BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secretin...BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay （IFA） indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.展开更多
AIM： To study the effect of oxymatrine-baicalin combination （OB） against HBV replication in 2.2.15 cells and α smooth muscle actin （ α SMA） expression, type I, collagen synthesis in HSC-T6 cells. METHODS： The ...AIM： To study the effect of oxymatrine-baicalin combination （OB） against HBV replication in 2.2.15 cells and α smooth muscle actin （ α SMA） expression, type I, collagen synthesis in HSC-T6 cells. METHODS： The 2.2.15 cells and HSC-T6 cells were cultured and treated respectively. HBsAg and HBeAg in the culture supernatants were detected by ELISA and HBV DNA levels were determined by fluorescence quantitative PCR. Total RNA was extracted from HSC-T6 cells and reverse transcribed into cDNA. The cDNAs were amplified by PCR and the quantities were expressed in proportion to β actin. The total cellular proteins extracted from HSC-T6 cells were separated by electrophoresis. Resolved proteins were electrophoretically transferred to nitrocellulose membrane. Protein bands were revealed and the quantities were corrected by β actin. RESULTS： In the 2.2.15 cell culture system, the inhibitory rate against secretion of HBsAg and HBeAg in the OB group was significantly stronger than that in the oxymatrine group （HBsAg, P = 0.043; HBeAg, P = 0.026; respectively）; HBV DNA level in the OB group was significantly lower than that in the oxymatrine group （P = 0.041）. In HSC-T6 cells the mRNA and protein expression levels of α SMA in the OB group were significantly lower as compared with those in the oxymatrine group （mRNA, P = 0.013; protein, P = 0.042; respectively）; The mRNA and protein expression levels of type I collagen in the OB group were significantly lower as compared with those in the oxymatrine group （mRNA, P 〈 0.01; protein, P 〈 0.01; respectively）.CONCLUSION： OB combination has a better effect against HBV replication in 2.2.15 cells and is more effective against α SMA expression and type I collagen synthesis in HSC-T6 cells than oxymatrine in vitro.展开更多
AIM： To investigate the correlation between hepatitis B virus surface antigen （HBsAg）, hepatitis C virus （HCV） expression in hepatocellular carcinoma （HCC）, the HAI score of the noncancerous region of the liver...AIM： To investigate the correlation between hepatitis B virus surface antigen （HBsAg）, hepatitis C virus （HCV） expression in hepatocellular carcinoma （HCC）, the HAI score of the noncancerous region of the liver and the serum Alpha fetoprotein （AFP） level. METHODS： The patterns of HBsAg and HCV in 100 cases of HCC and their surrounding liver tissues were studied on paraffin-embedded sections with immunohistochemistry, the histological status was determined by one pathologist and one surgeon simultaneously using the hepatitis activity index （HAIl score, and AFP was detected by radioimmunity. The study included 100 consecutive patients who underwent curative resection for HCC. Based on HBsAg and HCV expression, the patients were classified into 4 groups： patients positive for HBsAg （HBsAg group）, patients positive for HCV （HCV group）, patients negative for both HCV and HBsAg （NBNC group） and patients positive for both HBsAg and HCV （BC group）. RESULTS： The BC group had significantly higher HAI scores than the other three groups. （BC 〉 HCV 〉 HBsAg 〉 NBNC）. HBV and HCV virus infection was positively correlated with HAI （rs = 0.39, P = 0.00011. The positive rate of AFP （85.7%） and the value of AFP （541.2 ng/mL） in the group with HBV and HCV co-infection were the highest among the four groups. The positive rate （53.3%） of AFP and the value of AFP （ 53.3 ng/mL） in the group with none-infection of HBV and HCV were the lowest. HBV and HCV virus infection was positively correlated with AFP（rs = 0.38, P = 0.0001）. CONCLUSION： The AFP increase in patients with liver cancer was positively correlated with the infection of HBV and HCV. The-serum AFP elevation by the infection of HBV and HCV is one of mechanisms which lead to hepatocarcinogenesis, and the antivirus intervening treatment of hepatitis is significant for the prognosis of liver cancer. From our Spearman＇s rank correlation analysis, we can conclude that the severity of virally induced inflammation is correlated with HBsAg and HCV expression in HCC tissues and noncancerous tissues. Prior co-infection of HBV in HCV patients may be an adverse risk factor for intrahepatic inflammation.展开更多
