论文部分内容阅读
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 of peb1A gene of Campylobacter jejuni and to investigate the immune effect of DNA vaccine in different immunization programs. METHODS: The peb1A gene of Campylobacter jejuni was cloned into the eukaryotic expression vector pcDNA3.1 (-) using the recombinant plasmid pET28a (+) - peb1A as a template. After identification by restriction enzyme and sequencing, the recombinant plasmid was transfected into COS-7 cells The expression of protein was detected by Western blot. Kunming mice were randomly divided into control group, empty vector control group and pcDNA3.1 (-) - peb1A experimental group. The serum IgG and IgM Antibody level. Results: The recombinant plasmid pcDNA3.1 (-) - peb1A was confirmed by double enzyme digestion and PCR. The results of peb1A gene sequencing are consistent with the sequence of peb1A gene in GenBank. The recombinant plasmids were successfully transfected into COS-7 cells and correctly expressed the recombinant protein. The level of IgG in the nude DNA group was higher than that in the two control groups, with significant difference (P <0.05). The immunization procedure of the same dose for a short time (0, 10, 20 days) was superior to the immunization procedure for a long time (0, 15, 25 and 35 days) (P <0.05). CONCLUSION: The eukaryotic expression vector pcDNA3.1 (-) - peb1A of Campylobacter jejuni is constructed successfully. The recombinant plasmid pcDNA3.1 (-) - peb1A is immunized by intramuscular injection. It has good immunogenicity and good immunization effect in short time.