【作者】 刘良专；

【导师】 吴移谋；

【作者基本信息】 南华大学 ， 病原生物学， 2010， 硕士

【摘要】 目的:预测、筛选肺炎嗜衣原体(Chlamydophila pneumoniae,Cpn)III型分泌系统(Type III secretion system,T3SS)效应蛋白编码基因,并研究其免疫活性,探讨预测的T3SS效应蛋白重组蛋白在临床Cpn血清学诊断中的应用价值。方法:①通过生物信息学分析结合相关文献报道,预测Cpn T3SS效应蛋白编码基因;②以Cpn AR39菌株总RNA为模板,RT-PCR从转录水平分析Cpn T3SS效应蛋白编码基因在感染细胞的表达情况,并筛选出在感染后72小时有表达的基因;③PCR扩增预测Cpn T3SS效应蛋白编码基因,将其亚克隆至原核表达载体pGEX-6P-2中构建重组质粒pGEX-6P-2/目的片段,经PCR、测序鉴定后将其转化至表达宿主菌E.coli BL21中,IPTG诱导表达。采用SDS-PAGE和Western blot分析和鉴定表达产物;对温度、IPTG和时间等诱导表达条件进行系列的优化后,获得可溶性表达蛋白并大量诱导表达。GST纯化树脂纯化重组蛋白,用tris8.0将目的蛋白透析降低谷胱甘肽浓度,BCA法测定蛋白浓度;用重组蛋白包被微孔板,建立间接ELISA法检测临床Cpn患者血清,评价重组蛋白的抗原性;用重组蛋白经皮下注入免疫BALB/c小鼠,检测免疫小鼠血清中重组蛋白多克隆抗体的效价,分析重组蛋白的免疫原性,并以制备的多克隆抗体为一抗,应用间接免疫荧光检测预测蛋白在感染细胞的定位情况,以筛选Cpn分泌性蛋白。结果:通过生物信息学分析结合相关文献报道,预测了14个Cpn T3SS效应蛋白编码基因:Cpn0704,Cpn0710,Cpn1022,Cpn0810,Cpn0962,Cpn0960,Cpn0422,Cpn0425,Cpn0323,Cpn0322,Cpn0432,Cpn0431,Cpn0434,Cpn0661。RT-PCR分析筛选了11个在感染后72h有表达的基因: Cpn0704,Cpn0710,Cpn1022,Cpn0810,Cpn0962,Cpn0960,Cpn0422,Cpn0425,Cpn0323,Cpn0322,Cpn0431;PCR扩增得到其目的片段;构建的重组质粒经PCR和测序鉴定证实插入片段为目的基因,测序结果与Genbank上登录序列完全一致;SDS-PAGE结果显示,在IPTG诱导下,重组工程菌分别表达了相对分子量(Mr)与预测分子量相近的蛋白,对诱导表达条件进行系列的优化后,获得5个可溶性表达蛋白:Cpn1022,Cpn0960,Cpn0810,Cpn0704,Cpn0425,经GST纯化树脂纯化后,纯度在98%以上;以纯化的重组蛋白为包被抗原建立间接ELISA法,检测临床Cpn患者血清,我们收集了20例确诊为Cpn感染患者血清和10例正常人的血清进行ELISA分析,结果显示:Cpn0810能识别全部病人的血清,Cpn0425能识别17个病人的血清,Cpn0960与Cpn0704能与14个病人的血清发生反应,Cpn1022只识别11个病人的血清,而正常人的血清未出现上述反应。利用纯化的Cpn1022,Cpn0960,Cpn0810,Cpn0704,Cpn0425重组蛋白免疫新BALB/c小鼠,间接ELISA法测定兔免疫血清特异性抗体,效价分别为Cpn0810与Cpn0425达1:64000,Cpn0960与Cpn0704达1:32000,Cpn1022达1:16000;5种重组蛋白刺激BALB/c小鼠,皮肤出现发热、红肿,6~10d后逐渐消失,迟发型超敏反应(delayed type hypersensitivity,DTH)阳性。结论:1. Cpn0704,Cpn0710,Cpn1022,Cpn0810,Cpn0962,Cpn0960,Cpn0422,Cpn0425,Cpn0323,Cpn0322,Cpn0431在Cpn感染细胞后72h有转录;2.成功构建pGEX-6P-2/Cpn0704,Cpn0710,Cpn1022,Cpn0810,Cpn0962,Cpn0960,Cpn0422,Cpn0425,Cpn0323,Cpn0322,Cpn0431等11个原核表达载体,将其转化至E.coli BL21后表达出相应的重组蛋白;3. Cpn1022,Cpn0960,Cpn0810,Cpn0704,Cpn0425重组蛋白具有良好的免疫原性,能刺激BALB/c小鼠产生高效价的特异性抗体;4. Cpn1022,Cpn0960,Cpn0810,Cpn0704,Cpn0425重组蛋白具有较好的抗原性,能与Cpn阳性血清特异性结合。更多还原

