溶藻弧菌抗独特型抗体scFv的原核表达及免疫原性鉴定

Prokaryotic expression and immunogenicity characterization of the single chain variable fragment of anti-idiotype monoclonal antibody against Vibrio alginolyticus

目的构建溶藻弧菌抗独特型单链抗体(scFv)的原核表达载体,并对其表达的蛋白进行免疫原性鉴定。方法从分泌溶藻弧菌抗独特型单克隆抗体的杂交瘤细胞株(2F4)中已获得的该抗体重链可变区基因(VH)和轻链可变区基因(VL),通过基因重组构建scFv及原核表达载体pET32a-AL,转化大肠杆菌BL21后用IPTG诱导表达。表达后的重组蛋白利用动物免疫试验进行免疫原性鉴定。结果scFv基因序列全长747碱基对,编码249个氨基酸,符合小鼠免疫球蛋白可变区基因特征,含有框架区(FRs)、抗原互补决定区(CDRs)及抗体特征性的半胱氨酸残基。ELISA测定和动物免疫试验显示抗独特性抗体的scFv具有与溶藻弧菌一样的免疫原性。结论成功构建了抗溶藻弧菌独特型抗体scFv原核表达载体并表达于包涵体中,表达的融合蛋白具有与溶藻弧菌一样的免疫原性,为溶藻弧菌抗独特型单链抗体scFv成为鱼用基因工程疫苗奠定了初步基础。

Objective To construct recombinant prokaryotic expression plasmid and characterize immunogenicity of the single chain variable fragment （scFv） of anti-idiotype monoclonal antibody against Vibrio alginolyticus. Methods Total RNA was extracted from hybridoma cell 2F4 secreting mAb against Vibrio alginolyticus, while cDNA was amplified by RT-PCR. The prokaryotic expression vector pEq32a-AL was constructed for the recombinant single chain variable fragment expression. The transformed E. coli BI21 cells were propagated and induced by WIG. The immunogenicity of the fusion protein was determined by using immtmized Balb/c mice. Results The scFv gene contained 747 base pairs and encoded 249 amine acid residues; there were four FRs, three CDRs, and two characteristic cysteine residues in the Vn and VL gene, respectively, Immunogenicity of scFv was assayed after immunity. All results indicated that the scFv does share a similar immunogenicity with Vibrio alginolyticus. The antigenicity of the fusion protein was shown similar with parental antibody by ELISA. Conclusion The scFv of antiidiotype monoclonal antibody against shown similar with parental antibody ibrio alginolyticus is constructed successfully and expression products is found in the inclusion bodies. The antigenicity of the fusion protein does share a similar immunogenicity with Vibrio alginolyticus. This scFv may be a new genetically engineered vaccine of the anti-idiotype monoclonal antibody against Vibrio alginolyticus in fishery.