Specific immune responses induced by a multi-epitope antigen of hepatitis C virus in mice and rabbits

Five highly conserved and immunogenic epitopes of hepatitis C virus (HCV) have been chosen to form a multi-epitope antigen gene and fused with β-galactosidase gene to express a hybrid GZ-PCX antigen, which could be specifically recognized by human HCV sera. High level of anti-GZ-PCX IgG has been induced when mice or rabbits were immunized with GZ-PCX antigen emulsificated with complete Freund’s adjuvant or mixed with killed attenuatedSalmonella typhimurium SL3261. The specific anti-GZ-PCX IgG reached a high titer of 10^-6, which remained for several months. Specific cytotoxic T lymphocyte (CTL) effects, delayed type hypersensitivity reaction (DTH) and proliferation of peripheral lymphocytes have been induced by GZ-PCX antigen or synthetic peptides. High level of anti-GZ-PCX slgG has been detected in mice’s intestinal washing fluids, which indicates that the antigen induced mucosal immunity as well as systematic immunity. The studies show that the HCV multi-epitope antigen induces high level of specific immune responses without obvious toxicity, which might be able to provide protectivity to any HCV genotypes and isolates.     

Expression of E1 gene of a hepatitis C virus inE. coli and protein purification

The cDNA containing full encoding region of E1 antigen of HCV was cloned into an expression plasmid pRSETHisB. The recombinant plasmid pRSETE1 was introduced into the BL21 (DE3) strain ofE. coli. The engineering bacteria harbouring the pRSETE1 was cultivated in 2YT medium at 37°C. When the Expression of E1 protein was induced by 1 mmol IPTG, the bacteria was killed and the number of living cell was droped down from 10⁷ to 10³ cell/mL one hour post induction. Suggest that E1 protein is poisoned toE. coli. However, the 26kD polypeptide of E1 fussion protein still synthesized in appropriate condition. The expression level was about 10% of total protein 4 h after inducing. The E1 protin was purified by Ni²⁺-NTA-Agarose column chromatography to homogeneous. The purified E1 protein was sensitive and specific in reaction with anti-HCV antibody in sera. Supported by the Science Committec of Hubei Province Ye Linbai: born in Feb. 1948. Professor     

HCV多表位抗原基因的表达及其在小鼠中诱导的抗体应答研究

丙型肝炎病毒(HCV)是引起病毒性非甲非乙肝炎的主要病原．全世界约有1．7亿HCV的感染者,我国估计占4000万以上．发展有效的丙型肝炎疫苗刻不容缓．构建了3段串联重复的丙型肝炎病毒多表位抗原基因3如,将其克隆到His融合表达载体pET-28b( )上后在大肠杆菌BL21(DE3)中得到了高效表达．3PCX经Ni^2 -NTA—agarose柱纯化后免疫BALB／c小鼠,诱发了高水平的抗体免疫应答．     

Secretory expression of the fusion protein composed of human IL-2 and PreS antigen of hepatitis B virus in mammalian cells

In order to make entire HBV pmSAg secrete from mammalian cells, we conatmcted an eukaryotic expression vector by using leader sequence of human interleukin-2 (IL-2) as secretory signal peptide, and using high hydmphilic amino acids as the linker between IL-2 C end and preSAg N end. As a result, the IL-2preS fusing protein could be secreted from mamalian cells transfected with the reconstructed vector and the expression efficiency was identical to that of natural IL-2. It was considered that the retentive effect of preSlAg could be successfully bypassed. The results not only laid a theoretical and practical foundation for constructing specific gene vaccine against HBV persistent infection, but also supplied experimental evidence for studying modulation of protein secretory expression.     

雏鸭混合感染基因A型和基因C型鸭甲肝病毒研究

研究了2013年四川地区某鸭场暴发的基因A型鸭甲肝病毒(DHAV-A)和基因C型鸭甲肝病毒(DHAV-C)的混合感染病例.疾病发生于20~25日龄雏鸭,发病率25%~30%,死亡率为20%~30%,临死前出现明显的神经症状,病死雏鸭肝脏肿大,出血.利用双重RT-PCR从病死雏鸭的肝脏组织中同时检测出DHAV-A和DHAV-C.这是四川省养鸭密集区第一次发现雏鸭混合感染DHAV-A和DHAV-C的情况.此外,对2009~2012年四川省各养鸭密集区鸭甲肝炎流行情况做了调查.一共从四川省养鸭密集区采集312份疑似鸭肝炎样本中检测出120份鸭甲型肝病毒性肝炎.其中基因C型鸭肝炎阳性率为75%,基因A型鸭肝炎仅为25%,未发现基因A型和基因C型混合感染情况.结果说明2009~2012年我省鸭病毒性肝炎以基因C型鸭甲肝病毒为优势病原.2013年,开始出现DHAV-A和DHAV-C混合感染病例.因此如何采取有效措施应对其危害,这应该引起养鸭界及相关部门的高度重视.     