Hepatitis B surface antigen (HBsAg) is produced and secreted through a complex mechanism that is still not fully understood. In clinical fields, HBsAg has long served as a qualitative diagnostic marker for hepatitis B...Hepatitis B surface antigen (HBsAg) is produced and secreted through a complex mechanism that is still not fully understood. In clinical fields, HBsAg has long served as a qualitative diagnostic marker for hepatitis B virus infection. Notably, advances have been made in the development of quantitative HBsAg assays, which have allowed viral replication monitoring, and there is an opportunity to make maximal use of quantitative HBsAg to elucidate its role in clinical fields. Yet, it needs to be underscored that a further understanding of HBsAg, not only from clinical point of view but also from a virologic point of view, would enable us to deepen our insights, so that we could more widely expand and apply its utility. It is also important to be familiar with HBsAg variants and their clinical consequences in terms of immune escape mutants, issues resulting from overlap with corresponding mutation in the P gene, and detection problems for the HBsAg variants. In this article, we review current concepts and issues on the quantification of HBsAg titers with respect to their biologic nature, method principles, and clinically relevant topics.展开更多
Primary liver cancer is an important cause of cancer death, and hepatocellular carcinoma （HCC） accounts for 70%-85% of total liver cancer worldwide. Chronic hepatitis B virus （HBV） infection contributes to 〉 75% ...Primary liver cancer is an important cause of cancer death, and hepatocellular carcinoma （HCC） accounts for 70%-85% of total liver cancer worldwide. Chronic hepatitis B virus （HBV） infection contributes to 〉 75% of HCC cases. High serum viral load is the most reliable indicator of viral replication in predicting development of HCC. HBV genotype C is closely associated with HCC in cirrhotic patients aged 〉 50 years, whereas genotype B is associated with development of HCC in non-cirrhotic young patients and postoperative relapse of HCC. Different HBV subgenotypes have distinct patterns of mutations, which are clearly associated with increased risk of HCC. Mutations accumulate during chronic HBV infection and predict occurrence of HCC. Chronic inflammation leads to increased frequency of viral mutation via cellular cytidine deaminase induction. Mutations are negatively selected by host immunity, whereas some immuno-escaped HBV mutants are active in hepatocarcinogenesis. Inflammatory pathways contribute to the inflammation-necrosis-regeneration process, ultimately HCC. Their hallmark molecules can predict malignancy in HBV-infected subjects. Continuing inflammation is involved in hepatocarcinogenesis and closely related to recurrence and metastasis. HBV load, genotype C, viral mutations and expression of inflammatory molecules in HBV-related HCC tissues are significantly associated with poor prognosis. Imbalance between intratumoral CD8^＋T cells and regulatory T cells or Thl and Th2 cytokines in peritumoral tissues can predict prognosis of HBV-related HCC. These factors are important for developing active prevention and surveillance of HBV-infected subjects who are more likely to develop HCC, or for tailoring suitable treatment to improve survival or postpone postoperative recurrence of HCC.展开更多
[Objective] The aim was to study the prokaryotic expression of P1 gene of foot-and-mouth disease virus（FMDV）type Asia 1and the preparation of its antiserum.[Method]The P1 gene of FMDV type Asia 1 was obtained by gen...[Objective] The aim was to study the prokaryotic expression of P1 gene of foot-and-mouth disease virus（FMDV）type Asia 1and the preparation of its antiserum.[Method]The P1 gene of FMDV type Asia 1 was obtained by gene cloning techniques,and then cloned into pET-32a（＋）plasmid;subsequently the recombinant plasmid was transformed into E.coli BL21（DE3）;after the IPTG induction and protein purification,SDS-PAGE analysis was carried out;the ultrasonic wave was use to lyse the cultivated recombinant strain,and after the isolation and purification,this fusion protein was utilized to immunize New Zealand rabbits so as to prepare P1 protein antiserum.[Result]The positive clones were obtained;SDS-PAGE result showed that the target band was appeared at 105 kD;Western blot analysis showed that the antisera could bind to the expressed P1 fusion protein specifically;the ELISA titer of the rabbit anti-FMDV-P1 sera was approximately 1∶5 120.[Conclusion]This study had provided foundations for FMDV serological diagnostic methods and genetically engineered vaccine.展开更多
