猪圆环病毒2型衣壳蛋白优势B细胞表位重组蛋白免疫原性研究

猪圆环病毒2型(PCV-2)衣壳蛋白(Cap)是研制基因工程疫苗和血清抗体检测方法的主要候选抗原。为筛选免疫原性强的Cap抗原表位,将定位在Cap上的6个B细胞线性表位的编码基因序列重新组合,命名为R1234、R1235、R1236、R2345、R2346和R3456,然后克隆至pET32a原核表达载体,转化到大肠埃希菌中进行诱导表达。通过对表达和纯化条件的优化,最终获得6个多抗原表位串联体的重组蛋白。Western blot和间接ELISA结果显示,6组重组表位蛋白均能被PCV-2阳性血清特异性识别,具有良好的反应原性。将其与完整Cap分别免疫Balb/c小鼠,检测免疫后血清中特异性抗体和细胞因子水平以及脾脏淋巴细胞中CD4^+和CD8^+T淋巴细胞的比例。结果表明,R2346组合刺激机体产生的特异性抗体和细胞因子IFN-γ、IL-4的水平明显高于其他组,脾脏淋巴细胞中CD4^+和CD8^+T淋巴细胞含量与对照组相比显著增加(P<0.05),说明该重组蛋白能够刺激体液免疫和细胞免疫反应。因此,筛选获得的R2346重组表位蛋白可以作为研制PCV-2多表位基因工程疫苗和血清抗体诊断方法的候选抗原,用于PCV-2的预防和控制工作中。

Study on Immunogenicity of Porcine Circovirus Type 2 Cap Recombinant Proteins with Dominant B Cell Epitopes

The capsid protein(Cap)of porcine circovirus type 2(PCV-2)is the main candidate antigen for the development of genetic engineering vaccine and the detection of serum antibody.In order to screen Cap epitopes with strong immunogenicity,six coding gene sequences of B cell linear epitopes located on Cap protein were recombined,which were R1234,R1235,R1236,R2345,R2346 and R3456.Then they were cloned into the prokaryotic expression vector pET32 aand transformed into Escherichia coli to induce expression respectively.The results of Western blot and indirect ELISA showed that the six recombinant epitope proteins could be specifically recognized by PCV-2 positive serum with good reactivity.After that,the specific antibodies and cytokines in serum and the proportion of CD4^+and CD8^+T cells in spleen lymphocytes were detected after immunizing Balb/c mice with recombinant epitope proteins and intact Cap respectively.The results displayed that the levels of specific antibodies,IFN-γand IL-4 produced by R2346 group were significantly higher than those of other groups and the contents of CD4^+and CD8^+T cells in splenic lymphocytes of R2346 group were also increased significantly(P<0.05),which indicated that R2346 epitope protein could stimulate both humoral and cellular immune responses.Consequently,the R2346 epitope protein can be used as a candidate antigen to develop the PCV-2 multi-epitope genetic engineering vaccine and the serum antibody diagnosis kit in the prevention and control of PCV-2.