小菜蛾PGRP-SA的体外表达及介导酚氧化酶活力的研究

The research of PGRP-SA's expression in vitro and its involving in phenoloxidase activity from Plutella xylostella

肽聚糖识别蛋白(Peptidoglycan Recognition Proteins,PGRPs)是可以识别肽聚糖和含肽聚糖的细菌的一类模式识别受体,在介导酚氧化酶级联反应中起着重要的作用。本研究以实验室克隆的小菜蛾Px PGRP-SA(Gen Bank No.EU399240)为基础,RT-PCR克隆了其开放阅读框(ORF),在原核细胞中获得了高效重组表达,利用GST一步纯化的融合蛋白免疫新西兰大白兔,制备了抗血清,滴定效价为1∶51200。Western blot检测表明Micrococcus luteus和Serratia marcescens可以显著提高Px PGRP-SA在小菜蛾血淋巴中的含量。为了检测Px PGRP-SA与酚氧化酶PO的活力关系,本研究将Px PGRP-SA连接到真核表达载体p MT/Bip/V5/His A构建真核表达质粒p MTAPGRP,转染果蝇S2细胞中,获得稳定的细胞系,硫酸铜诱导获得表达后用anti-V5纯化获得重组蛋白Px PGRP-SA。将重组蛋白Px PGRP-SA与M.luteus和S.marcescens分别温育后,可以显著提高小菜蛾血浆中PO的活力。本研究结果阐明了重组蛋白Px PGRP-SA在小菜蛾体PO级联反应中的功能,为进一步研究Px PGRP-SA在小菜蛾体内免疫信号通路中的功能奠定了基础。

Peptidoglycan Recognition Protein（ PGRPs） are one family of pattern recognition receptors,which can recognize peptidoglycan,or bacteria containing peptidoglycan,and are playing an important role in mediating the phenoloxidase activity. In this study,based on the sequence encoding short type PGRP（ Gen Bank No. EU399240） which was obtained from Plutella xylostella in our lab,the open reading frame（ ORF） sequence of Px PGRP-SA was amplified by RT-PCR and cloned into prokaryotic plasmid. The fusion protein was highly expressed in prokaryotic cell and purified by GST tag. The purified fusion protein was immuned the New Zealand white rabbit to prepare the antiserum. The titre of the prepared antiserum is1 ∶ 51200. Western blot revealed that microbes including Micrococcus luteus and Serratia marcescens can induce the expression of protein Px PGRP-SA in P. xylostella. In order to clarify whether the protein Px PGRP-SA is involving in the activating of prophenoloxidase（ PPO） in P. xylostella. In the present study,Px PGRP-SA sequence is ligated into eukaryotic expression vector p MT / Bi P / V5 / His A,and was transiently transfected into Drosophila S2 cells and the stable cell lines containing recombinant Px PGRP-SA plasmidwere induced by Cu SO4. Recombinant protein Px PGRP-SA was purified from S2 cells by V5 tag. When the recombinant protein Px PGRP-SA was incubated with two microbes M. luteus and S. marcescens separately,the PO activity was increased significantly. This study illustrates the function of the recombinant protein Px PGRP-SA takes a vital role was involved in activating the PPO in P. xylostella,and our results,lay the foundation for the further research of Px PGRP-SA owning the immune function of signaling pathways in P.xylostella.