林氏扇头蜱酰胺酶型分泌蛋白表达及抗菌功能  

Expression and antibacterial function of amidase-type R.lnPGRP-SC1a in Rhipicephalus linnaei

作　　者：谢子芳 彭维祺 李晴[1,2] 黎明成 陈杰[1,2] 朱勇川 赵建国[1] 管庆丰[1,2] XIE Zifang;PEN Weiqi;LI Qing;LI Mingcheng;CHEN Jie;ZHU Yongchuang;ZHAO Jianguo;GUAN Qingfeng(School of Life and Health Sciences,Hainan University,Haikou,Hainan 570228,China;One Health Research Institute,Hainan University,Haikou,Hainan 570228,China)

机构地区：[1]海南大学生命健康学院,海南海口570228 [2]海南大学全健康研究院,海南海口570228

出　　处：《中国热带医学》2025年第2期161-165,共5页China Tropical Medicine

基　　金：海南省自然科学基金高层次人才项目(No.324RC446)。

摘　　要：目的 探究林氏扇头蜱中的酰胺酶型分泌蛋白(R.ln PGRP-SC1a)的抗菌功能,为蜱虫先天免疫中肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRPs)的功能研究提供参考依据。方法 依据编码区序列设计特异性引物,提取成蜱的RNA并反转录为c DNA,通过PCR扩增R.ln PGRP-SC1a蛋白基因片段,随后将该基因片段连接至质粒p ET32a+,从而构建重组表达载体p ET32a+-R.ln PGRP-SC1a。把该表达载体转入大肠杆菌BL21(DE3)感受态细胞,采用浓度为0.2 mmol/L的IPTG进行低温诱导,促使其在上清中表达,从而获得活性更强的上清蛋白。利用抑菌圈法评估上清蛋白对大肠埃希菌(E.coli)和金黄色葡萄球菌(S.aureus)2种常见致病细菌的抑菌效果。结果 扩增所得的基因片段长度为627 bp,成功构建了原核表达载体p ET32a+-R.ln PGRP-SC1a。经过低温诱导表达,结果显示该重组蛋白为可溶性蛋白,重组蛋白R.ln PGRP-SC1a的大小约为23.63 k D。抗菌结果表明,在同一浓度下,R.ln PGRP-SC1a蛋白对大肠埃希菌无抑制作用,而对金黄色葡萄球菌有抑制作用。R.ln PGRP-SC1a在浓度为5 mg/m L时开始呈现抑菌作用,且随着浓度的升高,抑菌作用增强。与各浓度卡那霉素的抑菌效果相比,其7 mg/m L的抑菌效果相当于1 mg/m L卡那霉素的抑菌效果。同时,R.ln PGRP-SC1a对金黄色葡萄球菌的起效时间为2 h,抑菌作用可持续48 h。结论 本研究成功构建了R.ln PGRP-SC1a重组表达载体,其表达产物具有持久性抗革兰阳性菌的活性,为生物活性抑菌剂的开发提供了可能。Objective To investigate the antibacterial properties of R.lnPGRP-SC1a in Rhipicephalus linnaei,and to provide a reference for the functional analysis of peptidoglycan recognition proteins(PGRPs)within the context of tick innate immunity.Methods Specific primers were meticulously designed based on the coding region sequence,followed by the extraction of tick RNA of adult ticks and its reverse transcription into cDNA.The R.lnPGRP-SC1a gene fragment was subsequently amplified via PCR and then ligated into the plasmid pET32a+,thereby constructing the recombinant expression vector pET32a+-R.lnPGRP-SC1a.This expression vector was then transferred into E.coli BL21(DE3)competent cells and induced with an IPTG concentration of 0.2 mmol/L at low temperature to enhance protein expression in the supernatant,thereby obtaining a soluble protein with stronger activity.Subsequently,the inhibitory effect of the supernatant protein against two common pathogenic bacteria,E.coli and S.aureus,was assessed using the agar diffusion method.Results The amplified gene fragment was 627 bp in length,and the prokaryotic expression vector pET32a+-R.lnPGRP-SC1a was successfully constructed.Low-temperature induction showed that the recombinant protein was soluble protein,with an approximate molecular weight of 23.63 kD.Antibacterial activity results indicated that,at the same concentration,R.lnPGRP-SC1a exhibited no inhibitory effect on E.coli but demonstrated significant inhibition against S.aureus.Specifically,antibacterial activity became evident at a concentration threshold of 5 mg/mL and increased with the protein concentration.Compared with the inhibitory effects of kanamycin at various concentrations,the inhibitory effect of R.lnPGRP-SC1a at 7 mg/mL was comparable to that of kanamycin at 1 mg/mL.Furthermore,the onset of R.lnPGRP-SC1a inhibitory effect against S.aureus was 2 hours,and the effect lasted for 48 hours.Conclusions This study successfully constructed the R.lnPGRP-SC1a expression vector,its expressed product exhibited persiste

关 键 词：林氏扇头蜱 酰胺酶型R.lnPGRP-SC1a蛋白 蛋白表达 抗菌作用 

分 类 号：R384.4[医药卫生—医学寄生虫学]