口蹄疫病毒VP1基因C末端串连表达及其抗体ELISA方法的初步建立

Gene Cloning and Expression of the VP1 and 2VP1 Gene of FMDV

口蹄疫病毒主要免疫原性基因VP1的B细胞和T细胞抗原表位位于其C末端,参照Taiwanse97(AJ294928)设计了1对特异性引物,扩增出VP1基因C末端。序列分析表明:其C末端长285bp,编码74个氨基酸,与O/HKN/12/91、O/PEN/TAW/99核苷酸和氨基酸同源性分别为95%、93%和96%、93%;利用同尾酶Xho 、Sal 将VP1基因C末端串连起来;再将单拷贝和双拷贝C末端基因插入到原核表达载体pGEX-KG后,转化BL21(DE3),在IPTG诱导下获得表达,经Westernblot检测证实表达产物具有活性。以表达产物包被ELISA板,初步建立了特异、敏感的ELISA诊断方法。同时用表达产物免疫Balb/c小鼠,能产生一定的抗O型口蹄疫病毒的抗体。

The C-terminal half of VP1 gene of FMDV was amplified by PCR. The PCR products were cloned into pMD18-T and sequenced. The squence result indicated that the C-terminal half of VP1 gene was about 285 bp and coded 75 amino acids.The homology analysis of the gene with O/HKN/12/91 and O/PEN/TAW/99 in GenBank indicated that it was relatively conserved. Then the PCR products were inserted into pGEX-KG and transformed BL21(DE3) bacteria. The expression of GST-2VP1 and GST-VP1 was induced with IPTG and confirmed by SDS-PAGE, the recombinant protein had a molecular weight of 45 ku and 37 ku respectively.Their activity were confirmed by Western blot. Based on expressed GST-2VP1 protein as antigen, the ELISA to detect antibody against VP1 was developed and was primarily used to detect serum samples.