家蝇肽聚糖识别蛋白（PGRP-SA）的克隆表达及细菌结合研究

doi:10.13560/j.cnki.biotech.bull.1985.2016.08.022

摘要/Abstract

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 kD,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。

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

Abstract: 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.

Key words: Musca domestica, innate immunity, peptidoglycan recognition proteins, prokaryotic expression, microorganism binding assay

罗嫚, 王宇, 胡亚, 修江帆, 王涛, 彭建, 尚小丽, 吴建伟. 家蝇肽聚糖识别蛋白（PGRP-SA）的克隆表达及细菌结合研究[J]. 生物技术通报, 2016, 32(8): 145-151.

LUO Man, WANG Yu, HU Ya, XIU Jiang-fan, WANG Tao, PENG Jian, SHANG Xiao-li, WU Jian-wei. Cloning,Expression and Bacterial Binding of Peptidoglycan Recognition Proteins-SA Gene from Musca domestica[J]. Biotechnology Bulletin, 2016, 32(8): 145-151.

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