空肠弯曲菌DNA疫苗构建及其疫苗免疫程序研究  

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

作　　者：杜联峰[1] 徐岗村[1] 孙万邦[1] 王宜峰[1] 王胜香[1] 罗军敏[1] 姚新生[1] 夏嫱[1] 杨瑞[1] 余妍[1] 

机构地区：[1]遵义医学院珠海校区/贵州省免疫学研究生教育创新基地,珠海519041

出　　处：《中国免疫学杂志》2012年第7期631-634,644,共5页Chinese Journal of Immunology

基　　金：贵州省优秀人才省长基金(200607);贵州省教育厅自然科学研究项目(20090105)资助

摘　　要：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。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 （-）-peblA. Methods：The PET28a（ ＋ ）-peblA as a template, peblA gene was amplified by PCR. Target gene and eukaryotic expression plasmid pcDNA3.1 （-） were digested by HindllI and EcoR I respectively, and then purified by DNA Extraction Kit. The plasmid of pcDNA3.1 （-）-peblA 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 （-）-peblA vaccine group. The latter included the pcDNA3.1 （-） （ 100 ug/100 ul） control group and normal saline（NS） control group. The levels of IgM and IgG in serum of each Kunming mice in pcDNA3.1 （-）-peblA vaccine group were detected with indirect ELISA. Results：Analyzed by restriction enzyme digestion, PCR and DNA sequencing, recombinant plasmid pcDNA3.1 （-）-peblA 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 （-）-pebl A compared with the two control groups （P 〈 0.05 ）. Conclusion：The recombinant pcDNA3.1 （-） -peblA has been successfully constructed and the PEB1 protein was suceessfully 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.

关 键 词：空肠弯曲菌 peblA基因 载体构建 免疫程序 

分 类 号：R392[医药卫生—免疫学]