HPV16型L1蛋白优势表位原核表达及其血清学验证  

Prokaryotic expression and serological verification of antigenic epitopes of human papillomavirus type 16 L1 protein

作　　者：侯江厚 张灵霞[2] 孙雯娜 刘艳华[2] 杨秉芬 孙卫国 HOU Jianghou;ZHANG Lingxia;SUN Wenna;LIU Yanhua;YANG Bingfen;SUN Weiguo(Kunming City Maternal and Child Health Hospital,Kunming 650013,Yunnan,China;Army Tuberculosis Prevention and Control Key Laboratory,Beijing Key Laboratory of New Techniques of Tuberculosis Diagnosis and Treatment,Institute for Tuberculosis Research,the Eighth Medical Center of Chinese PLA General Hospital,Beijing 100091,China)

机构地区：[1]昆明市妇幼保健院,云南昆明650013 [2]解放军总医院第八医学中心结核病研究所全军结核病防治重点实验室结核病诊疗新技术北京市重点实验室,北京100091

出　　处：《检验医学》2020年第9期928-932,共5页Laboratory Medicine

基　　金：国家传染病重大项目(2013ZX-10003-006)。

摘　　要：目的利用原核系统对人乳头瘤病毒(HPV)16型L1蛋白优势抗原表位进行重组表达与纯化,并检测血清学反应。方法对HPV16型L1蛋白优势抗原表位进行全基因合成,将核酸片段克隆至pET-DsbC原核表达载体,重组质粒转化大肠埃希菌Rosetta(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,采用亲和层析对表达产物进行纯化,采用免疫印迹法、酶联免疫吸附试验(ELISA)验证重组蛋白的免疫学活性。结果成功构建了pET-DsbC-HPV16 L1表达载体,并在大肠埃希菌Rosetta(DE3)中实现了稳定的可溶性表达。重组DsbC-HPV16 L1蛋白经亲和层析进行纯化,相对分子质量为45000,纯度为95%,重组蛋白纯化得率为22%。免疫印迹法结果显示,重组DsbC-HPV16 L1蛋白可与HPV16型阳性血清产生特异的抗原-抗体反应,与重组DsbC蛋白或健康人血清无交叉反应,具有良好的特异性。ELISA结果显示,重组DsbC-HPV16 L1蛋白与HPV16型阳性血清有较强的免疫反应性,A450 nm均值为0.56,高于健康人血清和磷酸盐缓冲液(PBS)阴性对照(A450 nm均值分别为0.24和0.21)。结论采用原核表达系统实现了HPV16型L1蛋白优势抗原表位的可溶性高表达,获得的重组蛋白具有良好的免疫原性和免疫学活性。Objective To obtain antigenic epitopes of human papillomavirus(HPV)type 16 L1 protein expression and purification in prokaryotic system,and to evaluate its serological reaction.Methods The nucleotide sequence of HPV type 16 L1 protein antigenic epitopes were synthesized and cloned into prokaryotic expression vector pET-DsbC.The constructed recombinant plasmid pET-DsbC-HPV16 L1 was transformed to Escherichia coli BL21 Rosetta(DE3)for expression under the induction of isopropyl-beta-D-thiogalactoside(IPTG).The L1 protein was expressed and purified by metal chelate affinity chromatography.The antigenicity and immunological activity of fusion protein were evaluated with clinical serum samples by western blotting and enzyme-linked immunosorbent assay(ELISA).Results The expression vector pET-DsbC-HPV16 L1 was successfully constructed and stably expressed in Escherichia coli Rosetta(DE3).The recombinant HPV16 L1 protein was purified by affinity chromatography,which had a relative molecular mass of 45000 and a purity of 95%.The purified rate of recombinant protein was 22%。The results of western blotting showed that the recombinant DsbC-HPV16 L1 protein could produce specific antigen-antibody reaction with HPV16 positive serum,but there was no cross reaction with recombinant DsbC protein or healthy human serum,which had good specificity.ELISA results also indicated that recombinant protein had a higher level of reactivity to serum from patients than serum from healthy or phosphate-buffered saline(PBS),the A450 values were 0.56,0.24 and 0.21,respectively.Conclusions HPV type 16 L1 protein antigentic epitopes are overexpressed solubly in prokaryotic system and have a high immunogenicity and immunological activity.

关 键 词：人乳头瘤病毒16型L1蛋白 原核表达 免疫原性 血清学反应 

分 类 号：R446.1[医药卫生—诊断学]