PEDV S基因的B细胞表位嵌入型乳杆菌S-层蛋白的原核表达及其鉴定

Prokaryotic expression and identification of PEDV S gene from B-cell epitope embedded type of Lactobacillus S-layer protein

利用生物软件和在线数据库中筛选出猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)S基因的B细胞表位,通过overlap PCR方法将其嵌入到嗜酸性乳杆菌的S-层蛋白基因中,获得融合基因SLP-EpitopeS。将该融合基因导入到原核表达载体pGEX-4T-3中,成功构建了原核表达载体pGEX-SLP-EpitopeS。经过双酶切和PCR方法以及基因测序方法验证融合表达载体pGEX-SLP-EpitopeS的正确性。应用SDS-PAGE和Western blot方法鉴定表达产物,其中SDS-PAGE结果显示:融合蛋白质的大小约为77000,与理论值相符;Western blot结果显示:融合蛋白SLP-EpitopeS被GST血清所识别,而B细胞表位分别被表位特异的鼠源单克隆抗体(McAb)和表位合成多肽所识别。本试验为进一步嵌入型蛋白SLP-EpitopeS的免疫原性研究奠定了基础。

B-cell epitopes on PEDV S gene were screened by biological software and online database.The fusion gene SLP-EpitopeS was obtained by embedding which into the S-layer protein of Lactobacillus acidophilus by overlap PCR.The fusion gene was introduced into the prokaryotic expression vector pGEX-4T-3,and the prokaryotic expression vector pGEX-SLP-EpitopeS was successfully constructed.The correctness of the fusion expression vector pGEX-SLP-EpitopeS was verified by double digestion,PCR and gene sequencing.The expression products were identified by monoclonal antibody technology,SDS-PAGE and Western blot.The results of SDS-PAGE showed that the size of the fusion protein was about 77000,which was consistent with the theoretical value.Western blot results showed that the fusion protein SLP-EpitopeS was recognized by GST serum,and the B cell epitope was recognized by epitope-specific murine monoclonal antibody(McAb)and epitope peptide positive serum.This experiment laid the foundation for further study on the immunogenicity of the embedded protein SLP-EpitopeS.