轮状病毒VP4蛋白酶切片段VP5*、VP8*在原核系统中的表达

Prokaryotic expression of protein VP5*,VP8*of rotavirus SA11

目的克隆表达轮状病毒结构蛋白VP4酶切片段VP5*、VP8*。方法以猴轮状病毒SA11株细胞培养物提取总RNA为模板,经RT-PCR方法获得VP5*、VP8*全长基因片段cDNA,将其重组于pET-30 a表达载体,转化大肠杆菌BL21(plyss),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达产物做SDS-PAGE和Western-blotting检测,目的产物用镍柱纯化。结果①重组载体在IPTG诱导下,工程菌表达的两个目的蛋白主要以包涵体形式存在;②SDS-PAGE检测表达产物与目的蛋白大小一致,VP5*为60 kD,VP8*为28 kD,Western-blotting检测发现两目的蛋白所带标签可与抗体反应;③镍柱纯化获得RV SA11 VP5*、VP8*蛋白,纯度达90%。结论①构建表达载体pET-30a-VP5*、pET-30a-VP8*;②表达、纯化VP4蛋白的VP5*、VP8*片段。

Objective To express enzyme digestion segments of VP4 protein in prokaryotic expression system. Methods Total RNA was extracted from MA104 cell infected with rotavirus. Total length eDNA of VP5 ＊ （1587bp） and VP8 ＊ （720bp） were amplified by RT-PCR. After sequencing, they were cloned into expression vector pET-30a. VP5 ＊ protein and VP8 ＊ protein were expressed in E. coli BL21 （ plyss） induced by IPTG and analyzed by SDS-PAGE and Western-blotting. VP5 ＊ protein and VP8 ＊ protein were purified by Ni-NTA. Results （1）VP5 ＊ and VP8 ＊ were expressed abundantly mainly in the form of incusion bodies. （2）Molecular weight of expressed products is 60 kD and 88 kD analyzed by SDS-PAGE. By consequence analysis of Western-blotting, we found proteins with a six-His tag can respond to anti-His antibody. （3）By Ni-NTA purification and dialysis, we acquired proteins with more than 90% purity. Conclusion The Rotavirus SA11 VP5 ＊ protein and VP8 ＊ protein were successfully expressed by prokaryotic expression system.