E型沙眼衣原体MOMP基因重组质粒的构建与表达

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Ct MOMP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Western blotting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。

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

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.