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

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

Abstract

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

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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