新城疫病毒血凝素-神经氨酸酶蛋白的可溶性超表达与免疫活性检测

Soluble overexpression and immunological activity of the Newcastle disease virus hemagglutinin-neuraminidase protein

目的检测新城疫病毒(NDV)血凝素-神经氨酸酶(HN)蛋白在大肠杆菌中的可溶性表达量,及其免疫活性。方法采用融合表达的方法将HN蛋白基因分别与Grifin、谷胱甘肽S移酶(GST)、麦芽糖结合蛋白(MBP)、Nus A、小泛素样相关修饰物(SUMO)、硫氧还蛋白(thioredoxin)、蛋白G(protein G)、γ-晶状体蛋白(γ-crystallin)、Ars C、Ppi B 10种融合标签基因采用柔性接头进行连接,构建10个重组表达质粒,经酶切及测序鉴定正确后,将这10个重组质粒分别转入大肠杆菌BL21中,用不同条件进行诱导表达,经SDS-PAGE检测并选出最佳诱导蛋白表达的条件,筛选出能够显著促进HN蛋白可溶性表达的标签,并用Western blotting对所表达蛋白的含量进行定性分析,同时纯化的重组蛋白用Western blotting和间接ELISA验证其免疫原性。结果成功构建了10个不同融合标签的HN融合蛋白表达载体,筛选出融合标签麦芽糖结合蛋白(MBP)能够显著促进HN蛋白可溶性表达,且Western blotting显示所表达重组蛋白的表达量是最多的。经Western blotting和间接ELISA验证重组HN蛋白具有良好的免疫原性。结论融合标签MBP可以显著提高HN蛋白在大肠杆菌中的可溶性表达量。

Objective To improve the soluble overexpression of Newcastle disease virus（ NDV） hemagylutininneuraminidase（ HN） protein in E. Coli and determine its immunological. Methods Ten plasmids that expressed NDV HN protein with-N-terminal Grifin,gultathione-S-transferase（ GST）,maltose biniding protein（ MBP）,Nus A,sanall ubiquitinrelated modifier（ SUMO）,thioredoxin,protein G,γ-crystallin,Ars C or Ppi B tags were constructed to test the effects of the various tags on the expression level and solubility of NDV HN protein in E. coli. The sequences of the ten plasmids were confirmed and the ten plasmids were transformed into E. coli BL21 strain. In order to select and screen the best expression conditions,different induced dose isopropy-β-D-thiogalactoside（ IPTG） final concentration,culture temperature,Amp final concentration and culture media were used. The expression level and solubility of fusion protein were analyzed by SDS-PAGE and Western blotting. The fusion proteins were purified by Ni-NTA chromatography to develop indirect ELISA. The purified fusion protein was detected by Western blotting and indirect ELISA. Results Ten HN fusion protein expression plasmids were constructed successfully. Maltose binding protein（ MBP） tag significantly increased the HN protein soluble overexpression. The expression level was the highest. The results from Western blotting and indirect ELISA showed that the purified HN protein had a good immunogenicity. Conclusion MBP tag enhances the expression and solubility of HN protein.The study will lay foundation for the research on the NDV mechanism,vaccine and diagnostic reagents.