斯氏按蚊肽聚糖识别蛋白(PGRP-LB)序列分析及其原核表达  

Sequencing and protein expression of a peptidoglycan recognition protein (PGRP-LB) in Anopheles stephensi

作　　者：姜欣伶 宋秀梅 王敬文 韩颖颖[1] JIANG Xin—ling;SONG Xiu—mei;WANG Jing—wen;HAN Ying—ying(School of Medical Instrument and Food Engineering,University of Shanghai for Science and Technology,Shanghai 200093,China;School of Life Science,Fudan University)

机构地区：[1]上海理工大学医疗器械与食品学院,上海200093 [2]复旦大学生命科学学院

出　　处：《中国病原生物学杂志》2020年第5期503-507,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31822051);美国NIH RO1(No.5R01AI129819)。

摘　　要：目的分析斯氏按蚊肽聚糖识别蛋白PGRP-LB的序列,表达和纯化PGRP-LB蛋白。方法采用生物信息学方法分析PGRP-LB蛋白的序列。以斯氏按蚊的cDNA为模板PCR扩增PGRP-LB,采用无缝克隆的方法将其克隆到原核表达载体pET28a中,然后转化入大肠埃希菌E.coli BL21(DE3)感受态细胞,利用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)进行目的蛋白的诱导表达。SDS-PAGE凝胶电泳及考马斯亮蓝染色检测PGRP-LB的表达情况。经镍离子亲和层析纯化后,Bradford法检测洗脱液的浓度。通过MEGA7软件构建系统进化树,进行PGRP-LB的进化分析。结果成功获得pET28a-PGRP-LB的重组质粒,37℃、0.5mmol/L的IPTG能诱导出大量的包涵体PGRP-LB重组蛋白,相对分子质量约为26×103,纯化后的蛋白浓度为0.5mg/ml。进化分析显示,斯氏按蚊PGRP-LB与同科物种间亲缘关系较近。结论成功使用大肠埃希菌原核表达系统表达斯氏按蚊PGRP-LB蛋白,为该蛋白在按蚊体内的定位及功能研究奠定了基础。Objectives To sequence,express,and purify the peptidoglycan recognition protein PGRP-LB in Anopheles stephensi.Methods The PGRP-LB gene was screened from an Anopheles stephensi cDNA library.The sequence of the PGRP-LB protein was analyzed bioinformatically.PGRP-LB was amplified using PCR with specific primers.After the vector plasmid was double-digested with EcoRⅠand Xhol,PGRP-LB was ligated into the prokaryotic expression vector pET28 aby seamless cloning and then transformed into E.coli BL21(DE3).After expression of the plasmid was induced with isopropylβ-D-thiogalactoside(IPTG),recombinant PGRP-LB was detected using SDS-PAGE gel electrophoresis and Coomassie blue staining.After purification via affinity chromatography with His-tag,the concentration of the eluate was measured using the Bradford method.Results The recombinant plasmid pET28 a-PGRP-LB was successfully obtained.At 37℃,0.5 mmol/L of IPTG induced the expression of a large amount of PGRP-LB recombinant inclusion body protein with a molecular weight of about 26×103.The recombinant protein at a concentration of 0.5 mg/mL can be obtained under the elution conditions of 8 mol/L of urea and 500 mmol/L of imidazole.Sequence alignment and phylogenetic tree analysis indicated that PGRP-LB is a transmembrane protein and is closely related in the same species.Conclusion A system for prokaryotic expression of E.coli was successfully used to express PGRP-LB in Anopheles stephensi,laying the foundation for further experimental research.

关 键 词：斯氏按蚊 PGRP-LB 原核表达 

分 类 号：R384.1[医药卫生—医学寄生虫学]