溶藻弧菌抗独特型抗体单链抗体在毕赤酵母中的表达

Immunogenicity of single chain variable fragment of anti-idiotype monoclonal antibody against Vibrio alginolyticus and its expression in Pichia pastoris

目的构建溶藻弧菌抗独特型抗体的单链抗体(scFv)基因的毕赤酵母分泌型表达载体,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抗体的杂交瘤细胞株(2F4)中克隆该抗体的重链可变区(VH)基因和轻链可变区(VL)基因(GenBank accession No:DQ011530和DQ011531),并通过基因重组为scFv基因。然后将其克隆入毕赤酵母菌分泌型表达载体pPIC9K中,经序列测定后,电转化入毕赤酵母菌SMD1168中,将经表型筛选和G418抗性筛选的重组酵母菌株进行PCR鉴定。以甲醇进行诱导表达,表达后的重组蛋白利用动物免疫试验进行免疫原性鉴定。结果获得毕赤酵母菌分泌型表达载体pPIC9K-scFv,经G418浓度梯度筛选及PCR鉴定得到高拷贝整合的重组酵母菌株,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。结论构建溶藻弧菌抗独特型抗体的scFv基因的真核表达载体并成功表达,为进一步研究其生物学活性奠定了基础。

Objective To construct recombinant Pichia pastoris secretion type expression vector and characterize immunogenicity of 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,and then cDNA was amplified by RT-PCR.Then the RT-PCR product was recombined and inserted into secretion type expression vector pPIC9K-scFv.Recombinant vector was transformed into Pichia pasto...