5株不同群型霍乱弧菌黏粒文库构建及JS9803株文库应用

Construction of genomic cosmid banks of 5 different serogroup and type of Vibrio cholerae strains and application of bank of JS9803 strain

目的研究5株不同群型霍乱弧菌(VC)黏粒文库的构建。方法染色体DNA提取、酶切,质粒DNA提取、酶切及去磷酸化,连接、包装、转染、铺板,克隆子鉴定和保存、菌落原位杂交,PCR和测序。结果成功构建吴江-2株(EVC流行株产毒株)、86015株(EVC流行株非产毒株)、JS32株(EVC非流行株非产毒株)、JS9803株(O139群VC产毒株)和GD98200株(O139群VC非产毒株)等5株霍乱弧菌的染色体基因组黏粒文库,文库克隆子中插入35～45kb的染色体DNA片段,库容量分别为3000～5000克隆子。从JS9803株文库中筛选出分别携带CTXφ或nct-CTXφ基因组的克隆子,后一克隆子携带的rstR-ig2基因为新类型。结论成功构建5株不同群型霍乱弧菌染色体基因组黏粒文库,并对JS9803株文库进行应用。

Objective To construct genomic cosmid banks of 5 different serogroups and type sof Vibrio cholerae （VC） strains. Methods Chromosome DNA were extracted and digested. Cosmid DNA were extracted, digested, and dephosphorylated. Chromosome DNA and cosmid DNA were ligated, packaged, adsorbed, and plated. Recombinants were identified and stored. Positive recombinants were choosed with dot-blotted, genes were polymerized and sequenced. Results Cosmid banks of WUJIANG2 strain （epidemic and toxic EVC）, 86015 strain （epidemic and non-toxic EVC）, JS32 strain （non-epidemic and non-toxic EVC）,JS9803 strain （toxic O139 serogroup VC）, and GD98200 strain （non-toxic O139 serogroup VC） were established successfully. Insertion fragments in cosmids of recombinants were 35-45 kb. Volumes of banks were 3000-5000. Clones including CTXφ or nct-CTXφ genome were chosen from cosmid bank of JS9803 strain, and rstR-ig2 of nct-CTXφ genome was new type. Conclusions Genomic cosmid banks of 5 different serogroups or types of Vibrio cholerae strains were constructed and that of JS9803 strain was applied.