基于自转运蛋白Ag43的HPV16L1蛋白的细菌表面展示  

Surface display of HPV16L1 by autotransporter Ag43

作　　者：蔡锟 王哲 黄丕英 韦良婉 王星媛 许雪梅[1] 储引娣 朱沛沛 范恩国 Cai Kun;Wang Zhe;Huang Piying;Wei Liangwan;Wang Xingyuan;Xu Xuemei;Chu Yindi;Zhu Peipei;Fan Enguo(State Key Laboratory of Medical Molecular Biology,Department of Microbiology and Parasitology,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100005,China;Department of Neurology,the Second Medical Center&National Clinical Research Center for Geriatric Diseases,Chinese PLA General Hospital,Beijing 100036,China)

机构地区：[1]中国医学科学院基础医学研究所北京协和医学院基础学院病原学系,医学分子生物学国家重点实验室,北京100005 [2]中国人民解放军总医院第二医学中心神经内科二病区,国家老年疾病临床医学研究中心,北京100036

出　　处：《中华微生物学和免疫学杂志》2022年第3期178-184,共7页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(31770901);国家重点实验室专项资金(2060204)。

摘　　要：目的:通过构建含有不同大小乘客结构域的Ag43表面展示载体,观察其将外源蛋白HPV16L1展示在细菌细胞表面的效率。方法:(1)利用基因工程手段将不同长度的Ag43基因序列连接到pET22b载体,获得4种Ag43表面展示载体:Ag43/138、Ag43/551、Ag43/552、Ag43/700;(2)克隆HPV16L1编码基因序列,利用基因工程手段将其分别连接到上述4种Ag43表面展示载体,获得4种重组蛋白表达载体;(3)HPV16L1-Ag43融合蛋白表达及SDS-PAGE分析;(4)利用胰蛋白酶消化验证HPV16L1蛋白在大肠埃希菌的表面展示。结果:琼脂糖凝胶电泳结果显示PCR扩增获得的Ag43和HPV16L1条带与预期大小一致,表面展示载体和重组蛋白表达载体的基因序列均通过基因测序验证无误。经IPTG诱导后,构建的4种Ag43表面展示载体均能表达HPV16L1蛋白。胰蛋白酶消化后,4种重组蛋白条带均减弱,且Ag43/700-HPV16L1条带减弱最明显。结论:成功构建了基于自转运蛋白Ag43的细菌表面展示载体,HPV16L1蛋白可通过构建的4种Ag43表面展示载体表达并展示在大肠埃希菌表面,且仅保留α-螺旋和β-桶状结构域部分的Ag43表达载体即Ag43/700的表面展示效果最优。Objective To construct a surface display system containing various lengths of the Ag43 passenger domain for an optimal bacterial surface display of foreign protein HPV16L1.Methods(1)Ag43 gene sequences of different lengths were inserted into pET22b vector to construct four Ag43 surface display vectors(Ag43/138,Ag43/551,Ag43/552 and Ag43/700)using PCR and subcloning strategy.(2)The generation of four HPV16L1-Ag43 fusion constructs was completed by PCR and subcloning methods.(3)HPV16L1-Ag43 fusion proteins were expressed and analyzed by SDS-PAGE.(4)The surface exposure of HPV-16L1 was verified using trypsin digestion.Results PCR analysis and sequencing results showed that Ag43 surface display vectors and HPV16L1-Ag43 fusions were constructed successfully.SDS-PAGE showed that the expression of HPV16L1-Ag43 fusion proteins could be induced with 0.2 mmol/L IPTG and the protein content was reduced after the cells were treated with trypsin,especially the content of Ag43/700-HPV16L1 that showed a drastic reduction.Conclusions The Ag43 surface display system was successfully constructed and could be used for a successful display of HPV16L1.This study also showed that Ag43/700 comprising only theα-helix and theβ-barrel of Ag43 provided an optimal surface display for HPV16L1.

关 键 词：自转运蛋白 细菌表面展示 HPV16L1 Ag43 

分 类 号：R378[医药卫生—病原生物学]