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目的原核表达并纯化呼吸道合胞病毒(respiratory syncytial virus,RSV)F1蛋白截短体(F212-489)。方法从质粒p MD-18T-f中PCR扩增截短f1基因(f212-489),经TA克隆测序正确后,将目的片段插入原核表达载体p ET-28a,构建重组表达质粒p ET-28a-f212-489,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经镍离子亲和层析纯化后,进行Western blot鉴定。结果重组表达质粒p ET-28a-f212-489经双酶切鉴定构建正确;表达的重组蛋白相对分子质量约为30 000,主要以包涵体形式表达,表达量约占菌体总蛋白的10%;纯化的重组蛋白纯度为85%,可与羊抗RSV多克隆抗体特异性结合。结论成功表达了RSV F1蛋白截短体(F212-489),纯化的重组蛋白反应原性良好,为更好地研究RSV的免疫机理及通过基因工程方法研制特定部位的亚单位或多肽疫苗奠定了基础。
Objective To express and purify the F1 syncytial virus (F212-489) recombinant of respiratory syncytial virus (RSV). Methods The truncated f1 gene (f212-489) was amplified by PCR from plasmid p MD-18T-f. After sequencing by TA cloning, the target fragment was inserted into prokaryotic expression vector p ET-28a to construct recombinant expression plasmid p ET-28a -f212-489, transformed into Escherichia coli BL21 (DE3), induced by IPTG. The expressed recombinant protein was purified by nickel ion affinity chromatography and identified by Western blot. Results The recombinant plasmid p ET-28a-f212-489 was identified by double enzyme digestion. The relative molecular mass of the expressed recombinant protein was about 30 000, which was mainly expressed in inclusion bodies. The expression level was about 10% The purity of purified recombinant protein was 85%, which could specifically bind with goat anti-RSV polyclonal antibody. Conclusion The RSV F1 protein truncated fragment (F212-489) was successfully expressed. The purified recombinant protein was found to be good for the study of the immunological mechanism of RSV and the subunits or peptide vaccines of specific sites through genetic engineering basis.