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目的:构建重组MyD88基因的铜绿假单胞菌(PA)表位核酸疫苗,并研究其在真核细胞中的表达。方法:将PA的OprF的三个中和性B细胞表位及一个外源通用T细胞表位串联,表位之间用赖氨酸间隔,采用重叠PCR方法合成全基因,并在此序列前插入组织纤溶酶原激活物(tPA)的信号肽基因,将获得的片段与MyD88基因分别克隆到pIRES载体的两个多克隆位点,构建真核双表达重组质粒pIRES-tPA-OprF-MyD88。电穿孔法将pIRES-tPA-OprF-MyD88转入COS-7细胞,通过Westernblot检测其细胞上清tPA-OprF蛋白及细胞内MyD88蛋白的表达。结果:构建的真核表达质粒pIRES-tPA-OprF-MyD88,经电转COS-7细胞后,在其培养的上清液及细胞内检测到目的蛋白。结论:成功构建pIRES-tPA-OprF-MyD88表达载体,并在转染的真核细胞中得以有效地表达,为研究PA预防性疫苗奠定了实验基础。
Objective: To construct the nucleic acid vaccine of Pseudomonas aeruginosa (PA) epitope of recombinant MyD88 gene and study its expression in eukaryotic cells. METHODS: Three neutralizing B-cell epitopes of OprF and one exogenous universal T-cell epitope of PA were connected in series. The epitopes were separated by lysine. The whole gene was synthesized by overlapping PCR. Before the sequence Inserting the signal peptide gene of tissue plasminogen activator (tPA), the obtained fragment and MyD88 gene were cloned into two multi-cloning sites of pIRES vector to construct eukaryotic double-expression recombinant plasmid pIRES-tPA-OprF-MyD88 . The pIRES-tPA-OprF-MyD88 was transfected into COS-7 cells by electroporation. The expression of tPA-OprF and MyD88 in cells were detected by Western blot. Results: The eukaryotic expression plasmid pIRES-tPA-OprF-MyD88 was constructed and the target protein was detected in the supernatant and cells after electroporation of COS-7 cells. CONCLUSION: The pIRES-tPA-OprF-MyD88 expression vector was successfully constructed and expressed in the transfected eukaryotic cells effectively, which laid the experimental foundation for the study of PA preventive vaccine.