淋球菌外膜蛋白NspA和耐辐射奇球菌SOD基因的克隆和在乳酸菌中表达的初步研究

发布时间：2018-01-09 15:20

本文关键词：淋球菌外膜蛋白NspA和耐辐射奇球菌SOD基因的克隆和在乳酸菌中表达的初步研究　出处：《四川大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的运用乳酸菌胞内表达载体pMG36e和分泌表达载体pVE5523，构建淋病奈瑟菌表面蛋白A(Neisseria surface protein A，NspA)和耐辐射奇球菌锰超氧化物歧化酶(Mn superoxide dismutase，Mn-SOD)两种目的基因的表达重组子，将重组子经电穿孔转化乳酸乳球菌，初步研究乳酸乳球菌表达融合NspA和SOD的情况，，为筛选目的基因的乳酸菌表达载体提供依据和思路：为后续开发淋球菌外膜蛋白的乳酸菌双功能活菌疫苗提供参考；为进一步研究SOD的表达对乳酸菌生物学特性的影响、开发可直接食用型SOD产品或微生态制剂奠定基础。方法本研究内容分两部分： 1．pMG36e-nspA和pMG36e-sod重组子的构建和在乳酸菌中表达 1) 优化CTAB法提取淋球菌和耐辐射奇球菌基因组的条件；设计引物，优化PCR扩增目的基因nspA和sod的条件；目的基因分别与乳酸菌胞内表达载体pMG36e连接，构建表达重组子pMG36e-nspA和pMG36e-sod，将表达重组子转化E．coli DH5a，利用红霉素抗性筛选阳性克隆，质粒小量提取后，双酶切和测序鉴定。
[Abstract]:Purpose Lactic acid bacteria intracellular expression vector pMG36e and secretory expression vector pVE5523 were used. Neisseria surface protein A was constructed from Neisseria gonorrhoeae. NspA) and manganese superoxide dismutase (mn superoxide dismutase Mn-SOD) were expressed as recombinant genes. The expression and fusion of NspA and SOD of Lactococcus lactis were preliminarily studied by electroporation of the recombinant plasmid into Lactococcus lactis. To provide the basis and train of thought for screening the lactic acid bacteria expression vector of the target gene: to provide the reference for the further development of the Lactobacillus bifunctional live vaccine of the outer membrane protein of Neisseria gonorrhoeae; In order to further study the effect of SOD expression on the biological characteristics of lactic acid bacteria, the development of direct edible SOD products or microecological preparations laid the foundation. Method This research is divided into two parts: 1. Construction and expression of pMG36e-nspA and pMG36e-sod recombinant in lactic acid bacteria 1) to optimize the conditions for extracting the genomes of Neisseria gonorrhoeae and Radiococci by CTAB method. Primers were designed to optimize the conditions for nspA and sod amplification by PCR. Objective to construct the recombinant pMG36e-nspA and pMG36e-sod, respectively, by ligating the genes with the intracellular expression vector pMG36e of lactic acid bacteria. The recombinant plasmid was transformed into E. coli DH 5a. The positive clones were screened by erythromycin resistance screening. The plasmid was extracted in a small amount and identified by double enzyme digestion and sequencing.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R371

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