截短型A组轮状病毒VP6在E.coli中的高效表达

Efficient expression of truncated Group A rotavirus VP6 in E.coli

目的：研究A组轮状病毒（RV）组特异性抗原（VP6）片段的表达，为RV免疫检测技术提供材料。方法：以pcDNA3．1．VP6为模板，通过PCR扩增得到VP6的截短突变体编码基因VP6．1，将其插入谷胱甘肽巯基转移酶（GST）融合表达载体pGEX-5X，在E．coli中用异丙基-β-D-硫代半乳糖苷（WIG）诱导表达。对表达产物进行SDS．PAGE和Western blot分析。通过改变宿主菌、培养基、IPTG浓度和培养温度等条件，提高目的蛋白的可溶性表达水平，使用GST-Sepharose 4B亲合层析进行初步纯化。结果：选定VP6氨基酸12-143片段为目的蛋白，PCR扩增其396bp的编码基因片段，构建出pGEX．5X．VP6．1。将该重组质粒转化E．coli后，SDS．PAGE和Western blotting分析均显示，含有截短VP6蛋白的重组融合蛋白GST：：VP6-1在E．coli中获得高效表达，约占菌体总蛋白的30％。该蛋白主要以包涵体形式存在，经条件优化，最终通过低浓度IPTG、低温诱导提高重组蛋白的可溶性表达水平，GST亲和层析可对其进行初步纯化。结论：VP6．1蛋白在E．coli中可被高效表达和纯化，为下一步制备抗VP6的特异性单克隆抗体并用于免疫检测打下基础。

Objective： To express the group specific antigen VP6 fragment of Group A rotavirus （RV） in E. coli and therefore to provide materials for the development of the rotavirus immunoassays. Methods： By bioinformatics analysis, one conserved domain named VP6-1 within VP6 with strong hydrophilicity and antigenecity was picked out. The gene fragment encoding the truncated form of VP6 was amplified with PCR by using the plasmid pcDNA3.1-VP6 as a template and was inserted into the Glutathione-S-transferase （GST） fusion expression vector pGEX5X following the confirmation of enzynmtic analysis and DNA sequencing. The resultant plasmid was then introduced into E. coli for IPTG induced expression. The expression products were validated by both SDS-PAGE and Westem blotting. The soluble expression of the recombinant protein in E. coli was further optimized by means of different host bacteria as well as different culture conditions including IPTG concentration, temperature, medium, etc. The protein was preliminarily purified with GST affinity chromatography. Results： The fragment comprised of aa 12-143 of the VP6 （nt 34-429） was selected as the target peptide for prokaryotic expression after comprehensive bioinformatics analysis. The 396bp fragment encoding the peptide was amplified by PCR and the recombinant plasmid pGEX-5XVP6.1 containing the VP6-1 gene was correcdy constructed. The efficient expression of the recombinant fusion protein GST： ： VP6-1 harboring the tnmcated VP6 in E. coli was shown by SDS-PAGE and Western blotting, which was performed with an anti-RV polyclonal antibody. The 41kD recombinant fusion protein which occupied about 30% of the total cell protein was mainly expressed in an inclusion body fashion. After a series of optimization procedures, the sol- uble expression of the GST： ：VP6-1 was finally increased to some extent through lower culture temperature in combination with lower IPTG concentration and could be well purified by GST-Sepharose 4B chromatography. Conclusions： The efficient expression and purification of the VP6-1 protein in E. coli makes it possible to prepare the specific monoclonal antibodies against VP6, and may lay a foundation for the development of RV specific immunoassays.