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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。
Objective To construct the prokaryotic expression system of cj0069 gene of Campylobacter jejuni, clone and express cj0069 protein, predict its antigenic structure according to the amino acid sequence of cj0069 and verify its antigenic characteristics. Methods The cj0069 protein was predicted by antigenic software using on-line software. The cj0069 gene was amplified by PCR from the genomic DNA of Chinese isolate ICDCCJ07001 and ligated into pMD18-T vector. The recombinant plasmid pMD18-T / cj0069 and expression vector pGEX 4T-1 double digestion, cj0069 and pGEX-4T-1 digested products were recovered and ligated, the ligation products were transformed into Escherichia coli BL21 (DE3), the recombinant plasmid pGEX-4T-1 / cj0069 were sequenced And digested with IPTG. The expressed product was analyzed by SDS-PAGE and identified by flight mass spectrometry. The antigenicity of cj0069 protein was analyzed by Western blot using the serum of Campylobacter jejuni rabbit. Results PCR amplification, DNA sequencing and restriction enzyme digestion confirmed that the recombinant plasmid pGEX-4T-1 / cj0069 was constructed successfully. The recombinant protein rGST-cj0069 was identified as a conserved Campylobacter jejuni protein by flight mass spectrometry. Structural prediction of antigenic peptide cj0069 protein epitopes and linear epitopes and other antigenic structural features. Western blot showed that the protein could not be recognized by Campylobacter jejuni immune rabbit serum. Conclusion The cj0069 gene recombinant expression vector pGEX-4T-1 / cj0069 was successfully constructed and expressed efficiently in Escherichia coli. The CJ0069 protein of Campylobacter jejuni has antigenic structural features but not antigenic reactivity against immune sera.