沙眼衣原体OmcBc基因的克隆、表达与抗原性研究  

Cloning and expression of Chlamydia trachomatis OmcBc gene and antigenicity analysis of the protein

作　　者：王洁[1] 张颖骞[2] 钟光明[2] 余平[1] 

机构地区：[1]中南大学基础医学院免疫学系,湖南长沙410078 [2]美国德克萨斯大学圣安东尼奥健康科学中心微生物学免疫学系

出　　处：《南方医科大学学报》2010年第7期1558-1561,共4页Journal of Southern Medical University

基　　金：美国国立卫生研究院基金(IR01AI47997-01)

摘　　要：目的探讨重组沙眼衣原体OmcB基因C端编码蛋白的抗原性,为衣原体感染性疾病的早期诊断和疫苗研究提供新的靶标。方法采用PCR技术从沙眼衣原体基因组中扩增OmcB的C端(T270-Y553)序列,并构建pGEX-6p-CtOmcBc原核表达质粒,转染大肠杆菌XL-1blue,IPTG诱导表达重组GST-OmcBc融合蛋白;收集120例Ct生殖道感染患者血清,以CtD血清型灭活的EB分别于皮下和腹腔免疫7只新西兰大白兔和5只Balb/C小鼠,以CtD血清型活的EB滴鼻感染9只Balb/C小鼠,分别收集这些动物免疫血清;间接免疫荧光法检测各血清中抗衣原体特异性抗体的效价;ELISA法检测重组GST-OmcBc融合蛋白与各免疫血清的反应性。结果成功构建了pGEX-6p-CtOmcBc原核表达质粒,经测序显示插入基因长度为852bp,编码284个氨基酸。IPTG成功诱导表达了重组GST-OmcBc融合蛋白,其分子质量约为57kDa;所制备和收集的各免疫血清均含高滴度抗衣原体特异性抗体;ELISA结果显示重组GST-OmcBc融合蛋白对各免疫血清均具有强的抗原性。结论 CtOmcBc是衣原体的一个免疫优势蛋白,重组GST-OmcBc融合蛋白的成功表达及其良好的抗原性为进一步开展衣原体感染早期诊断研究和疫苗的研发奠定了基础。Objective To investigate the antigenicity of recombinant Chlamydia trachomatis （Ct） OmcBc protein and search for the new target for early diagnosis of Chlamydia infection and Chlamydia vaccine development. Methods The C fragment of OmcB encoding the amino acids from T270 to T553 was amplified from Chlamydia serovar D genomic DNA. The pGEX-6p-Ct OmcBc expression plasmid was constructed and transformed into E.coli XL-1blue. The expression of recombinant Ct OmcBc protein was induced by IPTG. Serum samples were collected from 120 patients with urogenital Chlamydia infection. The antiserum samples were collected from 7 New Zealand white rabbits and 5 Balb/C mice immunized subcutaneously and intraperitoneally with Ct serovar D inactivated EB, respectively, and from 9 Balb/C mice intranasally infected with Ct serovar D live EB. The anti-Chlamydia specific antibody were titrated by an immunofluorescence assay （IFA）. The reactivity of the recombinant OmcBc protein with all the above antisera was detected by ELISA. Results The pGEX-6p-Ct OmcBc expression plasmid was successfully constructed. DNA sequencing showed that the inserted OmcBc was about 852 bp, encoding a protein with 284 amino acids. The expression of the recombinant GST-OmcBc was induced by IPTG, producing a fusion protein with a molecular weight of about 57 kD. The titer of the specific antibodies to Chlamydia in all the antisera was high. ELISA results showed strong reactivities of the recombinant GST-OmcBc fusion protein with all the above antisera. Conclusions OmcBc protein is an immunodominant protein of Chlamydia. The recombinant GST-OmcBc with strong antigenicity may provide a basis for further study of early diagnosis of chlamydia infection and development of Chlamydia vaccine.

关 键 词：沙眼衣原体 OmcB 抗原性 

分 类 号：R392[医药卫生—免疫学]