抗溶藻弧菌独特型单克隆抗体单链抗体ScFv的构建和表达

目的：克隆抗溶藻弧菌独特型单克隆抗体ScFv。方法：从分泌抗溶藻弧菌独特型单克隆抗体的杂交瘤细胞株（2F4）中提取总RNA，利用RT-PCR技术，克隆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VH-VL的表达载体pTAT-ALI，转化大肠杆菌BL21后用IPTG诱导表达。结果：VH基因序列全长369碱基对，编码123个氨基酸，VL基因序列全长339碱基对，编码113个氨基酸，二者均符合小鼠免疫球蛋白可变区基因特征，含有4个框架区（FRs）、3个抗原互补决定区（CDRs）及抗体特征性的两个半胱氨酸残基。ELISA测定显示基因工程抗体ScFv与原代抗体一样，具有较高的抗原亲和力。结论：成功构建了抗溶藻弧菌独特型抗体ScFv基因并表达于细菌壁膜间隙和包涵体中，为溶藻弧菌独特型抗体单链抗体ScFv成为新一代基因工程疫苗奠定了初步基础。

Construction and expression of single chain variable Fv of anti-idiotype monoclonal antibody against Vibrio alginolyticus

Objective:To clone the single chain variable Fv of anti-idiotype monoclonal antibody against vibrio alginolyticus.Methods:Total RNA was extracted from hybridoma cell 2F4 secreting MAb against vibrio alginolyticus and cDNA was amplified by retropolymerase chain reaction(RT-PCR),the expression vector pTAT-AL1 was constructed for the recombinant V_H-V_L expression.The transformed E.coli BL21 cells were propagated and induced by IPTG.Results:The V_H gene contained 369 base pairs and encoded 123 amine acid residues;The V_L gene contained 339 base pairs and encoded 113 amine acid residues;There were four FRs three CDRs and two characteristic cysteine residues in the V_H and V_L gene,respectively.ELISA results showed the ScFv retained almost the same antigen affinity and specificity as its parent monoclonal anitbody.Conclusion:The single chain variable Fv of anti-idiotype monoclonal antibody against vibrio alginolyticus was constructed successfully and expression products was found in the periplasmic space and inclusion bodies.This ScFv might be a new generation of gene engineering vaccine of the anti-idiotype monoclonal antibody against vibrio alginolyticus in fishery.