空肠弯曲菌DNA疫苗构建及其疫苗免疫程序研究

目的:构建空肠弯曲菌peb1A基因真核表达载体,探讨DNA疫苗不同免疫程序的免疫效果。方法:以重组pET28a(+)-peb1A质粒为模板,将空肠弯曲菌peb1A基因克隆入真核表达载体pcDNA3.1(-),经酶切和测序鉴定后,将重组质粒转染COS-7细胞中表达,Western blot鉴定表达蛋白;昆明小鼠随机分组:设空白对照组、空载体对照组、pcDNA3.1(-)-peb1A实验组,用ELISA法检测免疫后的昆明小鼠血清IgG和IgM抗体水平。结果:重组质粒pcDNA3.1(-)-peb1A经双酶切和PCR鉴定,构建正确。peb1A基因测序结果与GenBank中peb1A基因序列一致。重组质粒成功转染COS-7细胞,并能正确表达重组蛋白。裸DNA组IgG水平均高于两对照组,有显著性差异(P<0.05)。同一剂量短时间(0、10、20天)免疫程序优于长时间(0、15、25、35天)免疫程序(P<0.05)。结论:成功构建空肠弯曲菌真核表达重组质粒pcDNA3.1(-)-peb1A,其经肌肉注射免疫小鼠,具有较好的免疫原性,短时间免疫程序免疫效果好。

Construction of the Campylobacter jejuni DNA vaccine and investigation of its immunization schedule

Objective:To construct eukaryotic expression vector carrying Campylobacter jejuni peblA gene and express peblA protein in COS-7,then explore the immunogenicity of the different immunization program of CJ pcDNA3.1(-)-peb1A.Methods:The PET28a(+)-peb1A as a template,peblA gene was amplified by PCR.Target gene and eukaryotic expression plasmid pcDNA3.1(-) were digested by HindⅢ and EcoRⅠ respectively,and then purified by DNA Extraction Kit.The plasmid of pcDNA3.1(-)-peb1A was extracted and transfected into COS-7 cells with jetPEITM reagent.The recombinant protein was identified with Western blot.Healthy male KM mice were divided into experimental groups and control groups.The former contained naked pcDNA3.1(-)-peb1A vaccine group.The latter included the pcDNA3.1(-)(100 μg/100 μl) control group and normal saline(NS) control group.The levels of IgM and IgG in serum of each Kunming mice in pcDNA3.1(-)-peb1A vaccine group were detected with indirect ELISA.Results:Analyzed by restriction enzyme digestion,PCR and DNA sequencing,recombinant plasmid pcDNA3.1(-)-peb1A was correctly constructed.The recombinant plasmid had been successfully transfected into COS-7 cells,and the PEB1 protein was expressed,checked by Western blot.The level of serum IgG antibody was significantly increased in mice immunized with recombinant pcDNA3.1(-)-peb1A compared with the two control groups(P<0.05).Conclusion:The recombinant pcDNA3.1(-)-peblA has been successfully constructed and the PEB1 protein was successfully expressed in mammalian COS-7 cells.The recombinant pcDNA3.1(-)-peblA had good immunogenicity via intramuscular injection immunize mice.Short time immunization is better than long time immunization.