Expression of human clotting factor Ⅸ mediated by recom- binant lentiviral vector in cultured cells and hemophilia B mice

Hemophilia B, a serious bleeding disorder, is an inherited X chromosome-linked disease for the deficiency or inactivity of human clotting factor Ⅸ (hFⅨ). Though factor substitution therapy has greatly improved the lives of hemophiliac patients, there are still limitations to the current treatment, which have triggered interest in alternative treatments by gene therapy[1]. Based on preclinical studies in rabbits[2], our lab had first initiated an ex vivo gene therapy clinical trial whereby a…     

Expression of human clotting factor Ⅸ mediated by recom- binant lentiviral vector in cultured cells and hemophilia B mice

To explore the expression of human clotting factor Ⅸ (hFⅨ) cDNA in vitro and the feasibility of gene therapy for hemophilia B mice mediated by recombinant lentiviral vector, a recombinant hFⅨ lentiviral vector driven by ubiquitin-C promoter, FUXW, and by ABP liver specific promoter, FAXW, was constructed respectively. Recombinant lentivirus was harvested from 293T cells by calcium phosphate-mediated transient cotransfection of three plasmids (transgene vector, CMV腞8.2, VSV-G). hFⅨ expression was detected in supernatant of 293T, BHK and L-02 cells infected with FUXW virus, whereas higher expression of hFⅨ levels (630 ng/106 cells/48 h) was detected only in L-02 cells infected with FAXW virus. Serum hFⅨ antigen was detected in all hemophilia B mice treated with FAXW virus by tail vein injection, an efficiency level of hFⅨ was observed (45 ng/mL, approximately 1% of normal human levels), the expression lasted for more than 60 d. The results indicated that HIV-based lentiviral vectors offer a promising approach to the gene therapy of hemophilia B.     

Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the micro- particle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 μg/mg soluble proteins on average and the highest value was 2.497 μg/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR- Southern and total DNA hybridization. Prospect of kelp bioreactor producing high value materials such as edible HBV vaccine was discussed as well.     

Adeno-associated virus-mediated integration and expression of human P-globin gene with the core fragment of locus control region in erythroid cells

To improve the integration stability and expression of the transferred human p-globin gene, the two recombinant adeno-associated virus (AAV) vectors containing the human [3-globin gene with a single or multiple DNase I hypersensitive site (HS) core fragment of the LCR were constructed. These recombinants were respectively introduced into MEL cells via AAV-mediated gene transfer to investigate their integration and expression. The results suggested that following AAV vector-mediated gene transfer, the human [3-globin gene with the multiple HS core fragment of the LCR could steadily integrate into MEL cells and confer an expression level comparable with endogenous mouse a-globin gene.     

Expression of hepatitis B surface antigen gene (HBsAg) in Laminaria japonica (Laminariales, Phaeophyta)

A transformation model for Laminaria japonica was established from 1993 to 1998, on the basis of which the transgenic kelp with heterologous gene encoding hepatitis B surface antigen (HBsAg) was obtained by using the microparticle bombardment transformation method. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 μg/mg soluble proteins on average and the highest value was 2.497 μg/mg, implying that recombinant HBsAg had natural epitope. Further support for the integration of HBsAg gene into kelp genome was obtained by PCR-Southern and total DNA hybridization. Prospect of kelp bioreactor producing high value materials such as edible HBV vaccine was discussed as well.     

以社会主义核心价值体系为引领,深入探讨高校思想道德建设的有效途径

高校提高大学生思想道德素质,必须坚持以社会主义核心价值体系为主线,力求夯实大学生的政治理论基础,使之成为应对各种挑战的思想武器。为了提高教育的实效性,从加强＂思想政治理论课＂教学、创新教学模式、改革教学方法等8方面,探讨以社会主义核心价值体系引领高校思想道德建设的有效途径。