AIM： To compare the response of standard hepatitis B virus （HBV） vaccination between patients with chronic hepatitis C virus （HCV） infection and healthy individuals. METHODS： This is a prospective case-control s...AIM： To compare the response of standard hepatitis B virus （HBV） vaccination between patients with chronic hepatitis C virus （HCV） infection and healthy individuals. METHODS： This is a prospective case-control study. A total of 38 patients with chronic HCV infection and 40 healthy controls were included. Vaccination was performed by injection of 20μg recombinant HBsAg into the deltoid muscle at mo 0,1 and 6. Anti-HBs concentration was determined 3 mo after the last dose and compared between the two groups. The response pattern was characterized as （1） high-response when the anti-HBs antibody titer was 〉 100 IU/L, （2） low-response when the titer was 10-100 IU/L. and （3） no-response when the titer was 〈 10 IU/L. RESULTS： In the patient group, there were 10/38 （26.3%） non-responders, 8/38 （21.1%） Iow-responders and 20/38 （52.6%） high-responders. The corresponding values in the control group were 2/40 （5.0%）, 7/40 （17.5%） and 31/40 （77.5%）, respectively. The response pattern was statistically different between the two groups. In multivariate analysis, smoking was a significant confounder, while HCV infection lost its significant correlation with lower antibody response. CONCLUSION： Patients with chronic HCV infection tend to respond weakly to HBV vaccination compared to healthy individuals, though this correlation is not independent according to multivariate analysis.展开更多
基金Supported by National Natural Science Foundation of China(31302071)AgriculturalScience and Technology Independent Innovation Fund of Jiangsu Province(TechnicalInnovation)[CX(13)3065]~~
文摘BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay （IFA） indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.
文摘AIM： To study the effect of oxymatrine-baicalin combination （OB） against HBV replication in 2.2.15 cells and α smooth muscle actin （ α SMA） expression, type I, collagen synthesis in HSC-T6 cells. METHODS： The 2.2.15 cells and HSC-T6 cells were cultured and treated respectively. HBsAg and HBeAg in the culture supernatants were detected by ELISA and HBV DNA levels were determined by fluorescence quantitative PCR. Total RNA was extracted from HSC-T6 cells and reverse transcribed into cDNA. The cDNAs were amplified by PCR and the quantities were expressed in proportion to β actin. The total cellular proteins extracted from HSC-T6 cells were separated by electrophoresis. Resolved proteins were electrophoretically transferred to nitrocellulose membrane. Protein bands were revealed and the quantities were corrected by β actin. RESULTS： In the 2.2.15 cell culture system, the inhibitory rate against secretion of HBsAg and HBeAg in the OB group was significantly stronger than that in the oxymatrine group （HBsAg, P = 0.043; HBeAg, P = 0.026; respectively）; HBV DNA level in the OB group was significantly lower than that in the oxymatrine group （P = 0.041）. In HSC-T6 cells the mRNA and protein expression levels of α SMA in the OB group were significantly lower as compared with those in the oxymatrine group （mRNA, P = 0.013; protein, P = 0.042; respectively）; The mRNA and protein expression levels of type I collagen in the OB group were significantly lower as compared with those in the oxymatrine group （mRNA, P 〈 0.01; protein, P 〈 0.01; respectively）.CONCLUSION： OB combination has a better effect against HBV replication in 2.2.15 cells and is more effective against α SMA expression and type I collagen synthesis in HSC-T6 cells than oxymatrine in vitro.
