, but not with age, gender; white blood count or percentage of blast cells. Conclusions The PRAME gene is highly expressed in acute leukemia and could be a useful marker to monitor minimal residual disease. This gene is also a candidate target for the immunotherapy of acute leukemia.
基金This study was supported by the Science & Technology Plan (No. 2001C12001) of Guangdong Province,P.R. China
文摘To obtain the recombinant core domain of porcine zone pellucida 3β (cZP3β) for the further research on its functions Methods The nucleotide sequence region from 44 to 306 codons of pZP3β entire eDNA was obtained by PCR and then was cloned into pET-3c vector. After being identified, recon was transformed into E.coli BL21 (DE3) pLysS and then induced by IPTG. Results The recombinant cZP3β was expressed in E. coli up to 15% of total cellular proteins, and was made sure by Western blot analysis. Conclusion The research on expression of core domain of pZP3β could benefit to further investigation of its immunogenicity and the development of antigen preparation.
基金Supported by Project of Science and Technology Development of Jilin Province(20140204018YY)
文摘[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the GenBank,primers were designed for amplification of MPT-64 gene,and the recombinant plasmid pET-32a-MPT-64 was constructed.The recombinant plasmid was expressed in prokaryotic expression vector to obtain fusion protein.[Result]Protective antigen gene MPT-64 was successfully cloned.The recombinant plasmid pET-32a-MPT-64 was obtained.MPT-64 fusion protein was successfully expressed.[Conclusion]This study laid solid foundation for the prevention,diagnosis,treatment of tuberculosis and the development of tuberculosis vaccines.
文摘Objective:To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods:MAGE-A9 cDNA was cloned from human hepatocellular carcinoma tissue by using RT-PCR, and then subcloned into the plasmid pMD18-T. After sequencing, the MAGE-A9 was cloned into the prokaryotic expression vector pBAD/gⅢ to construct the recombinant expression vector pBAD/gⅢ - MAGE-A9, and was transformed into E. coli TOP10. The recombinant MAGE-A9 protein was expressed under induction of L-Arabinose, and was purified through Hitrap column. The anti-MAGE-A9 monoclonal antibody was generated. The expression of MAGE-A9 in hepatocellular carcinoma specimens was examined through ABC assay. Results:The cDNA sequence of the cloned MAGE-A9 gene was consistent with the reported sequence. By affinity column and SDS-PAGE, the purified MAGE-A9 fusion protein displayed a band of Mr 35,000, and subsequently the anti-MAGE-A9 monoclonal antibody was obtained. We found that MAGE-A9 expressed in the cytoplast of positive cells and MAGE-A9 antigen was detected in 8 cases out of 39 (21%) hepatocellular carcinoma specimens. Conclusion:MAGE-A9 antigen was expressed in a fair proportion of hepatocellular carcinoma specimens, these patients might be suitable candidates for immune involving antigen, encoded by the MAGE-A9 gene.
文摘The Epstein-Barr virus membrane antigen was constructed and inserted into vaccinia virus, Tian-tan strain in order to study the effect of this virus on EB infection and tumorogenesis. The EBV-derived membrane antigen was expressed under the control of a 7.5 K promoter of vaccinia virus. The antibody against the membrane antigen of EB virus was produced on rabbits vaccinated with recombinant vaccinia virus.
文摘The gene encoding the core and NS3 proteins of hepatitis C virus was amplified by PCR, respectirely. Two genes were fused and formed the recombinant plamid pHCV CN (Core+NS3) and pHCV NC (NS3+Core). The fused plasmid were expressed in E.coli 06. The pHC CN plasmid expressed fussion protein was shown by a major band with a expected molecular weight about 68 kD on SDS PAGE. However, the pHCV NC plasmid expressed fussion protein was 66 kD in molecular weight. The results indicate that the pHCV NC fussion protein differs from the pHCV CN fussion protein in mulecular weight. It suggests that the fusion way is important for the structure of recombinant proteins.
基金Supported by the National Natural Science Foundation of China(No.39970719).
文摘AIM: To evaluate the relationship between the expression of lipopolysaccharides (LPS) binding protein (LBP) and CD14 mRNA and the severity of liver injury in alcohol-fed rats. METHODS: Twenty Wistar rats were divided into two groups:ethanol-fed group (group E) and control group (group C). Group E was fed with ethanol(5-12 g x kg(-1) x d(-1)) and group C received dextrose instead of ethanol. Rats of the two groups were sacrificed at 4 weeks and 8 weeks. Levels of endotoxin and alanine transaminase (ALT) in blood were measured, and liver pathology was observed under light and electronic microscopy. Expressions of LBP and CD14 mRNA in liver tissues were determined by RT-PCR analysis. RESULTS: Plasma endotoxin levels were increased more significantly in group E(129+/-21) ng x L(-1) and (187+/-35) ng x L(-1) at 4 and 8 wk than in control rats(48+/-9) ng x L(-1) and (53+/-11) ng x L(-1), respectively (P【0.05). Mean values of plasma ALT levels were (1867+/-250) nkat x L(-1) and (2450+/-367) nkat x L(-1) in Group E. The values were increased more dramatically in ethanol-fed rats than in Group C after 4 and 8 weeks. In liver section from ethanol-fed rats, there were marked pathological changes (steatosis, cell infiltration and necrosis). In ethanol-fed rats, ethanol administration led to a significant increase in LBP and CD14 mRNA levels compared with the control group (P【0.05). CONCLUSION: Ethanol administration led to a significant increase in endotoxin levels in serum and LBP and CD14 mRNA expressions in liver tissues. The increase of LBP and CD14 mRNA expression might wake the liver more sensitive to endotoxin and liver injury.
