【摘要】 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。更多还原

【Abstract】 Objective: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.The BALB/c mice immunized with recombinant adenovirus and anti-MOMP specific IgG in serum were tested.Results: 1.1?kb gene fragment was amplified from genome DNA by PCR method as being anticipated.Recombinant pshuttle vectors,pDC316-MOMP were confirmed by enzyme digestion and nucleotide sequencing.Recombinant adenovirus Ad-MOMP was verified byPCR and RT-PCR.AntiMOMP specific IgG antibodies were induced in mice by recombinant adenovirus AdMOMP.Conclusion: The recombinant adenovirus Ad-MOMP has been successfully constructed,and it is able to induce Anti-MOMP specific antibodies in mice.更多还原