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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。
Objective To express and purify human papillomavirus type 16 (HPV16) L1 protein in Escherichia coli (E. coli), and to provide a new idea for the development of cervical cancer vaccine. Methods HPV16 genomic DNA was used as a template to amplify the coding region of HPV16 L1 gene and then cloned into the downstream of coding region of glutathione transferase (GST) in prokaryotic expression vector pGEX-4T-1. The recombinant plasmid was constructed and transformed into E. coli BL21 (DE3), induced by IPTG. The expressed product was identified by SDS-PAGE and Western blot. The recombinant fusion protein was purified by GST purification column, and then the GST tag was removed by thrombin. After purification by S100 molecular sieve, the VLP morphology of the purified protein was observed by electron microscopy. Results Recombinant plasmids were double-digested and sequenced to confirm that they were constructed correctly. The relative molecular weight of the expressed product was about 80,000, mainly in soluble form. Western blot showed that the target protein could specifically react with mouse anti-HPV16 L1 antibody. The purity of the purified protein was about 95%. The diameter of the VLP was about 50 ~ 60 nm under the electron microscope. Conclusion The soluble HPV16 L1 protein VLP was successfully expressed in Escherichia coli and purified. It laid the foundation for the development of cervical cancer vaccine and serodiagnostic reagents.