淋球菌外膜蛋白PI的克隆、序列分析及表达-营养与食品卫生学专业论文.docxVIP

四J|I大学硕+论文 四J|I大学硕+论文 淋球菌PI基因的克隆、序列分析及表达 得到阳性结果。用抗His．Tag单克隆抗体进行免疫印迹实验，检测重组PI蛋白， 仅诱导组有染色条带出现，证实成功表达了带His标签的重组蛋白；但用自制淋 球菌多克隆抗体检测进行免疫印迹实验，仅有弱反应条带出现，其原因有待探讨。 结论成功构建淋球菌PI基因的克隆重组子，测定我国淋球菌标准菌株(29400 和29403)PI基因序列，结合前期工作结果，首次向GenBank提交了上述标准菌 株和4株临床分离株PI基因序列；对我国淋球菌PI基因序列多序列同源性比较、 二级及三缴结构预测，为PI蛋白稳定性抗原靶位的筛选提供了有价值的资料；成 功构建淋球菌PT基因表达重组予，实现了浚基因在大肠杆菌中的高效表达，表达 蛋白在ELISA和免疫印迹实验中呈阳性结果，提示这些蛋白具有免疫反应性。上 述研究为寻找能刺激机体产生保护性免疫反应的外膜蛋白抗原靶位奠定了基础， 对后续研究PI蛋白的免疫功能和研制预防淋病的基因工程疫苗具有重要的意义。 关键词淋球菌PI基因疫苗克隆序列分析表达 璺型奎堂堡：!堡茎 璺型奎堂堡：!堡茎 燮壁堕堕薹旦盟塞堕：壁型坌堑墨壅垄 Cloning，Sequence Analysis and Expression of the Outer Membrane Protein I of Neissria gonorrhoeae ABSRACT Posgraduate：Wang Dongli Mentor：Prof．Pei Xiaofang Objective To construct T-A cloning recombinant plasmids of PI genes of the Neisseria gonorrhoeae reference strains，and analyze the outer membrane protein PI gene difference between the reference strains and clinical isolates；to construct recombinant expression plasmids of PI gene，induce the recombinant strains to produce PI protein，and study their expression characteristic and the function ofthe recombmant PI protein． Methods There are four parts in this dissertation． 1．Construction of cloning recombinant strains of PI genes of Neisseria gonorrhoeae reference strains：First，the genome DNA of Neisseria gonorrhoeae reference strains 29400 and 29403 was prepared according to the method described by J．Lewington et a1．PI genes were then amplified by PCR．The products of PCR were purified and added Adenine at the end of the sequences．These PI genes were ligated with pBS—T vector and transformed into competent cell E．Coli DH5但Positive clones selected by踟picillin resistance and blue—white selection were incubated and extracted plasmids，which were digested by restrictive endonuclease EcoR I and Xho I to confirm whether the PI genes were inserted in． 2．Sequence analysis on PI genes：Sequence the PI genes obtained in step 1，and compare them with sequences in GenBank database so as to find their similar 4 四川火学硕+论文