Sequence Analysis And Recombinant Expression Of The Pattern Recognition Receptor PGRP-A From The Chinese Oak Silkworm, Antheraea Pernyi

Insect immunity is different from vertebrate immunity; they have no T cell, B cell and complement system. Although they don’t have the adaptive immune system, they can resist rapidly and effectively to infection of various pathogens through innate immune system. Peptidoglycan recognition proteins (PGRPs) can recognize peptidoglycan of microbial surface and play an important role in immune regulatory pathways. PGRPs have been found in many insects and other animals, although these PGRPs are conservative in the structure, their function and expression patterns is different.Antheraea pernyi is a kind of wild silk-producing insect, which blongs to Lepidoptera Saturniidae Antheraea. A. pernyi has good officinal and edible economical values except for utilizing in spinning industry as raw material. We cloned a PGRP in A. pernyi and named it PGRP-A in this study, At the meanwhile we analyzed the sequence characteristic and expression in Escherichia coli by using the method of molecular biology and bioinformatics. The major results are summarized as follows;1. Cloning and sequence analysis of PGRP-A cDNAA new peptidoglycan recognition protein (PGRP-A) was isolated from the A. pernyi used RT-PCR and RACE-PCR. The cDNA sequence of PGRP-A,961bp in length, contains a40bp5-untranslated sequence (5’-UTR), an open reading frame of582nucleotides encoding a putative protein of193amino acid residues with predicted molecular weight of22.0kDa and isoelectric point of7.368, and a339bp3’-untranslated region (3’-UTR). Phylogenetic analysis indicated that A. pernyi PGRP-A gene had the highest identity with Antheraea mylitta PGRP.2. Expression analysis of PGRP-AThe ORF was amplified by RT-PCR, ligated to the expression vector pET-28a(+), and then transformed the recombinant plasmid into E. coli Transetta (DE3), induced by different concentrationsof IPTG. Through SDS-PAGE and Western blotting analysis, the recombinant protein obtained stable expression in E. coli Transetta (DE3). The induced cells were harvested and subjected to nickel affinity column chromatography to purify recombinant proteins.