甲型副伤寒沙门菌噬菌体LSPA1启动子筛选方法的建立

Establishment of a method to screen promoter of Salmonella paratyphi A phage LSPA1

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。

Aim：To screen promoter of Salmonella paratyphi A phage LSPA1 through promoter trap vector pCAT 3. Methods：CalⅠrestriction site on plasmid pCAT 2 backbone was deleted by PCR nonsense mutation , and then the CAT gene with new CalⅠon the 5′end was inserted into pCAT2 to get promoter trap vector pCAT3.The digested fragment of LSPA1 phage genome was inserted to pCAT 3 and then transformed into E.coli DH5α.The bacteria phage LSPA1 promotor library was screened using plates containing chloramphenicol .After plasmid was extracted from a randomly picked bacteri-al, the information of inserted sequences was gained by sequencing .According to the results , primers were designed to am-plify the possible promoter sequence and inserted to plasmid p-3lysm-egfp.After this plasmid was transformed into DH 5α, the recombinant bacteria were observed under fluorescence microscopy .Results：Promoter trap vector pCAT3 was success-fully constructed .About 100 bacteria containing promoter were gained , one of which was presumed to be ORF 4 promoter of phage LSPA1.Recombinant bacteria DH5α/p-3lysm-egfp carried the insert DNA fragments was obviously observed green fluorescence under fluorescence microscope .Conclusion： This library can effectively screen phage LSPA 1 promoter, which would be helpful for further research of phage LSPA 1.