家蝇肽聚糖识别蛋白(PGRP-SA)的克隆表达及细菌结合研究  

Cloning,Expression and Bacterial Binding of Peptidoglycan Recognition Proteins-SA Gene from Musca domestica

作　　者：罗嫚[1] 王宇[1,2] 胡亚[1] 修江帆[1] 王涛[1] 彭建[1] 尚小丽[1] 吴建伟[1] LUO Man WANG Yu HU Ya XIU Jiang-fan WANG Tao PENG Jian SHANG Xiao-li WU Jian-wei(Department of Parasitology, Guizhou Medical University, Guiyang 550004 Guizhou Provincial Center for Disease Control and Prevention, Guiyang 550004)

机构地区：[1]贵州医科大学寄生虫学教研室,贵阳550004 [2]贵州省疾病预防控制中心,贵阳550004

出　　处：《生物技术通报》2016年第8期145-151,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(81360254);贵州省农业攻关项目(NY[2014]3054)

摘　　要：对家蝇PGRP-SA基因进行克隆表达以及研究其重组蛋白与细菌结合能力。从构建的家蝇(Musca domestica)幼虫cDNA质粒文库中筛选到PGRP-SA基因,以cDNA质粒为模板设计引物,通过PCR扩增,获得PGRP-SA基因完整编码序列。运用生物信息学方法对该基因及其编码蛋白进行预测和分析。构建pET-28a(+)-PGRP-SA重组质粒,转化到大肠杆菌BL21(DE3)中进行诱导表达及蛋白纯化。利用半定量RT-PCR检测PGRP-SA在家蝇3龄幼虫不同组织中的表达量差异。PGRP-SA重组蛋白进行微生物结合实验。结果表明,PGRP-SA基因ORF全长615 bp,编码204个氨基酸,理论分子量22.8 k D,等电点9.11,具有保守的PGRP结构域。成功构建了pET-28a(+)-PGRP-SA重组质粒,蛋白经IPTG诱导后在大肠杆菌中获得表达,经亲和层析柱纯化获得目的蛋白,利用Western blot检测证明纯化蛋白与预期大小相符。PGRP-SA在家蝇3龄幼虫血淋巴、脂肪体、前肠、中肠、气管、马氏管都有表达,血淋巴组织中表达量最高,后肠无表达,由此说明PGRP-SA基因的表达具有一定的组织性。PGRP-SA重组蛋白能与金黄色葡萄球菌和大肠杆菌结合,与白色念珠菌不能结合。成功表达及纯化家蝇PGRP-SA蛋白,证实家蝇PGRP-SA能与金黄色葡萄球菌和大肠杆菌结合。This work is to clone and express the Musca domestica peptidoglycan recognition proteins-SA（PGRP-SA）gene and research the microbial binding activity of it. The PGRP-SA gene was isolated from M. domestica larvae cDNA library,then primers were designed using cDNA plasmid as the template,and the complete encoding sequence of PGRP-SA was acquired by PCR. The bioinformatics methods were employed to predict and analyze the gene and its encoded proteins. The recombinant plasmid pET-28a（＋）-PGRP-SA was expressed in Escherichia coli for induced expression and protein purification. RT-PCR was used to test the varied transcription levels of PGRP-SA gene in different tissues. The microbial binding activity of PGRP-SA protein was studied by the microorganism binding assay. The results indicated that the ORF full-length of PGRP-SA gene was 615 bp,encoding 204 amino acids. The molecular weight was 22.8 kD and pI 9.11 and had the conserved PGRP domain. After the recombinant plasmid pET-28a（＋）-PGRP-SA was successfully constructed,it was expressed in E. coli by IPTG. SDS-PAGE and Western blot analysis showed that the functional protein purified by Ni2＋ affinity chromatography was in consistent as the predicted in size. PGRP-SA was expressed in three instars hemolymph,fat body,foregut,midgut,trachea,malpighian tube except hindgut,the highest in the hemolymph,indicating that the expression of PGRP-SA was tissue-specific. Recombinant PGRP-SA protein bound to Staphylococcus aureus and E. coli,but not Monilia albica. Conclusively,the M. domestica PGRP-SA protein was successfully expressed and purified,and also it was confirmed that PGRP-SA protein from M. domestica bound to S. aureus and E. coli.

关 键 词：家蝇 先天免疫 肽聚糖识别蛋白 原核表达 微生物结合实验 

分 类 号：R392[医药卫生—免疫学] Q78[医药卫生—基础医学]