D型沙眼衣原体质粒蛋白pgp3的克隆、表达、鉴定及其免疫原性初探

Plasmid-encoded pgp3 protein of Cldamydla trachomatis serovar D： cloning, expression, identification, andevaluation of immunogenicity

目的获得D型沙眼衣原体质粒蛋白pgp3基因及其蛋白，并鉴定其免疫原性。方法PCR扩增目的基因，将其定向插入原核表达载体pGEX-6P-1中，连接产物转化入大肠埃希菌DH5c~，提取质粒进行酶切、测序、PCR鉴定。再将重组质粒转化人大肠埃希菌BL21并诱导表达，Western印迹鉴定目的蛋白，谷胱甘肽巯基转移酶（GST）磁珠纯化pgp3-GST融合蛋白。用纯化后的蛋白免疫新西兰家兔，Western印迹检测抗体与pgp3蛋白的结合，酶联免疫吸附试验（ELISA）测定抗体效价。结果克隆目的基因序列长度为795bp，与基因库中一致，Western印迹显示目的蛋白相对分子质量为54000，并得到纯化蛋白。所得兔血清可识别pgp3蛋白，ELISA检测多克隆抗体效价达1：25600。结论成功表达具有强免疫原性的pgp3-GST融合蛋白，为进一步研究奠定基础。

Objective To express and identify the plasmid-encoded pgp3 protein of Chlamydia trachomatis （Ct） serovar D, and to evaluate its immunogenieity. Methods The pgp3 gene of Ct serovar D was amplified by PCR and directly inserted into the prokaryotic expression plasmid vector pGEX-6P-1 through the pZeroBack vector. Then, the recombinant plasmid pGEX-6P-1/pgp3 was transformed into E.coli DH5et followed by enzymatic digestion, sequencing and PCR identification. After the identification, the recombinant plasmid was transformed into E.coli BL21 followed by isopropyl-13-D-thiogalactoside （IPTG） induction. The expressed protein was identified by Western blot, and purified by glutathione S-transferase （GST） MagBeads. Male New Zealand rabbits were immunized with the purified protein for three times. Blood samples were collected from these rabbits one week after the last immunization and subjected to the qualification and quantification of anti-pgp3 polyclonal antibodies by Western blot and enzyme linked immunosorbent assay （ELISA） respectively. Results The length of the cloned gene was 795 bp, which was consistent with the sequence of the pgp3 gene in GenBank. The recombinant fusion protein, whose relative molecular weight was 54 000, was successfully purified. The serum of immunized rabbits could react with the pgp3 protein, with the titer of anti-pgp3 polyclonal antibodies being 1 ： 25 600. Conclusions The pgp3-GST fusion protein with high immunogenicity is successfully expressed and purified, which may lay a foundation for further studies.