家蚕整合素β2的表达、纯化及其免疫功能  

Expression, Purification and Immunologic Function of Integrin β2 in the Silkworm(Bombyx mori)

作　　者：张奎[1] 李重阳[1] 苏晶晶[1] 谈娟[1] 徐曼[1] 崔红娟[1] ZHANG Kui;LI ChongYang;SU JingJing;TAN Juan;XU Man;CUI HongJuan(State Key Laboratory of Silkworm Genome Biology,Southwest University,Chongqing 400716)

机构地区：[1]西南大学家蚕基因组生物学国家重点实验室,重庆400716

出　　处：《中国农业科学》2019年第1期181-190,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31802142);中国博士后科学基金(2017M620408);重庆市自然科学基金(cstc2016jcyjA0425);重庆高校创行团队建设基金(CXTDX201601010)

摘　　要：【目的】分析家蚕(Bombyx mori)整合素β2的序列和结构特征及其在家蚕感染病原菌后血细胞中的表达变化,检测其重组蛋白对不同病原相关模式分子识别和病原菌的凝聚作用,为进一步探究家蚕整合素β2的蛋白功能打下基础。【方法】利用生物信息学对整合素β2的序列和结构特征进行分析,利用实时荧光定量PCR检测家蚕分别注射大肠杆菌(Escherichia coli)和金黄色葡萄球菌(Staphylococcus aureus)后整合素β2在血细胞中的表达变化。通过PCR技术扩增获得整合素β2完整的胞外域片段,构建至pET22b原核表达载体后转化至E.coli Rosetta(DE3)表达菌株。经IPTG诱导获得重组蛋白,利用Ni-NTA亲和层析得到纯化的体外重组蛋白,利用SDS-PAGE和Western blot对纯化获得的体外重组蛋白的纯度和质量进行检测。利用ELISA检测重组蛋白与两种不同病原相关分子模式LPS和PGN的结合情况,运用Western blot检测重组蛋白与不同病原微生物的结合情况,通过凝集试验检测重组蛋白对金黄色葡萄球菌的凝集能力,最后在个体水平探索重组蛋白在体内细菌清理中的作用。【结果】家蚕整合素β2具有典型的β整合素亚基保守结构特征,即由一个较长的胞外域、一个单次跨膜结构域和一个较短的胞内域构成。家蚕整合素β2具有金属离子结合位点MIDAS、EGF结构域、半胱氨酸重复基序和NPxY等整合素典型的结构特征。实时荧光定量PCR检测结果表明家蚕在受到细菌感染后,整合素β2的表达发生显著变化。通过原核表达和蛋白纯化获得纯度较高的重组蛋白,SDS-PAGE和Western blot检测结果表明纯化的重组蛋白纯度较高,可以用于后续试验。ELISA试验表明重组蛋白对LPS和PGN等病原相关分子模式具有较强的结合能力。细菌结合试验结果显示重组蛋白能够结合多种细菌,但与革兰氏阳性菌的结合能力要高于革兰氏阴性菌。细菌凝聚试验【Objective】The objective of this study is to analyze the gene sequence and structural characteristics of integrin β2 in silkworm(Bombyx mori), and its expression profile in hemocytes following the larval exposure to different bacterial pathogens, investigate the binding and agglutination properties of the recombinant integrin β2 protein to various pathogen-associated molecular patterns(PAMPs) and bacteria, which will lay a foundation for further exploring the protein function of integrin β2 in B. mori. 【Method】Bioinformatics tools were used to determine the sequence and structural characteristics of integrin β2, and real-time fluorescent quantitative PCR(RT-qPCR) assay was executed to evaluate expression profile in hemocytes after microbial(Escherichia coli and Staphylococcus aureus) challenge. The cDNA fragment of integrin β2 was amplified using PCR, and the fragment containing the extracellular domain was inserted into a prokaryotic expression vector(pET22 b). The insertion was confirmed in the recombinant plasmid and transformed into E. coli Rosetta(DE3), and then induced by IPTG to produce recombinant protein. The recombinant protein was purified using Ni-NTA affinity chromatography and analyzed by SDS-PAGE and Western blot. ELISA and Western blot were executed to determine the binding abilities of the recombinant protein to PAMPs(LPS and PGN) and different bacteria. Moreover, the agglutination ability and bacterial clearance assay were performed to understand the specific biological roles of integrin β2 in immunity.【Result】B. mori integrin β2 contains typical integrin β subunits, which comprises a long extracellular domain, a single transmembrane region and a short cytoplasmic tail. Further, it has several conserved motifs such as the MIDAS, EGF domain, Cys-repeat sequences and NPxY motifs. The RT-qPCR analysis showed that the integrin β2 expression varied significantly in hemocytes after infection with bacteria. High purity recombinant protein was obtained by prokaryotic expression and

关 键 词：家蚕 整合素β2 细菌感染 原核表达 细菌凝聚 

分 类 号：S881.2[农业科学—特种经济动物饲养]