犬瘟热病毒截短融合蛋白原核表达及其单克隆抗体制备

Prokaryotic Expression of Canine Distemper Virus F Protein and Preparation of Monoclonal Antibody

为制备犬瘟热病毒(canine distemper virus,CDV)融合蛋白(F)单克隆抗体,研究通过PCR扩增CDV F蛋白胞外区基因,将其克隆至pET-28a原核表达载体,并构建pET-28a-CDV-F重组质粒;将其转化到BL21(DE3)感受态细胞,经终浓度为0.2 mmol·L^(-1)的IPTG在37℃诱导6 h后,SDS-PAGE检测表达产物,结果显示在约33 kDa处出现目的条带,且该蛋白主要以包涵体形式表达。Western blot结果表明,重组F蛋白可特异性识别抗His标签单克隆抗体和CDV阳性血清。将重组F蛋白采用切胶纯化法纯化后,以此为免疫原免疫BALB/c小鼠4次,取脾细胞与SP2/0细胞融合,以重组F蛋白为包被抗原,应用间接ELISA检测方法与有限稀释法筛选阳性杂交瘤细胞,最终筛选得到2株单克隆抗体(命名为1B6、1G8);经抗体亚类试剂盒鉴定,2株单抗均属于IgM/κ型;Western blot结果表明2株单克隆抗体均能与重组F蛋白特异性结合。在CDV感染的细胞上进行间接免疫荧光试验,2株单克隆抗体均能识别CDV3疫苗株和LN(10)1野毒株。综上,CDV F蛋白单克隆抗体的制备为CDV的检测奠定基础。

In order to prepare monoclonal antibody(mAb)against canine distemper virus F protein,the extracellular region of CDV F protein was amplified by RT-PCR,cloned into pET-28a prokaryotic expression vector,and pET-28a-CDV-F recombinant plasmid was constructed.The recombinant plasmid was expressed in BL21(DE3)and induced by IPTG at a final concentration of 0.2 mmol·L^(-1) at 37℃for 6 h.The expression product was detected by SDS-PAGE.The results showed that the target band appeared at about 33 kDa,and the protein was mainly expressed in the form of inclusion bodies.Western blot results showed that the recombinant F protein could specifically recognize anti-His tag monoclonal antibody and CDV positive serum.The recombinant F protein was purified by gel cutting purification method and used it as immunogen to immunize BALB/c mice for 4 times.The spleen cells were fused with SP2/0 cells.The recombinant F protein was used as the coating antigen to screen the positive hybridoma cells by indirect ELISA and limited dilution method.Finally,two monoclonal antibodies(named 1B6 and 1G8)were screened.The two monoclonal antibodies were identified by antibody subclass kit,and the results showed that the two monoclonal antibodies belonged to IgM,κtype;Western blot analysis of the specificity of two monoclonal antibodies showed that the two monoclonal antibodies specifically recognized recombinant CDV F protein.Both mAbs could react specifically with CDV3 vaccine strain and LN(10)1 wild-type strain by IFA assay.The preparation of monoclonal antibodies against CDV F protein lays a foundation for the detection of CDV.