口蹄疫病毒VP1-3免疫表位基因与猪γ-干扰素基因的融合表达

Fusion expression of immunological epitopes from VP1-3 of foot-and-mouth disease virus with porcine interferon-gamma

将人工合成的口蹄疫病毒VP1-3上的抗原表位基因和猪γ-干扰素基因串联入原核表达载体pGEX-KG中,经酶切鉴定及测序表明重组载体中二者以接头融合的形式构建成重组表达质粒。将该质粒转化BL21后经IPTG诱导,实现了重组融合蛋白的高效表达,表达产物经SDS-PAGE和Western blot分析,重组蛋白分子量约为66 Ku,与预期大小相符。薄层扫描分析显示表达的重组蛋白占菌体总蛋白的37.4%,且主要以包涵体的形式存在。包涵体以SKL变性后,经PEG4000、氧化型谷胱苷肽和还原型谷胱苷肽复性。粗提产物以CPE_(50)测得重组蛋白在MDBK细胞上的抗水泡性口炎病毒(Vesicular stomatitis virus,VSV)的活性达到1600 U/mL,说明表达产物具有干扰素的生物学活性,为下一步基因工程疫苗的研究奠定了基础。

Synthetic immtmological epitopes from VP1-3 of foot-and-mouth disease virus （FMDV） were inserted into prokaryotic expression vector （pGEX-KG） in cascade connection with porcine interferon- γ. The recombinant plasmids were confirmed by restriction digestion and sequencing, and transformed into BL21 cells. Protein expression was expressed by IPTG induction and analyzed by SDS-PAGE and Western blot. The results showed that the fusion protein, as expected, has a molecular weight of 66 Ku. The protein was expressed in high yield accounting for 37.4 % of the total protein and mainly existed in inclusion bodies. The protein was extracted by SKL treatment of inclusion bodies, and re-natured by PEG4000 and tathion. The crude product has an anti-VSV （Vesicular stomatitis virus, VSV） activity of 1600 U/mL of in MDBK cells by CPE50 method, indicating that the expressed protein had the biologic activity of interferon. This study established foundation for future work on genetically engineered vaccines.