The aim of this study is to investigate the feasibility and mechanism of hIL-2-preS DNA vaccine as prevention and therapeutic approach against Hepatitis B. Eukaryon expression vector involving hIL-2 and preS gene was ...The aim of this study is to investigate the feasibility and mechanism of hIL-2-preS DNA vaccine as prevention and therapeutic approach against Hepatitis B. Eukaryon expression vector involving hIL-2 and preS gene was constructed with recombinant technique and transferred into normal BALB/c mice and HBV transgenic mice (Tg-Mice) respectively. Then a series of detection were performed: detection of anti-preS2, HBs antibody and HBsAg in BALB/c mice and Tg-mice with ELISA, quantification of HBV DNA copies in HBV Tg-mice serum with real-time PCR, determination of hepatitis degree with immunopathological HE staining and detection of liver function. Anti-preS1 can be detected at 4 th , 6 th and 10 th week in inoculated BALB/c mice. Injection with gene gun gained an advantage over muscular and subcutaneous injection since it acquired just 1/10 inoculation quantity (10 μg/mouse). Highest expression of IgG2a at 4 th week suggested Th1-mediated immune response, which facilitated HBV cleaning. Of all inoculated HBV Tg-mice, 80% of them showed anti-preS2, HBs antibody positive and HBV DNA decreased, and 20% showed negative for HBsAg. HE staining to hepatic tissue showed obvious infiltration of inflammatory cells, swelling and granular degeneration of hepatocytes. In our study, IL-2-preS DNA vaccine which can provoke the humoral and cellular immune response and break the immune tolerance supports the designation and construction of new vaccine against HBV and specific immune remedy for HBV continuous infection.展开更多
目的研究乙型肝炎病毒(hepatitis B virus,HBV)preC/C区基因突变与原发性肝癌患者预后的关系。方法收集81例乙型肝炎病毒相关性肝细胞肝癌(hepatitis B virus associated hepatocellular carcinoma,HBV-HCC)患者的癌组织并提取基因组DNA...目的研究乙型肝炎病毒(hepatitis B virus,HBV)preC/C区基因突变与原发性肝癌患者预后的关系。方法收集81例乙型肝炎病毒相关性肝细胞肝癌(hepatitis B virus associated hepatocellular carcinoma,HBV-HCC)患者的癌组织并提取基因组DNA,对HBVpreC/C区基因进行扩增和测序,并根据NCBI数据库鉴定出其突变位点,运用Kaplan-Meier和Cox回归等方法分析HBV-HCC患者的临床资料、突变位点与其术后生存期之间的关系。结果门脉瘤栓、肿瘤分期和肿瘤大小是与HBV-HCC患者术后生存相关的独立危险因素。1915、2134、2176、2221和2260突变位点被确定为预测HBV-HCC患者生存相关的独立危险因子,1979和2245突变位点与HBV-HCC患者生存具有临界统计学差异。结论门脉瘤栓、肿瘤分期和肿瘤大小以及HBVpreC/C区基因的以上7个突变位点被确定为与肝癌患者术后预后相关的独立危险因素。展开更多
Hepatitis B virus(HBV)is characterized with high mutations,which is attributed to the lack of proof-reading of the viral reverse transcriptase and host immune pressure.In this study,31 HBV chronic carriers from 14 fam...Hepatitis B virus(HBV)is characterized with high mutations,which is attributed to the lack of proof-reading of the viral reverse transcriptase and host immune pressure.In this study,31 HBV chronic carriers from 14 families were enrolled to investigate the evolution of the same original HBV sources in different hosts.Sequences of pre-C and C(pre-C/C)genes were analyzed in eight pairs of HBV-infected mothers with longitudinal sera(at an interval of 6.0–7.2 years)and their children(5.5–6.7 years old),and in 15 adults(21–78 years old)from six families with known intrafamilial HBV infection.The pre-C/C sequences had almost no change in eight mothers during 6.0–7.2 years and their children who were in immune tolerant phase.The pre-C/C sequences from the 15 adults of six families,mostly in the immune-clearance phase or the low replicative phase,showed various diversified mutations between individuals from each family.Compared to a reference stain(GQ205441)isolated nearby,the pre-C/C in individuals in immune tolerant phase showed 98.56%–99.52%homology at nucleotide level and 99.5%–100%homology at amino acid level.In contrast,multiple mutations were developed in the immune-clearance phase or the low replicative phase,affecting immune epitopes in core gene and G1896 in pre-C gene.The results indicate that the evolution of new HBV variants is not mainly resulted from the spontaneous error rate of viral reverse transcription,but from the host immune pressure.展开更多
