摘要
目的构建并表达抗人红细胞血型A抗原单链小分子抗体(single-chainFv)蛋白。方法设计合成抗重、轻链可变区引物,通过重叠延伸拼接PCR技术,在50AmAbV和VL基因间引入连结短肽,体外构建50AScFv基因并进行序列分析;将其克隆入表达载体pET-28a中,在大肠杆菌中诱导表达;并以免疫印迹测定重组蛋白的生物学活性。结果序列分析表明,50AScFv基因全长747bp,编码249个氨基酸;重组体表达产物聚丙烯酰胺凝胶电泳显示,融合蛋白的相对分子质量(Mr)为29000左右,与预期的结果一致,表达产物主要以不溶性包涵体形式存在;光密度扫描结果表明,重组蛋白占菌体总蛋白的16.2%,免疫印迹试验显示,重组小分子抗体保留了亲本mAb的特异性和力。结论成功地构建50AScFv基因,并在原核细胞中获得较高水平的表达,为进一步研制基因工程血型检定抗体奠定了基础。
Aim To construct and express ScFv fusion protein against human blood group A substance. Methods The restriction enzyme sites were added to the previously cloned VH and VL genes fo 50A by add on PCR. The 50A ScFv genes constructed by the splicing overlap extension (SOE) PCR using a (Gly4Ser)3 linker were sequenced by Sanger's method and then inserted into fusion expression vector pET 28a. The recombinant proteins were examined by immunoblot. Results The nucleotide sequencing indicated that the cloned genes coded the light and heavy chain variable domains in mouse antibody. The 50A ScFv gene consisted of 747 bp, encoding 249 amino acids. After induction, a new protein band with relative molecular mass(Mr) of 29 000 appeared on SDS PAGE gel and amounted to 16.2% of total bacteria protein in a form of inclusion body. Immuno blot analysis identified that the recombinant protein retained the specific affinity of the intact 50A mAb. Conclusion The 50A ScFv gene has been constructed successfully and expressed in E.coli BL 21(DE3). This provided a foundation for studying engineering antibody as blood grouping reagent.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2001年第3期271-273,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助
No.39670645
"九五"全军医药科研基金资助
No.96M025