文摘AIM： To investigate the correlation between hepatitis B virus surface antigen （HBsAg）, hepatitis C virus （HCV） expression in hepatocellular carcinoma （HCC）, the HAI score of the noncancerous region of the liver and the serum Alpha fetoprotein （AFP） level. METHODS： The patterns of HBsAg and HCV in 100 cases of HCC and their surrounding liver tissues were studied on paraffin-embedded sections with immunohistochemistry, the histological status was determined by one pathologist and one surgeon simultaneously using the hepatitis activity index （HAIl score, and AFP was detected by radioimmunity. The study included 100 consecutive patients who underwent curative resection for HCC. Based on HBsAg and HCV expression, the patients were classified into 4 groups： patients positive for HBsAg （HBsAg group）, patients positive for HCV （HCV group）, patients negative for both HCV and HBsAg （NBNC group） and patients positive for both HBsAg and HCV （BC group）. RESULTS： The BC group had significantly higher HAI scores than the other three groups. （BC 〉 HCV 〉 HBsAg 〉 NBNC）. HBV and HCV virus infection was positively correlated with HAI （rs = 0.39, P = 0.00011. The positive rate of AFP （85.7%） and the value of AFP （541.2 ng/mL） in the group with HBV and HCV co-infection were the highest among the four groups. The positive rate （53.3%） of AFP and the value of AFP （ 53.3 ng/mL） in the group with none-infection of HBV and HCV were the lowest. HBV and HCV virus infection was positively correlated with AFP（rs = 0.38, P = 0.0001）. CONCLUSION： The AFP increase in patients with liver cancer was positively correlated with the infection of HBV and HCV. The-serum AFP elevation by the infection of HBV and HCV is one of mechanisms which lead to hepatocarcinogenesis, and the antivirus intervening treatment of hepatitis is significant for the prognosis of liver cancer. From our Spearman＇s rank correlation analysis, we can conclude that the severity of virally induced inflammation is correlated with HBsAg and HCV expression in HCC tissues and noncancerous tissues. Prior co-infection of HBV in HCV patients may be an adverse risk factor for intrahepatic inflammation.
基金Supported by The Grant of the Bilateral International Collaborative R&D Program from the Ministry of Knowledge Economythe Good Health R&D Project from the Ministry for Health,Welfare and Family Affairs,South Korea (A050021)
文摘Hepatitis B surface antigen (HBsAg) is produced and secreted through a complex mechanism that is still not fully understood. In clinical fields, HBsAg has long served as a qualitative diagnostic marker for hepatitis B virus infection. Notably, advances have been made in the development of quantitative HBsAg assays, which have allowed viral replication monitoring, and there is an opportunity to make maximal use of quantitative HBsAg to elucidate its role in clinical fields. Yet, it needs to be underscored that a further understanding of HBsAg, not only from clinical point of view but also from a virologic point of view, would enable us to deepen our insights, so that we could more widely expand and apply its utility. It is also important to be familiar with HBsAg variants and their clinical consequences in terms of immune escape mutants, issues resulting from overlap with corresponding mutation in the P gene, and detection problems for the HBsAg variants. In this article, we review current concepts and issues on the quantification of HBsAg titers with respect to their biologic nature, method principles, and clinically relevant topics.