[目的]构建丙型肝炎病毒(hepatitis C virus,HCV)串联多中和抗原表位与乙型肝炎病毒(hepatitis B virus,HBV)S抗原嵌合基因真核表达质粒,并在293T细胞中进行表达。[方法]将HCV基因中高度保守且具有广谱交叉中和活性的三个表位和一个HVR...[目的]构建丙型肝炎病毒(hepatitis C virus,HCV)串联多中和抗原表位与乙型肝炎病毒(hepatitis B virus,HBV)S抗原嵌合基因真核表达质粒,并在293T细胞中进行表达。[方法]将HCV基因中高度保守且具有广谱交叉中和活性的三个表位和一个HVR1模拟表位串连。串联表位嵌合于HBV S基因的氨基端胞外区,形成嵌合基因MEpS;将基因克隆至真核表达载体pCI-neo中,构建重组质粒pCI-MEpS。将质粒转染至293T细胞中,间接免疫荧光及Western Blot检测嵌合基因的表达情况。[结果]重组质粒经双酶切证实构建正确;pCI-MEpS转染的293T细胞胞浆内可见较强的绿色荧光,Western Blot显示pCI-MEpS在相对分子量约38 kDa处可见蛋白条带。[结论]构建了HBV S抗原嵌合HCV串联多中和抗原表位的重组质粒,成功在293T细胞中表达,为制备嵌合HCV串联多中和抗原表位的HBV S抗原VLPs,研究嵌合VLPs免疫动物后产生的中和抗体的保护作用奠定了实验基础。展开更多
基金This study was supported by a grant from the National Natural Science Foundation of China (No. 30271213)
文摘The aim of this study is to investigate the feasibility and mechanism of hIL-2-preS DNA vaccine as prevention and therapeutic approach against Hepatitis B. Eukaryon expression vector involving hIL-2 and preS gene was constructed with recombinant technique and transferred into normal BALB/c mice and HBV transgenic mice (Tg-Mice) respectively. Then a series of detection were performed: detection of anti-preS2, HBs antibody and HBsAg in BALB/c mice and Tg-mice with ELISA, quantification of HBV DNA copies in HBV Tg-mice serum with real-time PCR, determination of hepatitis degree with immunopathological HE staining and detection of liver function. Anti-preS1 can be detected at 4 th , 6 th and 10 th week in inoculated BALB/c mice. Injection with gene gun gained an advantage over muscular and subcutaneous injection since it acquired just 1/10 inoculation quantity (10 μg/mouse). Highest expression of IgG2a at 4 th week suggested Th1-mediated immune response, which facilitated HBV cleaning. Of all inoculated HBV Tg-mice, 80% of them showed anti-preS2, HBs antibody positive and HBV DNA decreased, and 20% showed negative for HBsAg. HE staining to hepatic tissue showed obvious infiltration of inflammatory cells, swelling and granular degeneration of hepatocytes. In our study, IL-2-preS DNA vaccine which can provoke the humoral and cellular immune response and break the immune tolerance supports the designation and construction of new vaccine against HBV and specific immune remedy for HBV continuous infection.
文摘目的研究乙型肝炎病毒(hepatitis B virus,HBV)preC/C区基因突变与原发性肝癌患者预后的关系。方法收集81例乙型肝炎病毒相关性肝细胞肝癌(hepatitis B virus associated hepatocellular carcinoma,HBV-HCC)患者的癌组织并提取基因组DNA,对HBVpreC/C区基因进行扩增和测序,并根据NCBI数据库鉴定出其突变位点,运用Kaplan-Meier和Cox回归等方法分析HBV-HCC患者的临床资料、突变位点与其术后生存期之间的关系。结果门脉瘤栓、肿瘤分期和肿瘤大小是与HBV-HCC患者术后生存相关的独立危险因素。1915、2134、2176、2221和2260突变位点被确定为预测HBV-HCC患者生存相关的独立危险因子,1979和2245突变位点与HBV-HCC患者生存具有临界统计学差异。结论门脉瘤栓、肿瘤分期和肿瘤大小以及HBVpreC/C区基因的以上7个突变位点被确定为与肝癌患者术后预后相关的独立危险因素。
基金supported by the National Natural Science Foundation of China(81672002)the Science and Technology Department of Jiangsu Province(BK20161105)the Jiangsu Provincial Department of Health(H201537),China。
文摘Hepatitis B virus(HBV)is characterized with high mutations,which is attributed to the lack of proof-reading of the viral reverse transcriptase and host immune pressure.In this study,31 HBV chronic carriers from 14 families were enrolled to investigate the evolution of the same original HBV sources in different hosts.Sequences of pre-C and C(pre-C/C)genes were analyzed in eight pairs of HBV-infected mothers with longitudinal sera(at an interval of 6.0–7.2 years)and their children(5.5–6.7 years old),and in 15 adults(21–78 years old)from six families with known intrafamilial HBV infection.The pre-C/C sequences had almost no change in eight mothers during 6.0–7.2 years and their children who were in immune tolerant phase.The pre-C/C sequences from the 15 adults of six families,mostly in the immune-clearance phase or the low replicative phase,showed various diversified mutations between individuals from each family.Compared to a reference stain(GQ205441)isolated nearby,the pre-C/C in individuals in immune tolerant phase showed 98.56%–99.52%homology at nucleotide level and 99.5%–100%homology at amino acid level.In contrast,multiple mutations were developed in the immune-clearance phase or the low replicative phase,affecting immune epitopes in core gene and G1896 in pre-C gene.The results indicate that the evolution of new HBV variants is not mainly resulted from the spontaneous error rate of viral reverse transcription,but from the host immune pressure.
