摘要
目的:探讨质粒编码的分泌蛋白Pgp3在沙眼衣原体(Ct)感染宿主过程中的作用及机制,为防治Ct引起的泌尿生殖道感染提供实验依据。方法:构建pGEX-6p/Pgp3原核表达重组质粒并转化XL1-Blue大肠杆菌,IPTG诱导表达GSTPgp3融合蛋白,融合蛋白经亲和层析纯化及GST标签切割后制备Pgp3蛋白。将抗菌肽LL37和Pgp3蛋白共同孵育Ct后感染HeLa细胞,间接免疫荧光技术检测不同处理组Ct包涵体数量。GST pull-down法研究Pgp3与LL37的作用方式及Pgp3的活性片段。结果:LL37与Ct内原体(EB)孵育后感染HeLa细胞,感染率从50%降至10%,不同浓度Pgp3与LL37预先孵育后处理Ct EB再感染HeLa细胞,感染率较LL37组显著升高(P<0.05),且具有剂量依赖性。GST pull-down实验结果显示,LL37可与Pgp3结合,且结合部位位于Pgp3中间段(67~132 aa)。结论:Pgp3在Ct感染宿主过程中通过与人类抗菌肽LL37结合,抑制LL37对Ct的杀伤作用,导致Ct感染。
Objective:To investigate effects and mechanisms of secreted protein Pgp3 encoded by chlamydia trachomatis(Ct) plasmid during chlamydia trachomatis infecting host and to provide experimental gist for prevention and treatment of urogenital tract infection caused by Ct.Methods:Build pGEX-6 p/Pgp3 recombinant plasmids expressed in prokaryote and transform to XL1-Blue Escherichia coli.Fusion protein GST-Pgp3 was induced and expressed by IPTG,and fusion protein was purified using affinity chromatography method and cut off the GST tag and Pgp3 was obtained.Ct was co-incubated with antimicrobial peptide LL37 and Pgp3 to infect HeLa cells and number of inclusion bodies of Ct in different treatment groups was detected with indirect immunofluorescence.GST pull-down method to study action mode of Pgp3 and LL37 and active fragment of Pgp3.Results:Infection rate of Ct decreased from50%to 10%with Ct elementary body(EB)incubated by LL37 to infect HeLa cells,and infection rate of Ct was higher than that of LL37 group with Ct EB incubated by different concentrations of Pgp3 and LL37 infected HeLa cells(P<0.05),which had a dose-dependent effect.GST pull-down experiment results showed LL37 could bind to Pgp3,whose binding site was in middle segment of Pgp3(67~132 aa).Conclusion:Pgp3 inhibits killing effect of LL37 on Ct during process of Ct infecting host by binding human antimicrobial peptide LL37,which leading to Ct infection.
作者
张晗
孟宪勇
武海霞
杨建明
吴苗苗
董晓华(指导)
ZHANG Han;MENG Xian-Yong;WU Hai-Xia;YANG Jian-Ming;WU Miao-Miao;DONG Xiao-Hua(Hebei Key Laboratory of Neuropharmacology,Hebei North University,Zhangjiakou 075000,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2021年第8期917-921,共5页
Chinese Journal of Immunology
基金
河北省教育厅重点课题项目(ZD2018231)。
