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目的探讨假结核耶尔森菌侵染素(invasin)羧基端397个氨基酸(Inv397)对抗原免疫原性的影响,以评价其作为DNA疫苗佐剂的效果。方法利用PCR扩增Inv397编码基因(inv397),分别构建编码猪丹毒丝菌表面抗原氨基端蛋白(spaA-N)和Inv397蛋白的重组真核表达质粒pcDNA3.1-spaA-N,pcDNA3.1-spaA-N-inv397。同时构建重组载体pET-30a-inv397,并在大肠杆菌BL21中诱导表达,经过Ni Sepharose 6 Fast Flow亲和层析纯化重组Inv397蛋白。将纯化后重组Inv397蛋白与包含spaA-N的重组真核质粒滴鼻免疫ICR小鼠,ELISA法检测血清特异性抗体水平,细胞增殖实验分析T淋巴细胞增殖反应。结果 Inv397蛋白与spaA-N重组质粒滴鼻免疫组的血清spaA-N特异性IgG水平明显高于spaA-N其它对照组(P<0.01),且T细胞增殖水平也高于其他各组,但无显著差异。结论 Inv397蛋白具有一定程度的免疫增强作用,有可能成为一种新的DNA疫苗佐剂。
Objective To investigate the effect of 397 amino acids (INV397) on the immunogenicity of antigen of Yersinia pilots in order to evaluate the effect of DNA vaccine adjuvant. Methods Inv397 gene was amplified by PCR. The eukaryotic expression plasmids pcDNA3.1-spaA-N and pcDNA3.1-spaA-N encoding spaA-N and Inv397 proteins were constructed. spaA-N-inv397. At the same time, recombinant vector pET-30a-inv397 was constructed and induced in E. coli BL21. Recombinant Inv397 protein was purified by Ni Sepharose 6 Fast Flow affinity chromatography. ICR mice were immunized intranasally with the purified recombinant Inv397 protein and recombinant eukaryotic plasmid containing spaA-N. Serum-specific antibody levels were detected by ELISA. The proliferation of T lymphocytes was analyzed by cell proliferation assay. Results The serum level of spaA-N specific IgG of the immunized group with Inv397 protein and spaA-N recombinant plasmid was significantly higher than that of the other control groups (P <0.01), and the T cell proliferation was also higher than that of the other groups No significant difference. Conclusion Inv397 protein has a certain degree of immune enhancement and may become a new DNA vaccine adjuvant.