【Abstract】 Objective: This study attempts to predict and screen effector proteins secreted via type III secretion system of Chlamydophila pneumoniae , and study on characterization and intracellular localization of these putative Chlamydia pneumoniae effector proteins.Methods: Genes of effector proteins secreted via type III secretion system of Chlamydophila pneumoniae were predicted according to the bioinformatics analysis and references. RT-PCR was used to examine the transcription of these genes in Cpn infected cells at 72h pi. Eleven Pairs of Primers were synthesized according to these gene sequenees of Chlamydia pneumoniae strain AR39. PCR was used to amplify these genes,And then these genes were cloned into pGEX -6P-2 vector to induce the expression of corresponding recombinant proteins with IPTG in E.coli BL21,and then purified using Merck GST*tag assay kit. their concentrations were determined by BCA assay, Indirect ELISA were developed by coating microwell plates with the purified proteins, their reliability test and stability test were carried out after optimization.The purified fusion proteins were used to immunize BALB/c mice to produce polyclonalantibodies.Antibodies were used to loealize these proteins in Cpn infected cells at 72h pi.Results: Cpn0704,Cpn0710,Cpn1022,Cpn0810,Cpn0962,Cpn0960,Cpn0422,Cpn0425,Cpn0323,Cpn0322,Cpn0432,Cpn0431,Cpn0434,Cpn0661 were predicted as genes of effector proteins secreted via Cpn T3SS according to the bioinformatics analysis and references, Cpn0704,Cpn0710,Cpn1022,Cpn0810,Cpn0962,Cpn0960,Cpn0422,Cpn0425,Cpn0323,Cpn0322,Cpn0431 transcripted by RT-PCR analysis in Cpn infected cells at 72h pi,Then these genes fragments were amplified by PCR; Restriction enzyme digestion and sequencing showed that the inserted target genes were the purpose fragments, compared with genes reported by GenBank, it had 100% similarity.These fusion proteins was expressed after induced with IPTG,Cpn1022,Cpn0960,Cpn0810,Cpn0704,Cpn0425 were purified. Western blot proved that the recombinant proteins could specifically react with anti-GST polyclonalantibody. Indirect ELISA was successfully developed to detect the antibody to Cpn in human sera, all the positive sera were reacted with the Cpn0810;17 of the positive sera reacted with the Cpn0425;14 of the positive sera reacted with the Cpn0960 and Cpn07704;Only 11 of the positive sera reacted with the Cpn1022. While all the proteins were not reacted with uninfected human sera. Specific humoral response were elicited by recombinant proteins in BALB/c mice and the specific antibody titer was 1∶1 6000, 1∶32 000 and 1∶64 000 respectively. five kinds of recombinant proteins stimulated the BALB/c mice to show fever red skin in leg, disappearanced after 6~10d and delayed-type hypersensitivity were positive.Conclusion:1. Cpn0704,Cpn0710,Cpn1022,Cpn0810,Cpn0962,Cpn0960,Cpn0422,Cpn0425,Cpn0323,Cpn0322,Cpn0431 transcripted in Cpn infected cells at 72h pi;2. Prokaryotic expression vector pGEX-6P-2 / Cpn0704,Cpn0710,Cpn1022,Cpn0810,Cpn0962,Cpn0960,Cpn0422,Cpn0425,Cpn0323,Cpn0322,Cpn0431 were successfully constructed, and the corresponding recombinant proteins were expressed in E.coli BL21;3. Cpn1022,Cpn0960,Cpn0810,Cpn0704,Cpn0425 recombinant proteins showed excellent immunongenicity and could induce the humoral responses efficiently;4. Cpn1022,Cpn0960,Cpn0810,Cpn0704,Cpn0425 recombinant proteins showed better immunoreactivity, could specifically react with Cpn IgG positive sera;更多还原