人乳头瘤病毒16型L1蛋白VLP的原核表达及纯化

目的在大肠杆菌中表达人乳头瘤病毒16型(HPV16)L1蛋白病毒样颗粒(VLP),并进行纯化,为研制宫颈癌疫苗提供新的思路。方法以HPV16基因组DNA为模板,PCR扩增HPV16L1基因的编码区,定向克隆至原核表达载体pGEX-4T-1中谷胱甘肽转移酶(GST)编码区下游,构建重组表达质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot鉴定后,采用GST纯化柱纯化重组融合蛋白,并用凝血酶切去GST标签,再经S100分子筛纯化,浓缩后,电镜观察纯化蛋白的VLP形态。结果重组表达质粒经双酶切和测序,证明构建正确。表达产物的相对分子质量约为80000,主要以可溶性形式存在。Western blot显示,目的蛋白可与小鼠抗HPV16L1抗体发生特异性反应。纯化蛋白的纯度约为95%,在电镜下可见直径约为50～60nm的VLP。结论已成功地在大肠杆菌中表达了可溶性的HPV16L1蛋白VLP,并进行了纯化,为宫颈癌疫苗及血清学诊断试剂的研制奠定了基础。

Prokaryotic Expression and Purification of Virus-like Particles of Major Cap-sid Protein L1 of Human Papillomavirus Type 16

Objective To express the virus-like particles(VLPs)of major capsid protein L1 of human papillomavirus(HPV) type 16 in E.coli, purify the expressed product and lay a foundation of development of cervical cancer vaccine.Methods The encoding region of HPV16 L1 gene was amplified by PCR using the genomic DNA of HPV16 as a template, and directionally cloned downstream to the encoding region of glutathione S-transferase(GST)in prokaryotic expression vector pGEX-4T-1.The constructed recombinant plasmid pGEX-4T-1-HPV16L1 was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot and purified by GST affinity chromatography.After removal of GST label by cleavage with thrombin, the expressed fusion protein was further purified by S100 molecular sieve chromatography, then concentrated and observed by electron microscopy.Results Restriction analysis and sequencing proved that recombinant plasmid pGEX-4T-1-HPV16L1 was constructed correctly.The expressed fusion protein, with a relative molecular mass of about 80 000, main-ly existed in a soluble form and showed specific reaction with murine McAb against HPV16 L1.The purified fusion protein reached a purity of about 95%, and the VLPs at sizes of about 50～60 nm were observed by electron microscopy.Conclusion The VLPs of soluble HPV16 L1 protein were successfully expressed in E.coli and purified, which laid a foundation of developing cervical cancer vaccine and serological diagnostic kit.