E型沙眼衣原体MOMP基因重组腺病毒的构建及免疫原性研究

目的:构建E型沙眼衣原体(Ct)主要外膜蛋白（MOMP）基因重组腺病毒，为沙眼衣原体腺病毒疫苗的研究奠定基础。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MOMP基因片段，克隆至pcDNAII载体，测序后连接入腺病毒穿梭载体pDC316。穿梭载体pDC316 MOMP与含腺病毒基因组的辅助质粒PBHGlox△E1,3Cre共转染至HEK293细胞，在Cre loxP重组酶作用下进行重组，包装成重组腺病毒颗粒，用PCR和RT PCR方法进行鉴定。并用动物免疫试验检测重组腺病毒的免疫原性。结果:从E型Ct基因组DNA中扩增出约1.1?kb的特异MOMP基因片段，酶切鉴定及DNA序列测定证实穿梭载体pDC316 MOMP构建正确。穿梭载体pDC316 MOMP与含腺病毒基因组的辅助质粒PBHGlox△E1,3Cre共转染至293细胞，出现明显细胞病变效应。收集重组腺病毒，PCR法证实重组腺病毒含有MOMP基因，RT PCR证实重组腺病毒在293细胞能表达MOMP基因。重组腺病毒免疫小鼠可诱导特异性抗体产生，证明重组腺病毒具有良好免疫原性。结论:成功构建了E型沙眼衣原体MOMP基因重组腺病毒，该重组腺病毒可诱导小鼠产生特异性抗体。

Construction and immunogenicity of recombinant adenovirus carrying the major outer membrane protein gene of Chlamydia trachomatis seovar E

To construct a recombinant adenovirus Ad MOMP carrying the major outer membrane protein (MOMP) gene of Chlamydia trachomatis serovar E, and provide the basis for further research on Chlamydia trachomatis adenovirus vaccine. 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 then were subcloneded into the pshuttle vector pDC316. The pshuttle vector pDC316 MOMP and the genomic plasmids PBHGlox△E1, 3Cre were co transfected into 293 cells for package and amplification of recombinant adenovirus Ad MOMP, which was identified by PCR and RT PCR.