目的:探讨n-3多不饱和脂肪酸(n-3 poly unsaturated fatty acid,n-3 PUFA)对NOD小鼠T细胞免疫学功能的影响.方法:取野生型Balb/c小鼠和已出现典型的I型糖尿病症状的NOD小鼠脾脏细胞,磁珠分选后获得小鼠CD4+T细胞,将其分为4组:野生型小鼠为Wild type组;NOD小鼠分为未处理组、二十二碳六烯酸(docosahexaenoic acid,DHA)处理组、二十碳五烯酸(eicosapentaenoic acid,EPA)处理组.DHA、EPA处理24 h,PMA和Ionomycin刺激活化后,采用CCK-8法检测T细胞增殖、流式细胞仪检测Th1/Th2极化、ELISA法检测T细胞细胞因子的分泌水平变化.结果:DHA、EPA对T细胞增殖具有显著抑制作用,促进NOD小鼠Th2细胞分化,抑制Th1细胞分化,经ELISA检测,DHA和EPA能够抑制T细胞IL-6、IL-17的分泌.结论:n-3多不饱和脂肪酸通过抑制T细胞增殖,平衡Th1/Th2比例和抑制IL-6、IL-17的分泌,对NOD小鼠的T淋巴细胞起到免疫抑制作用.

To express and purify herpes simplex virus type I(HSV I)glycoprotein B(gB)in eukaryote cells with the purpose of analyzing the antigenicity and immunogenicity.At the beginning,the extracellular domain fragment gene of gD1 was synthesized by chemical method and cloned into eukaryotic expression vector pCEP4 to construct the recombinant plasmid pCEP4-gD1.After transfection of HEK293 cells with the recombinant plasmid,the expressed protein was characterized by Western blot and purified through Ni affinity chromatography.Then antigenicity of the protein was detected by ELISA.Finally,the purified protein was used to immunize Kunming mice in 1,3,5 weeks respectively,and antiserum were collected in 3,5 and 7 weeks.The antibody titer was detected by indirect ELISA for immunogenicity analysis.Gene sequencing analysis demonstrates that the recombinant plasmid pCEP4-gB1 was constructed successfully.Western blot analysis indicated one major protein band,which molecular weight is approximate 85 kDa corresponding to the truncated forms of gB1 protein,was observed.In addition,ELISA detection showed that expressed gB1 has good antigenicity.After the third immunization,antibody titer of the mouse anti-gB1 was 5×103.The successful expression of the recombinant protein gD1,which can induce humoral immune response,lays a foundation for serological diagnosis and vaccine study of HSV.

Envelope proteins of herpes simplex virus (HSV) plays a vital role not only in the infection process of adsorption and invasion but also in the stimulation to the organism that gives rise to immune response. Among the envelope proteins, glycoprotein D (gD), which can induce specific immune response, are the primary targets of humoral and cellular immunity of the host. In order to analyze the antigenicity and immunogenicity of HSV-gD1, we chemically synthesized the extracellular domain fragment gene of gD1, cloned it into eucaryotic expression vector pCEP4, and transfected the HEK293 cells with the recombinant vector. Then we identified the recombinant protein by Western blotting, and detected antigenicity of the protein by ELISA. Finally, we used the purified gD1 protein to immunize Kunming mice in 1, 3, 5 weeks, and collected antiserum in 3, 5 and 7 weeks. We titrated the sera for the detection of anti gD1 using an ELISA assay. Gene sequencing analysis demonstrated that the recombinant plasmid pCEP4-gD1 was constructed successfully. Western blotting analysis indicated one major protein band, which molecular weights is approximate 46 kDa corresponding to the truncated forms of gD1 protein, was observed. ELISA assay showed that the expressed recombinant protein gD1 had good antigenicity. After the third immunization, antibody titer of the mouse anti-gD1 was at least 5 x10(3). The successful expression of the recombinant protein gD1, which can induce humoral immune response, lays a foundation for serological diagnosis and vaccine study of HSV.

In order to improve the soluble expression of recombinant Hepatitis C virus RNA-dependent RNA polymerase(RdRp) in E.coli,several factors influencing RdRp expression level and solubility were optimized by using single factor tests.The expressed RdRp was detected by SDS-PAGE.The results showed that amplification cultural time,IPTG concentration,induction temperature and induction time have significant effects on solubility of the expressed RdRp.The best condition for expressing soluble RdRp was obtained,when OD600 of the culture medium reached about 1.6,and the engineered E.coli was induced with 0.20mmol/L IPTG at 25℃ for 16h.This study has established the foundation for assaying RdRp enzymatic activity in vitro and screening the anti-HCV drugs targeting RdRp.