空肠弯曲菌cj0069蛋白的克隆表达及抗原性分析

Cloning,expression and antigenicity analysis of cj0069 from Campylobacter jejuni

目的构建空肠弯曲菌cj0069基因原核表达系统,克隆表达cj0069蛋白,根据cj0069的氨基酸序列进行抗原性结构预测分析,并验证其抗原性特征。方法利用在线软件对cj0069蛋白进行抗原性结构预测分析;用PCR方法从中国分离菌株ICDCCJ07001的基因组DNA中扩增cj0069基因,连接入pMD18-T载体,将重组质粒pMD18-T/cj0069和表达载体pGEX-4T-1双酶切,回收cj0069和pGEX-4T-1酶切产物后连接,连接产物转化入大肠埃希菌E.coli BL21(DE3),对重组质粒pGEX-4T-1/cj0069进行测序及酶切鉴定,IPTG诱导其表达,表达产物进行SDS-PAGE分析及飞行质谱鉴定;利用空肠弯曲菌兔免疫血清,采用Western blot分析cj0069蛋白的抗原反应性。结果 PCR扩增、DNA测序及酶切鉴定证实重组质粒pGEX-4T-1/cj0069构建成功,重组表达蛋白rGST-cj0069经飞行质谱鉴定为保守空肠弯曲菌蛋白。结构预测获得cj0069蛋白的抗原性肽段及线性表位等抗原性结构特征。Western blot检测显示该蛋白不能被空肠弯曲菌免疫兔血清识别。结论成功构建cj0069基因重组表达载体pGEX-4T-1/cj0069,并在大肠埃希菌中高效表达,获得的空肠弯曲菌cj0069蛋白具有抗原性结构特征,但针对免疫血清不具有抗原反应性。

Objectives To construct a prokaryotic expression vector of Cj0069 from Campylobacterjejuni and predict and verify its antigenic features. Methods cj0069 was amplified by PCR from the genomic DNA of C. jejuni strain ICDC- CJ07001. The antigenic features of Cj0069 were predicted using web-based software. The PCR products were cloned into the expression vector pGEX-4T-1 and the recombinant plasmid was transformed to Escherichia coli BL21. Expression of the recombinant protein rGST-Cj0069 was induced with IPTG. The antigenicity of rGST-Cj0069 was assessed by Western blotting with anti C. jejuni immune rabbit sera. The expressed recombinant protein was visualized with SDS-PAGE and the protein was identified with a MALDI TOF-TOF 4700 Proteomics Analyzer. Results The recombinant plasmid pGEX-4T 1/cj0069 was successfully constructed and the expressed recombinant protein rGST C]0069 was identified as a conserved hypothetical protein from C. jejuni by mass spectroscopy. The antigenic peptide and linear epitope of Cj0069 were obtained by predicting the antigenic structure. Western blot analysis indicated the recombinant rGST-Cj0069 would not react with anti-C, jejuni sera. Conclusion The recombinant plasmid pGEX-4T-1/cj0069 was successfully construc- ted and highly expressed in E. coli BL21. The recombinant protein rGST Cj0069 had predicted antigenic features but did not react specifically with anti-C, jejuni rabbit sera in this study.