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在从患病大口鲇中分离到普通变形杆菌TWN-3株的基础上,将该菌总DNA用EcoRI不完全酶切后,分离5kb左右的片段,连接入pUC18质粒,转化E.coli BL21,构建基因组文库,得到3.6×103个重组子,远大于理论计算的1831个重组子,随机挑选重组子经EcoRI酶切鉴定重组率为100%,文库构建成功;将该菌裂解液与佐剂混合,免疫雄性新西兰大白兔,免疫程序结束后,抽血并分离血清,对用异丙基-β-D-硫代半乳糖苷(IPTG)诱导后的文库进行免疫印迹筛选,最终得到8个阳性克隆子。本实验为该菌的DNA疫苗以及重要基因表达研究打下基础。
Based on the isolation of Proteus vulgaris TWN-3 strain from diseased mouth, the total DNA of this strain was incompletely digested with EcoRI, and a 5 kb fragment was isolated and ligated into pUC18 plasmid for transformation into E. coli BL21. The genomic library was constructed and 3.6 × 103 recombinants were obtained, much larger than the 1831 recombinants calculated theoretically. The recombinants were randomly selected for EcoRI digestion to confirm the recombination rate was 100%, and the library was constructed successfully. The bacterial lysate was mixed with the adjuvant , And immunized male New Zealand white rabbits. After the immunization program was completed, the blood was drawn and the serum was separated. The libraries induced by isopropyl-β-D-thiogalactoside (IPTG) were screened by immunoblotting and eventually got 8 positive Clone. This experiment laid the foundation for the study of DNA vaccine and important gene expression of the bacteria.