抗人大肠癌单链抗体基因的克隆与表达

背景与目的：单链抗体相对于完整抗体具有免疫源性低、对肿瘤组织穿透力强的特点,日益成为肿瘤诊断和治疗的良好导向载体。本研究的目的是将抗人大肠癌单克隆抗体ND－1的重链可变区VH和轻链可变区VL基因借助一短肽序列（Gly4Ser)3进行重组,构建单链抗体基因ND－1scFv,并使其在大肠杆菌中表达。方法：采用RT－PCR技术从能够分泌ND－1单抗的鼠杂交瘤细胞中扩增VH和VL基因,通过重叠延伸拼接PCR在VH和VL基因间引入连接短肽,体外构建ND－lscFv基因,经过常规转化和筛选,将其克隆至PET－28a( )表达载体,由IPTG诱导在大肠杆菌BL21中表达为ND－lscFv与His-Tag的融合蛋白。表达产物用Ni-NTA resin亲和层析方法纯化,并采用ELISA方法检测其免疫活性。结果：序列分析表明,ND－lscFv基因全长732bp,VH354bp位于上游;VL330bp位于下游。SDS－PAGE显示,重组蛋白相对分子量30kDa,与预期结果一致。scFv表达产物以不溶性包涵体形式存在,经亲和层析纯化后蛋白纯度达94％。ELISA结果显示scFv保留了与亲本抗体ND－1相似的免疫活性。结论：成功地构建了抗人大肠癌单链抗体ND－lscFv,并在大肠杆菌中获得了较高水平的功能性表达。

Cloning and expression of single chain Fv gene against human colorectal carcinoma

Background &Objective:Single chain Fv(scFv)has been employed as a favorable targ eting carrier in the therapy and diagnosis of tumors d ue to its advantages in relatively lo w immunogenity and stronger penetra nce to tumor tissues over intact mAb.This study was designed to recombine the genes fr om the variable regions of light chain and heavy chain of ND-1,a monoclonal antibody against human colorectal carcinoma ,by a short peptide(Gly 4 Ser) 3 to construct the ND-1scFv gene.The ND-1scFv protein was expressed in Escherichia coli.Methods:V H and V L gene were amplified from hybridoma cell IC-2,secreting monoclonal antibody ND-1,by RT-PCR,and then were connected to each other by a linker peptide using extension overlap spl icing PCR to obtain the ND-1scFv gene.The latter was cloned into the expre ssion vector PET-28a(+)and induced by IPTG to express a fusio n protein scFv and His-tag in E.coli BL-21.The expressed product was purified by affinity chromatography using Ni-NTA resin and its immun oactivity was analyzed using ELISA.Results:Sequence analysis showed that scFv g ene consisted of 732bp,among them,354bp for heavy chain gene,located up stream of scFv gene,and 330bp for the light chain gene,located donwstream.SDS-PA GE analysis showed that the relative molecular weight of fusion protein is 30kDa whi ch was consistent with the theoretic ally predicted value.scFv expression was in the form of an inclusion body,and SDS-PA GE analysis of the purified scFv show ed 94%purity.ELISA analysis revealed that scFv had equal immunoreactivity to the parent ND-1antibody.Conclusions:ND-1scFv gene against human colorectal carcinoma was successfu lly constructed,and functionally expressed in E.coli.