10种预测的鹦鹉热衣原体Ⅲ型分泌系统效应蛋白的定位分析

Localization of 10 Putative Effector Proteins Secreted via TypeⅢSecretion System of Chlamydia psittaci

旨在分析预测的10种鹦鹉热衣原体(Chlamydia psittaci,Cps)Ⅲ型分泌系统效应蛋白在细胞内的定位,为下一步研究其结构与功能奠定基础。本研究利用计算机辅助的生物信息学方法(Effective T3和BPBAac tool)预测Cps T 3SS效应蛋白。利用分子克隆技术构建重组质粒pGEX-6P-1/目的基因,诱导表达、纯化相应重组蛋白。皮下免疫BALB/c小鼠,制备各重组蛋白的多克隆抗体,ELISA测定其效价。以制备的多克隆抗体为一抗,应用间接免疫荧光法检测假定的效应蛋白在感染细胞中的定位。本研究通过应用Effective T3、BPBAac tool交叉验证,预测了14个Cps T 3SS效应蛋白编码基因:CPSIT_0357、CPSIT_0429、CPSIT_0461、CPSIT_0463、CPSIT_0490、CPSIT_0594、CPSIT_0785、CPSIT_0844、CPSIT_0846、CPSIT_0019、CPSIT_0020、CPSIT_0968、CPSIT_0969、CPSIT_0942,并进行分析。构建了14个靶基因的重组质粒,经IPTG诱导,除CPSIT_0357、CPSIT_0429、CPSIT_0968、CPSIT_0969外,其他10个重组菌分别表达了相对分子质量(M r)与预测M r相近的可溶性蛋白。经GST树脂纯化后,将重组蛋白免疫雌性BALB/c小鼠,血清抗体效价为(1∶32000)^(1∶64000)。IFA细胞内定位观察,发现CPSIT_0844和CPSIT_0846位于包涵体膜,而CPSIT_0461、CPSIT_0463、CPSIT_0490、CPSIT_0594、CPSIT_0785、CPSIT_0019、CPSIT_0020、CPSIT_0942位于包涵体内。本研究鉴定了2种定位于衣原体包涵体膜的效应蛋白和8种定位于衣原体包涵体中的效应蛋白。

This study aimed to investigate the intracellular localization of 10 putative effector proteins via typeⅢsecretion system(T 3 SS)in Chlamydia psittaci,laying the basis for the next structure and function analysis.In this study,computer-aided bioinformatics methods such as Effective T3 and BPBAac were used to select the possible T 3 SS effector proteins of C psittaci.The recombinant plasmid pGEX-6P-1-target genes were constructed and transformed into E.coli BL21,and the recombinant proteins were induced and purified.Then BALB/c mice were immunized with the recombinant proteins to produce polyclonal antibodies,and the titre of antibodies were detected by ELISA.Indirect immunofluorescence assay(IFA)was used to detect the subcellular localization of these proteins.In our study,fourteen possible Cps T 3 SS effector protein-coding genes were predicted:e.g.CPSIT_0357,CPSIT_0429,CPSIT_0461,CPSIT_0463,CPSIT_0490,CPSIT_0594,CPSIT_0785,CPSIT_0844,CPSIT_0846,CPSIT_0019,CPSIT_0020,CPSIT_0968,CPSIT_0969,and CPSIT_0942.Ten target recombinant proteins were successfully prepared except CPSIT_0357,CPSIT_0429,CPSIT_0968 and CPSIT_0969.After immunization of BALB/c mice,the specific antibodies were prepared,and the titers were(1:32000)-(1:64000).Moreover,the IFA results showed that CPSIT_0844 and CPSIT_0846 were located in the inclusion membrane,while CPSIT_0461,CPSIT_0463,CPSIT_0490,CPSIT_0594,CPSIT_0785,CPSIT_0019,CPSIT_0020 and CPSIT_0942 were located in whole the inclusion bodies.In conclusion,two effector proteins localized in the Chlamydia inclusion membrane and eight effector proteins localized in the chlamydia inclusion were identified.