基金Supported by National Natural Science Foundation of China,No. 81025015 and No. 30921006
文摘Primary liver cancer is an important cause of cancer death, and hepatocellular carcinoma （HCC） accounts for 70%-85% of total liver cancer worldwide. Chronic hepatitis B virus （HBV） infection contributes to 〉 75% of HCC cases. High serum viral load is the most reliable indicator of viral replication in predicting development of HCC. HBV genotype C is closely associated with HCC in cirrhotic patients aged 〉 50 years, whereas genotype B is associated with development of HCC in non-cirrhotic young patients and postoperative relapse of HCC. Different HBV subgenotypes have distinct patterns of mutations, which are clearly associated with increased risk of HCC. Mutations accumulate during chronic HBV infection and predict occurrence of HCC. Chronic inflammation leads to increased frequency of viral mutation via cellular cytidine deaminase induction. Mutations are negatively selected by host immunity, whereas some immuno-escaped HBV mutants are active in hepatocarcinogenesis. Inflammatory pathways contribute to the inflammation-necrosis-regeneration process, ultimately HCC. Their hallmark molecules can predict malignancy in HBV-infected subjects. Continuing inflammation is involved in hepatocarcinogenesis and closely related to recurrence and metastasis. HBV load, genotype C, viral mutations and expression of inflammatory molecules in HBV-related HCC tissues are significantly associated with poor prognosis. Imbalance between intratumoral CD8^＋T cells and regulatory T cells or Thl and Th2 cytokines in peritumoral tissues can predict prognosis of HBV-related HCC. These factors are important for developing active prevention and surveillance of HBV-infected subjects who are more likely to develop HCC, or for tailoring suitable treatment to improve survival or postpone postoperative recurrence of HCC.
基金Supported by National Transgenic Major Program of China(2009ZX08007-006B)the National Natural Science Foundation of China(31072160)+2 种基金Science and Technique Foundation of Shandong Province(2009GG20002032)Natural Science Foundation ofShandong Province(Y2008D20)an Open Issue of State Key Laboratory of Veterinary Biotechnology Fund(SKLVBF200806)~~
文摘[Objective] The aim was to study the prokaryotic expression of P1 gene of foot-and-mouth disease virus（FMDV）type Asia 1and the preparation of its antiserum.[Method]The P1 gene of FMDV type Asia 1 was obtained by gene cloning techniques,and then cloned into pET-32a（＋）plasmid;subsequently the recombinant plasmid was transformed into E.coli BL21（DE3）;after the IPTG induction and protein purification,SDS-PAGE analysis was carried out;the ultrasonic wave was use to lyse the cultivated recombinant strain,and after the isolation and purification,this fusion protein was utilized to immunize New Zealand rabbits so as to prepare P1 protein antiserum.[Result]The positive clones were obtained;SDS-PAGE result showed that the target band was appeared at 105 kD;Western blot analysis showed that the antisera could bind to the expressed P1 fusion protein specifically;the ELISA titer of the rabbit anti-FMDV-P1 sera was approximately 1∶5 120.[Conclusion]This study had provided foundations for FMDV serological diagnostic methods and genetically engineered vaccine.
文摘AIM： To compare the response of standard hepatitis B virus （HBV） vaccination between patients with chronic hepatitis C virus （HCV） infection and healthy individuals. METHODS： This is a prospective case-control study. A total of 38 patients with chronic HCV infection and 40 healthy controls were included. Vaccination was performed by injection of 20μg recombinant HBsAg into the deltoid muscle at mo 0,1 and 6. Anti-HBs concentration was determined 3 mo after the last dose and compared between the two groups. The response pattern was characterized as （1） high-response when the anti-HBs antibody titer was 〉 100 IU/L, （2） low-response when the titer was 10-100 IU/L. and （3） no-response when the titer was 〈 10 IU/L. RESULTS： In the patient group, there were 10/38 （26.3%） non-responders, 8/38 （21.1%） Iow-responders and 20/38 （52.6%） high-responders. The corresponding values in the control group were 2/40 （5.0%）, 7/40 （17.5%） and 31/40 （77.5%）, respectively. The response pattern was statistically different between the two groups. In multivariate analysis, smoking was a significant confounder, while HCV infection lost its significant correlation with lower antibody response. CONCLUSION： Patients with chronic HCV infection tend to respond weakly to HBV vaccination compared to healthy individuals, though this correlation is not independent according to multivariate analysis.
