空肠弯曲菌NCTC11168基因组表达文库的构建及鉴定

Construction and identification of the genomic expression library from Campylobacter jejuni strain NCTC11168

将空肠弯曲菌NCTC11168基因组用Sau3AⅠ部分酶切,回收400～3 000bp的片段。用BamHⅠ酶切原核表达载体pET30a(b,c),用SAP去磷酸化后,切胶回收线性载体片段。将基因组片段与载体按摩尔比例10∶1连接,转化DH5α感受态细胞,PCR分析插入片段大小的分布和插入的正确率。从转化的平板上提取质粒,转化表达宿主菌BL21(DE3),获得基因组表达文库。用IPTG诱导BL21(DE3),SDS-PAGE鉴定蛋白表达情况。提取基因组,以0.5U/μg Sau3AⅠ于37℃部分酶切基因组,回收400～3 000bp片段。载体用2.5U/μg BamHⅠ线性化后,用0.8U/μg SAP将其完全去磷酸化。基因组片段与载体连接转化DH5α感受态细胞,获得了基因组表达文库,库容达52 700个克隆,可以覆盖弯曲菌全基因组4次以上,片段插入正确率为83.3%～91.7%,片段大小为400～3 000bp,且均匀分布,IPTG诱导表达文库后蛋白呈现高效表达,表明成功构建了空肠弯曲菌的基因组表达文库。

The genomic DNA extracted from Campylobacter jejuni strain NCTC11168 was digested using Sau3AⅠ,then the fragments ranging from 0.4 kb to 3 kb were purified and ligated into pET30a（b,c）expression vectors,which were digested by BamHⅠ and dephosphorylated by SAP.The ligated products were transformed into Escherichia coli DH5α,and the sizes of fragments inserted into the vectors as well as the insertion efficiency were analyzed by PCR with pET30 vectors specific primers.Then the plasmids were extracted from recombinant E.coli DH5α and further transformed into BL21（DE3） to develop genomic expression library. The genomic expression library was induced with IPTG,and the protein expression was identified using SDS-PAGE. The genomic DNA was extracted by conventional methods,and was digested using 0.5U/μg Sau3AⅠ at 37℃ for 30min,and the DNA fragments ranging from 400 to 3000bp were purified. The pET30a（b,c） vectors were digested using 2.5U/μg BamHⅠ and dephosphorylated using 0.8U/μg SAP. The ligation was performed according to the genome fragments and vectors mole ratio of 10∶1,and the ligated products were transformed into competent DH5α to construct the genomic expression library of C.jejuni strain NCTC11168.The reservoir capacity of constructed library was 52700 clones,which equated to 4 times of NCTC11168 genome,and the insertion efficiency was 83.3% to 91.7%,the range of inserted fragments were 400 to 3000bp,and the fragments uniform distributed,and the protein was expressed efficiently. The results revealed that the genomic expression library of C.jejuni strain NCTC11168 was constructed successfully.