空肠弯曲菌peb1A与大肠埃希菌ltB基因融合基因的构建、表达及鉴定

Construction,expression,and identification of a fusion gene from an outer membrane protein(peb1A)gene of Campylobacter jejuni and the heat-labile enterotoxin B subunit(ltB)gene of Escherichia coli

目的采用重叠PCR方法将大肠埃希菌不耐热肠毒素B亚单位ltB基因和空肠弯曲菌外膜蛋白peb1A基因进行连接,构建ltB-peb1A融合基因原核表达系统,对其进行诱导表达与鉴定。方法利用PCR技术扩增ltB与peb1A基因,用重叠PCR法将ltB与peb1A基因进行融合,然后插入pET-28a(+)中,构建原核表达载体pET28a(+)-LtB-peb1A。用重组体转化E.coli BL21(DE3),IPTG诱导表达重组蛋白LTB-PEB1,SDS-PAGE分析重组蛋白表达情况,ELISA法鉴定其与牛Gm1活性,Western blot鉴定重组蛋白的反应原性。结果 PCR及测序证实ltB-peb1A融合基因原核表达载体pET28a(+)-LtB-peb1A构建成功,SDS-PAGE分析目的蛋白最高表达量占全菌总蛋白的28%左右,其分子质量单位为40.7ku。ELISA法鉴定纯化的重组蛋白与牛Gm1有结合活性,Western blot分析重组蛋白能被兔抗空肠弯曲菌识别。结论成功构建了ltB-peb1A融合基因原核表达载体,并获得高效表达的目的蛋白,为空肠弯曲菌黏膜免疫疫苗的研制奠定了基础。

Objectives To use overlapping PCR to construct a prokaryotic expression system for expression of the fusion gene ltB-peb1 Afrom the heat-labile enterotoxin B subunit（ltB）gene of Escherichia coli and an outer membrane protein（peb1A）gene of Campylobacter jejuni,to induce its expression,and to identify that gene. Methods The genes ltB and peb1 A were amplified with PCR.The ltB gene was fused with the peb1 Agene using overlapping PCR and the fusion gene was inserted into a pET-28a（＋）prokaryotic expression vector.The recombinant was transformed into E.coli BL21（DE3）,and expression of the recombinant protein LTB-PEB1 was induced with IPTG.SDS-PAGE was used to analyze expression of the recombinant protein,ELISA was used to identify its adjuvant activity,and Western blotting was used to determine the immunogenicity of the recombinant protein. Results The recombinant vector was identified with PCR and sequencing verified that a prokaryotic expression vector for the fusion gene ltB-peb1 Awas successfully constructed.SDSPAGE indicated that the highest level of expression of the recombinant protein accounted for about 28% of the total bacterial protein.The recombinant protein had a molecular mass of 40.7ku.ELISA identified the purified recombinant protein and indicated its binding activity with bovine Gm1.Western blotting of the recombinant protein indicated that it was recognized by rabbit anti-Campylobacter jejuni antibodies. Conclusion A prokaryotic expression vector for the fusion gene ltB-peb1 Awas successfully constructed and the target protein was efficiently expressed.This work has laid the foundation for further study of a vaccine to confer mucosal immunity from C.jejuni.