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目的:通过构建p CAT3启动子陷阱载体筛选甲型副伤寒沙门菌噬菌体LSPA1启动子。方法:PCR无义突变去除p CAT2载体骨架上CalⅠ酶切位点,并在CAT片段的5’端引入CalⅠ的酶切位点,构建启动子陷阱载体p CAT3。TaqⅠ酶切噬菌体LSPA1基因组,插入p CAT3载体中,转化至大肠杆菌DH5α,通过氯霉素抗性平板筛选获得噬菌体LSPA1基因文库菌;随机挑取1株提取质粒,测序及比对分析插入序列;根据分析结果设计引物,扩增可能的启动子序列,并插入验证载体p-3lysm-egfp,转化DH5α,荧光显微镜观察。结果:成功构建p CAT3启动子陷阱载体,获得约100启动子文库菌,其中1株文库菌质粒中随机插入片段可能对应噬菌体LSPA1的ORF4启动子;插入待验证DNA片段的p-3lysm-egfp载体重组菌在荧光显微镜下能见到明显绿色荧光。结论:该启动子文库可有效筛选噬菌体LSPA1启动子,为进一步深入研究噬菌体LSPA1提供帮助。
OBJECTIVE: To screen the promoter of LSPA1 of Salmonella paratyphi A by constructing p CAT3 promoter trap vector. METHODS: PCR-based nonsense mutation was used to remove the Cal Ⅰ restriction site on the p CAT2 vector backbone. Cal I restriction enzyme site was introduced at the 5 ’end of the CAT fragment to construct a promoter trap vector p CAT3. Taq Ⅰ enzyme-digested bacteriophage LSPA1 genome, inserted into p CAT3 vector, transformed into E. coli DH5α, and the bacteriophages LSPA1 gene library was obtained by chloramphenicol resistance screening; randomly picked 1 strain to extract the plasmid, sequencing and comparison analysis of the insert; According to the results of the analysis, primers were designed and the possible promoter sequences were amplified and inserted into the verification vector p-3lysm-egfp, transformed into DH5α and observed under a fluorescence microscope. RESULTS: The p CAT3 promoter trap vector was successfully constructed, and about 100 promoter library strains were obtained. Among them, 1 randomly inserted fragment of library plasmid might correspond to the ORF4 promoter of bacteriophage LSPA1. The p-3 lys-egfp vector inserted into the DNA fragment to be verified Recombinant bacteria can see a clear green fluorescence under a fluorescence microscope. Conclusion: This promoter library can effectively screen the bacteriophage LSPA1 promoter, which may be helpful for further study on bacteriophage LSPA1.