E型沙眼衣原体MOMP基因重组质粒的构建与表达

Construction and expression of recombiant eukaryotic plasmid of the majer outer membrane protein from Chlamydia trachomatis seovar E

目的:构建E型沙眼衣原体(Ct)主要外膜蛋白(MOMP)基因重组真核表达质粒pVAX1-MOMP,为探索安全的E型CtDNA疫苗奠定基础。方法:根据Genebank中E型CtMOMP基因序列设计引物,用高保真PCR方法从E型Ct基因组DNA中扩增得到约1.1kb的MOMP片段,克隆至pcDNAII载体,测序后亚克隆至表达载体pVAX1,构建卡那霉素抗性真核重组表达质粒pVAX1-MOMP,并以重组质粒转染COS-1细胞进行表达,用Westernblotting方法鉴定表达产物。结果:从E型Ct基因组DNA中扩增出特异的MOMP基因片段,酶切鉴定及DNA序列测定证实pVAX1-MOMP重组质粒构建正确,能在COS-1细胞内表达,表达产物能与抗MOMP单克隆抗体特异性结合。结论:成功构建E型沙眼衣原体MOMP真核表达质粒pVAX1-MOMP,并在真核细胞内获得表达。

Objective： To construct a recombiant eukaryotic expression plasmid pVAX1-MOMP carrying the major outer membrane protein （MOMP） gene of Chlamydia trachomatis serovar E, make an expression of MOMP gene and study the antigenicity of the expressed product. Methods： The MOMP gene fragments of Chlamydia trachomatis serovar E were amplified from genome DNA by PCR method. They were cloned into pcDNAⅡ vector and subcloneded into the vector pVAX1 to construct the recombiant eukaryotic expression plasmid pVAX1-MOMP. The recombinant expression plasmids were transfected into COS-1 cells. The expression of MOMP protein was detected by Western blotting. Results： 1.1 kb gene fragments was amplified from genome DNA by PCR method as anticipated. Recombinant plasmids were confirmed by enzyme digestion and nucleotide sequencing, and MOMP proteins were detected in COS-1 cells. Conclusion： Recombinant plasmids have been successfully constructed. MOMP gene can be expressed in COS-1 cells.