文摘INTRODUCTIONFas/ FasL system has been identified as a keymediator of apoptosis in tumor cells[1-4]. Theoccurrence and development of neoplasm are closelyrelated to apoptosis[5-7] Most chemotherapeuticdrugs kill cancer cells mainly by inducingapoptosis[8-14].'
基金Supported by Grants FONDECYT #1030894 and 1085232 and FONDEF DO2I-1067 from CONICYT,Chile
文摘AIM:To evaluate how widely Helicobacter pylori (H. pylori ) HopE and HopV porins are expressed among Chilean isolates and how seroprevalent they are among infected patients in Chile.METHODS: H. pylori hopE and hopV genes derived from strain CHCTX-1 were cloned by polymerase chain reaction (PCR), sequenced and expressed in Escherichia coli AD494 (DE3). Gel-purified porins were used to prepare polyclonal antibodies. The presence of both genes was tested by PCR in a collection of H. pylori clinical isolates and their expression was detected in lysates by immunoblotting. Immune responses against HopE, HopV and other H. pylori antigens in sera from infected and non-infected patients were tested by Western blotting using these sera as f irst antibody on recombinant H. pylori antigens.RESULTS: PCR and Western blotting assays revealed that 60 and 82 out of 130 Chilean isolates carried hopE and hopV genes, respectively, but only 16 and 9, respectively, expressed these porins. IgG serum immunoreactivity evaluation of 69 H. pylori-infected patients revealed that HopE and HopV were infrequently recognized (8.7% and 10.1% respectively) compared to H. pylori VacA (68.1%) and CagA (59.5%) antigens. Similar values were detected for IgA serum immunoreactivity against HopE (11.6%) and HopV (10.5%) although lower values for VacA (42%) and CagA (17.4%) were obtained when compared to the IgG response.CONCLUSION: A scarce expression of HopE and HopV among Chilean isolates was found, in agreement with the infrequent seroconversion against these antigens when tested in infected Chilean patients.
基金Supported by Science and Technology Planning Project of Zhejiang Province,No.LGF20H160001.
文摘BACKGROUND The relationship between hepatitis B surface antigen(HBsAg)-positive carrier status and liver cancer has been extensively studied.However,the epigenetic changes that occur during progression from HBsAg-positive carrier status or cirrhosis to liver cancer are unknown.The epigenetic modification of DNA hydroxymethylation is critical in tumor development.Further,5-hydroxymethylcytosine(5hmC)is an important base for DNA demethylation and epigenetic regulation.It is also involved in the assembly of chromosomes and the regulation of gene expression.However,the mechanism of action of 5hmC in HBsAgpositive carriers or patients with cirrhosis who develop liver cancer has not been fully elucidated.AIM To investigate the possible epigenetic mechanism of HBsAg-positive carriers and hepatocellular carcinoma(HCC)progression from cirrhosis.METHODS Forty HBsAg-positive carriers,forty patients with liver cirrhosis,and forty patients with liver cancer admitted to the First People's Hospital of Yongkang between March 2020 and November 2021 were selected as participants.Free DNA was extracted using a cf-DNA kit.cfDNA was extracted by 5hmC DNA sequencing for principal component analysis,the expression profiles of the three groups of samples were detected,and the differentially expressed genes(DEGs)modified by hydroxymethylation were screened.Bioinformatic analysis was used to enrich DEGs,such as in biological pathways.RESULTS A total of 16455 hydroxymethylated genes were identified.Sequencing results showed that 32 genes had significant 5hmC modification differences between HBsAg carriers and liver cancer patients,of which 30 were upregulated and 2 downregulated in patients with HCC compared with HBsAg-positive carriers.Significant 5hmC modification differences between liver cirrhosis and liver cancer patients were identified in 20 genes,of which 17 were upregulated and 3 were downregulated in patients with HCC compared with those with cirrhosis.These genes may have potential loci that are undiscovered or unelucidated,which contribute to the development and progression of liver cancer.Analysis of gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes showed that the major signaling pathways involved in the differential genes were biliary secretion and insulin secretion.The analysis of protein interactions showed that the important genes in the protein-protein interaction network were phosphoenolpyruvate carboxykinase and solute carrier family 2.CONCLUSION The occurrence and development of liver cancer involves multiple genes and pathways,which may be potential targets for preventing hepatitis B carriers from developing liver cancer.