表达乙型肝炎表面抗原及前 S2蛋白120～146片段重组酵母菌的构建及全菌体免疫效应评价  

Construction of recombinant yeast expressing PreS2120-146 and hepatitis B surface antigen and evaluation of the immune effects of whole yeast cells

作　　者：陈向敏[1] 张跃进[2] 田晓娟[3] 夏平 潘唯文 夏天 俞陈慧 张丽芳[3] 薛向阳[3] 

机构地区：[1]温州医科大学生物学实验教学中心,325035 [2]温州医科大学诊断学实验室,325035 [3]温州医科大学分子病毒与疫苗研究所微生物与免疫学教研室,325035 [4]仁济学院

出　　处：《中华传染病杂志》2014年第11期660-665,共6页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金资助项目（81001343）;浙江省自然科学基金资助项目（Y2100909）;温州市科技局资助项目（Y20120159）

摘　　要：目的：构建表达 HBsAg 及前 S2蛋白120～146片段（PreS2120-146）-HBsAg 的重组酵母菌，并评价其全菌体免疫效应。方法根据酵母密码子偏爱性优化 PreS2120-146区及 HBsAg 基因序列，串联后克隆到毕赤酵母 pPIC3．5K 表达载体，构建 pPIC3．5K／PreS2120-146-HBsAg 质粒，重组质粒经 BgkⅡ酶切线性化后，电转化至 GS115菌株中，筛选 PreS2120-146-HBsAg 重组毕赤酵母，通过 SDS-PAGE、Western 印迹、ELISA 分析目的蛋白的表达；以表达目的蛋白的灭活酵母全菌体免疫 BALB／c 小鼠，采用 ELISA 检测其产生的 HBsAg 特异性抗体；以 HBsAg CTL 表位肽刺激免疫小鼠脾细胞，通过实时PCR 检测γ干扰素表达，分析其诱导的 CTL 反应。采用独立样本 t 检验。结果PCR、酶切和测序分析表明成功构建了 pPIC3．5K／PreS2120-146-HBsAg 重组质粒。重组毕赤酵母甲醇诱导后，SDS-PAGE、Werstern 印迹和 ELISA 证实 PreS2120-146-HBsAg 目的蛋白表达。与 HBsAg 纯抗原免疫比较，灭活重组酵母菌免疫小鼠产生抗 HBsAg 特异性 IgG 抗体水平相当（t＝0．946，P ＝0．381）。CTL 反应检测显示， HBsAg CTL 表位肽刺激灭活重组酵母菌免疫组小鼠的脾细胞产生更高水平的γ干扰素（t＝2．305，P ＝0．044）。结论利用整合型重组毕赤酵母表达系统成功表达了 PreS2120-146-HBsAg 目的蛋白，全菌体免疫小鼠后能诱导产生特异的体液免疫和细胞免疫反应。Objective To construct the recombinant yeast expressing PreS2 120-146-hepatitis B surface antigen （HBsAg）,and to evaluate the immune effects of whole yeast cells.Methods PreS2 120-146 and HBsAg gene sequence were optimized according to the yeast cell codon preference,and were recombined and cloned into pPIC3.5K yeast expression vector to construct pPIC3.5K/PreS2 120-146 plasmid.After digested and linearized by Bgk Ⅱ restriction enzyme,pPIC3.5K/PreS2 120-146-HBsAg recombinant plasmid was electrotransformed into GS115 strain to screen PreS2 120-146-HBsAg-recombinant Pichiapastoris .The expression of PreS2 120-146-HBsAg was identified by sodium doclecyl sulfate polyacrylamide gel electrophogesis （SDS-PAGE）,Western blot and enzyme linked immunosorbent assay （ELISA）analysis. BALB/c mice were vaccinated by inactivated whole recombinant yeast cells expressing target protein. Specific antibodies to HBsAg were detected by ELISA.Cytotoxic T lymphocyte （CTL）response induced by interferon （IFN）-γ was detected by reverse transcription-polymerase chain reaction （RT-PCR）when immune spleen cells of mice were stimulated by CTL epitope on HBsAg.Independent sample t test was used. Results Data of PCR detection,restriction enzyme digestion and sequencing analysis showed that recombinant pPIC3.5K/PreS2 120-146-HBsAg plasmid was successfully constructed.SDS-PAGE,Western blot and ELISA verified the expression of PreS2 120-146-HBsAg in the lysate of the recombinant Pichiapastoris induced by methanol.Levels of specific anti-HBsAg IgG antibodies produced by inactivated yeast cells vaccinated mice were comparable to purified HBsAg immunization （t =0.946,P =0.381 ）. Analysis of HBsAg-specific CTL responses revealed that the level of IFN-γwas significantly higher when the immune spleen cells of mice were stimulated by CTL epitope peptides on HBsAg （t =2.305 ,P =0.044）.Conclusions PreS2 120-146-HBsAg target protein is successfully expressed by construction of recombinant Pichiapastoris . The specific humora

关 键 词：肝炎表面抗原 乙型 PRES2 毕赤酵母 免疫反应 

分 类 号：R512.62[医药卫生—内科学]