空肠弯曲菌共同抗原基因文库的建立与基因克隆

Construction of Genomic Library and Gene Cloning of Campylobacter jejuni Common Antigen

从鸡源空肠弯曲菌中提取共同抗原成分 ,用 SDS-PAGE和 Western blot进行分析。结果表明 ,抗空肠弯曲菌共同抗原的单克隆抗体 4 A7可与共同抗原中 62 0 0 0的蛋白带发生特异免疫反应。为获得编码这种蛋白的特异基因 ,从空肠弯曲菌中提取和纯化染色体基因组 DNA,用 Eco R 和 Hind 双酶切 ,从酶切产物的琼脂糖电泳中回收 1～ 2 kb的 DNA片段 ,与用相同酶切的 p Bluescript KS( +)质粒进行重组连接 ,共挑选出 4 2 0个插入外源片段大小较为适中的重组子 ,从而初步建立了可能含有编码空肠弯曲菌共同抗原 62 0 0 0蛋白成分基因片段的重组质粒基因文库。从该文库中用双酶切获取扩增的插入外源基因片段 ,与双酶切的p ET2 8a( +)质粒进行连接 ,转化受体菌后 ,以 IPTG诱导表达 ,用 4 A7单抗作探针进行免疫筛选 ,共筛选出 6个可与该单抗发生特异反应的阳性克隆。

The common antigen components extracted from the isolated C.jejuni strain were examined by SDS PAGE and western blot analysis. The results showed that the 4A7 McAb probe appeared positive reaction with about 62 000 protein band of the CA.The genomic DNA of C.jejuni was partially digested with EcoRⅠ and Hind Ⅲ restriction endonuclease.Selected DNA fragments of 1 2.0 kb were isolated from agarose gels and ligated into the EcoRⅠ and Hind Ⅲ site of pBluescriptⅡKS(+).The ligation mixture was transformed into E.coli DH5α cells by the calcium chloride procedure. A genomic library of C.jejuni was constructed in pBluescriptⅡKS(+) plasmids. pET28a(+) were used for expression of specific protein. For screening the recombinanted E.coli DE3 colonies, it was transferred to nitrocellulose filters by replica blotting, the filters were then placed on new Luria Bertani agar plates supplemented with 30 mg/L of kanamycin,80 μg x gal and IPTG 80 μg per plate and incubated at 37℃.The recombinants were lysed in situ by soaking the filters in lysis buffer for 4 h and probed with the 4A7 McAb. Six recombinant E.coli clones carrying EcoRⅠ, Hind Ⅲ inserts of genomic DNA of C.jejuni in pET28a(+), expressed proteins recognized by the 4A7 McAb.