正交试验在人乳头瘤病毒16型E2蛋白原核表达优化中的应用  

Application of Orthogonal Analysis to the Optimization of HPV16 E2 Protein Expression

作　　者：商庆龙[1] 马延秀[1] 郭志伟[1] 李立群[1] 郝美丽[1] 孙宇辉[2] 魏兰兰[1] 谷鸿喜[1] 

机构地区：[1]哈尔滨医科大学微生物学教研室,黑龙江省感染与免疫重点实验室,黑龙江省高校病原学重点实验室,哈尔滨150081 [2]哈尔滨医科大学附属第一医院妇产科,哈尔滨150001

出　　处：《生物医学工程学杂志》2011年第5期988-991,共4页Journal of Biomedical Engineering

基　　金：黑龙江省教育厅面上基金资助项目(11551236)

摘　　要：鉴定人乳头瘤病毒(HPV)16型E2蛋白的重组表达菌株,通过正交试验快速优化诱导表达条件,增加目的蛋白的表达量。应用SDS-PAGE及Western blot分析鉴定表达后的蛋白。选取诱导时间、诱导剂浓度、细菌密度OD600和诱导温度共4个主要外部因素。采用四因素两水平正交试验设计,以单位体积内HPV16 E2目的蛋白含量作为检测指标,结果用SPSS 13.0软件进行统计分析。结果表明,HPV16 E2菌株鉴定正确。HPV16 E2蛋白表达主要以不溶性蛋白为主,主要影响因素是诱导剂浓度和诱导温度,最佳诱导条件为37℃、1.0 mmol/L IPTG和OD600为1.0的条件下诱导7 h,正交试验对分子生物学反应的影响因素分析和条件优化有借鉴意义。This study was aimed to identify pET21b-HPV16EZ/BL2I （DE3） strain and to optimize the expression of human papillomavirus type 16（HPV16）E2 protein by orthogonal analysis. Four influence factors on two levels were selected to increase the target protein quantity. The four factors were induction time, induction temperature, inductor concentration and cell density. The quantity of HPV16 E2 protein was used as the evaluation parameter. Induced by IPTG, HPV16 E2 protein was analyzed by SDS-PAGE and Western Blot. Target protein was analyzed by GIS im- aging system to quantify the protein level. SPSS13.0 software was applied to analyze the result. Data showed that the expression strain pET21b-HPV16 E2/BL21（DE3） was identified correctly. HPV16 E2 protein expressed mainly at insoluble form. The 42KD protein band was identified by SDS-PAGE and Western blot. Orthogonal test was ap- plied on influence factor analysis and expression optimization successfully. Main influence factors were inductor con- centration and induction temperature. The optimimum condition of maximum expression quantity was 37°C, 7h, 1.0 mmol/L IPTG and ODo00 1.0. In this experiment, orthogonal test could not only be used to analyze the influential factors and promote the target protein expression, but also be used to provide a better experiment method for molecular biological study.

关 键 词：人乳头瘤病毒16型E2蛋白 正交试验 原核表达 条件优化 

分 类 号：R373[医药卫生—病原生物学]