D型沙眼衣原体多形外膜蛋白I的分段克隆表达及其免疫原性分析

Cloning, expression and identification of the different fragments of polymorphic membrane protein I and its immunogenicity analysis of Chlamydia trachomatis serovar D

目的获得D型沙眼衣原体多形外膜蛋白（Pmp）I的全长（PmpI—FL）和羧基端蛋白（PmpI—C），并鉴定蛋白免疫原性。方法用PCR扩增目的基因PmpI—FL和PmpI—C，分别将其定向插入原核表达载体pGEX-6P-1中，连接产物转化人大肠埃希菌DH5a，提取质粒进行酶切、PCR、测序鉴定。再将重组质粒分别转化人大肠埃希菌BL21并诱导表达，考马斯亮蓝染色和免疫印迹鉴定目的蛋白，谷胱甘肽巯基转移酶（GST）磁珠分别纯化PmpI—FL—GST融合蛋白和PmpI-C-GST融合蛋白。用纯化后的蛋白分别免疫BALB／c小鼠，ELISA测定抗体效价。结果克隆目的基因PmpI—FL和PmpI—C的长度分别为2659和1195bp，序列均与基因库中一致，考马斯亮蓝染色和免疫印迹显示目的蛋白相对分子质量分别约为122000和69000；并得到纯化的蛋白。免疫小鼠所得血清经ELISA检测多克隆抗体效价达1：12800（抗PmpI-FL）和1：6400（抗PmpI-C）。结论成功表达具有强免疫原性的PmpI-FL-GST和PmpI-C—GST两种融合蛋白，为进一步研究其功能奠定基础。

Objective To obtain the full length （FL） and C-terminal fragment of polymorphic membrane protein I （PmpI） of Chlamydia trachomatis serovar D, and to study the immunogenicity of these proteins. Methods The target genes of PmpI-FL and PmpI-C were amplified by polymerase chain reaction （PCR） and inserted into the prokaryotic plasmid vector pGEX-6P 1. The recombinant plasmids pGEX-6P-1/PmpI-FL and pGEX-6P-1/PmpI-C were separately transformed into Escherichia. coli （E. coli） DH5a and were identified by enzyme digestion, sequencing and PCR. After the identification, the recombinant plasmids were separately transformed into E. coli BL21 and induced to express the proteins. The expected proteins were identified by Coomassie brilliant blue staining and Western blot, then purified by glutathione S-transferase （GST） MagBeads. The purified proteins were then injected into BALB/c mice to prepare the polyclonal antibodies against PmpI-FL or PmpI-C. Enzyme-linked immune sorbent assay （ELISA） was used for the quantitative detection of the specific antibody. Results The lengths of cloned target genes PmpI-FL and PmpI-C were 2 659 bp and 1 195 bp, respectively, and the sequences were consistent with those of Chlamydia trachomatis serovar D in GenBank. The molecular masses of target proteins were 122 000 and 69 000, respectively, which were confirmed by Coomassie brilliant blue staining and Western blot and then purified. The titers of the antibodies （anti-PmpI-FL and anti-PmpI-C） in sera of immunized mice detected by ELISA were 1 ： 12 800 and 1 ： 6 400, respectively. Conclusion The PmpI-FI; GST and PmpI-C-GST fusion proteins with high immunogenicity are successfully expressed and purified, which lays the foundation for further study.