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目的原核表达并纯化人呼吸道合胞病毒(Human respiratory syncytial virus,HRSV)兰州株黏附蛋白(G)片段。方法利用PCR技术从HRSV兰州株(A亚型)中扩增G基因AA75-225片段,克隆至原核表达载体pET-42b(+)中,构建重组表达质粒pET-42b-G,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经镍离子亲和层析柱纯化后,进行SDS-PAGE和Western blot分析。结果 PCR扩增获得453 bp的DNA片段;重组表达质粒pET-42b-G经双酶切及测序鉴定证明构建正确;表达的重组蛋白相对分子质量为50 230,表达量占菌体总蛋白的25%,以包涵体和可溶性两种方式存在;纯化后重组蛋白纯度可达95%以上,可与抗RSV-G蛋白单克隆抗体特异性结合。结论已成功地在大肠杆菌BL21(DE3)中表达了HRSV兰州株G片段,为后续RSV疫苗的研制、RSV感染的血清学诊断及试剂盒的研发提供了材料。
Objective To prokaryotic express and purify the Lanzhou strain adhesion protein (G) fragment of human respiratory syncytial virus (HRSV). Methods The gene fragment of AA75-225 of G gene was amplified from Lanzhou strain (A subtype) of HRSV by PCR and cloned into prokaryotic expression vector pET-42b (+). The recombinant plasmid pET-42b-G was constructed and transformed into E.coli BL21 (DE3), induced by IPTG. The expressed product was purified by nickel ion affinity chromatography and analyzed by SDS-PAGE and Western blot. Results A 453 bp DNA fragment was amplified by PCR. The recombinant plasmid pET-42b-G was confirmed by double enzyme digestion and sequencing. The recombinant protein was expressed with a molecular weight of 50 230 and a total content of 25 %, In the presence of inclusion bodies and soluble in two ways; Purified recombinant protein purity of up to 95%, with anti-RSV-G protein monoclonal antibody specific binding. Conclusion The G fragment of Lanzhou strain HRSV was successfully expressed in Escherichia coli BL21 (DE3), which provided material for the development of subsequent RSV vaccine, serological diagnosis of RSV infection and the development of the kit.