摘要
目的:从分泌抗人类天冬氨酰基β-羟化酶(HAAH)单克隆抗体(mAb)的杂交瘤细胞G3/F11中,克隆出鼠源an-ti-HAAH mAb重、轻链可变区基因,构建Anti-HAAH的单链抗体(scFv),并进行scFv基因的蛋白表达。方法:提取杂交瘤细胞G3/F11的总RNA,通过RT-PCR扩增出VH和VL基因;再利用重叠延伸PCR(SOE-PCR),通过设计在引物上的linker序列,将VH和VL拼接为完整anti-HAAHscFv基因。将测序正确的scFv基因克隆入pHEN1载体,并利用E.coliHB2151进行蛋白表达;通过SDS-PAGE,Western blot分析其表达状况,ELISA鉴定其抗原结合活性。结果:测序结果显示,本实验成功地构建出鼠源anti-HAAHVH-linker-VLscFv基因,且VH、VL均具有完整正确的小鼠抗体骨架区和互补决定区结构,所得的scFv基因片段全长744bp,编码248个氨基酸。SDS-PAGE和Westernblot分析表明,pHEN1-anti-HAAH在E.coliHB2151可表达为Mr约27000的可溶性scFv蛋白,表达量为7.8%。间接ELISA检测显示,可溶性鼠源anti-HAAHscFv蛋白具有较高的抗原结合活性。结论:成功扩增出的鼠源anti-HAAHmAbVH区和VL区基因,并构建为anti-HAAHscFv基因。然后,利用pHEN1载体对anti-HAAHscFv基因进行成功表达,为进一步研究其生物活性及应用奠定了基础。
AIM：Construction and expression of anti-HAAH single chain variable fragment （scFv） by cloning of the variable region genes from anti-HAAH hybridoma cells G3/F11.METHODS：Total RNA was extracted from hybridoma cells G3/F11.By RT-PCR,murine VH and VL genes of mAb were amplified respectively.Then,They were assembled into VH-linker-VL scFv template by SOE-PCR and anti-HAAH scFv was express in E.coli by constructed pHEN 1-Anti-HAAH vector.The expression of Anti-HAAH scFv were detected by SDS-PAGE and Western blotting and the binding activity were demonstrated by ELISA.RESULTS：The analysis of DNA sequencing shown that the full-length of constructed scFv gene was 744 bp,encoding 248 amino acids.Moreover,the VH and VL genes were functional antibody variable region genes,as there were four FRs and three CDRs in both of them.By SDS-PAGE and Western blotting,the expression level of Anti-HAAH scFv were detected.The expression level of pHEN 1-Anti-HAAH scFv,which was expressed in E.coli HB2151,was 7.8% in total E.coli protein and were existed in soluble protein mainly.By indirect ELISA detcetion with HAAH protein,the binding activity of soluble anti-HAAH scFv was very well.CONCLUSION：The murine VH and VL genes of mAb against HAAH have been cloned successfully and anti-HAAH scFv have been constructed and expressed.Besides,the scFv could be further studied about their biological activity and application,due to their high affinity shown in preliminary detection.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2010年第5期467-470,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(30600564
30700030
30700710)
