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pCAT3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pCAT2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pCAT3。TaqⅠ酶切噬菌体LSPA1基因组，插入pCAT3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pCAT3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 pCAT3. Methods: CalⅠ restriction site on plasmid pCAT2 backbone was deleted by PCR nonsense mutation, and then the CAT gene with new CalⅠ on the 5′end was inserted into pCAT2 to get promoter trap vector pCAT3. The digested fragment of LSPA1 phage genome was inserted to pCAT3 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 bacterial, the information of inserted sequences was gained by sequencing.According to the results, primers were designed to amplify the possible promoter sequence and inserted to plasmid p？？3lysm？？egfp. After this plasmid was transformed into DH5α, the recombinant bacteria were observed under fluorescence microscopy. Results: Promoter trap vector pCAT3 was successfully constructed. About 100 bacteria containing promoter were gained, one of which was presumed to be ORF4 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 LSPA1 promoter, which would be helpful for further research of phage LSPA1