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目的可溶性表达牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)E2蛋白。方法参考BVDV NADL株序列设计1对特异性引物,PCR扩增BVDV NADL株E2全长编码区片段,目的片段经限制性内切酶克隆入质粒p ET28a,经双酶切及测序鉴定后,阳性重组质粒转化E.coli Rosetta(DE3)宿主菌,37℃IPTG诱导表达后,进行SDS-PAGE及Western blot分析;表达的重组蛋白经Ni-NTA Purification System纯化后进行SDS-PAGE鉴定。结果表达的重组E2蛋白相对分子质量约45 000,主要以可溶性形式存在,表达量占菌体总蛋白的38.9%,可与BVDV阳性血清发生特异性反应,纯度达95%以上,浓度约3.8 mg/ml,产量可达4.96 mg/L细菌培养物。结论实现了可溶性重组BVDV E2蛋白在原核表达系统中的表达,并具有较好的生物学活性。
Objective To express soluble E2 protein of bovine viral diarrhea virus (BVDV). Methods A pair of specific primers was designed according to the sequence of NADL strain of BVDV. The full-length coding region of BVDV NADL strain was amplified by PCR. The target fragment was cloned into plasmid pET28a by restriction enzyme and identified by double enzyme digestion and sequencing The recombinant plasmids were transformed into E.coli Rosetta (DE3) host strain and induced by IPTG at 37 ℃. The recombinant plasmids were analyzed by SDS-PAGE and Western blot. The expressed recombinant proteins were purified by Ni-NTA Purification System and identified by SDS-PAGE. Results The expressed recombinant E2 protein had a relative molecular mass of about 45 000 and existed mainly in soluble form. The expressed recombinant protein accounted for 38.9% of the total bacterial proteins and reacted specifically with BVDV positive serum with a purity of over 95% at a concentration of about 3.8 mg / ml, yield up to 4.96 mg / L bacterial culture. Conclusion The expression of soluble recombinant BVDV E2 protein in prokaryotic expression system has been achieved and has good biological activity.