抗人转铁蛋白单链抗体的研制及部分性能研究

研制人转铁蛋白的单链抗体。以纯化人转铁蛋白免疫小鼠,制备小鼠脾脏 mRNA,RT-PCR 扩增重轻链可变区基因;Linker 连接及引入 Sfi Ⅰ和 NotⅠ酶切位点构建单链抗体基因;同载体 pCANTAB 5E 连接,转化 E.coliTG1细胞,以 M13K07挽救制备噬菌体抗体呈现文库,筛选抗原阳性重组噬菌体粒子(Panning);感染 E.coliHB2151菌株,制备可溶抗体并检测其性质;测定抗体基因序列并分析同源性及结构特征。制备出4株分泌抗人转铁蛋白可溶抗体的 E.coli HB2151菌株。抗体基因只由小鼠抗体的轻重链可变区基因构成,开放读码,无终止子,并紧接一末尾为一琥珀终止密码的 E 末端序列,其对应氨基酸序列 V_H 和 V_L 部分均遵从抗体可变区特征。实验证明,可把基因工程抗体技术应用于法医血清学,以制备新型抗体试剂。

Preparation and partial characterization of ScFv antibody to human transferrin

To prepare human transferrin ScFv antibodies.The mRNA from the spleens of mice im- munized by the purified human transferrin was isolated.The genes of the variable region of either heavy or light chain were amplified by RT-PCR followed by linking with a linker and then the restriction sites of the Sfi Ⅰ and Not Ⅰ enzymes were introduced.The genes of the single chain variable fragment which is called ScFv was constructed.After that,the ScFv products were linked with the vector pCANTAB 5Es and then transformed E coli TG 1 cells.At last,through rescue by M13K07 helper phoges the phage-displayed re- combinant antibodies repertoires were established.The antigen-positive recombinant phage particles were screened and enriched.This process is called panning.E coli HB2151 cells were infected to secrete soluble antibodies which were partially characte rized characteriged.The sequence of the ScFv gene was deter- mined.The homologous analysis against the heavy and the light chains of the mouse's immunoglobulin was done in the Kabat Database by Internet.Four strains of E coli HB2151 secreting soluble human transferrin antibodies were obtained.The ScFv gene was only constructed with genes of variable region of mouse's heavy and light chains and was terminated by an E-tag with an amber stop codon at its end.The ScFv gene had an open reading frame,without stop codon.The relevant amino acid sequence completely conform with the variable regions of the immunoglobulin.In order to prepare a new kind of antibody reagent,the genetic engineering antibody technique could be used in the forensic serology field.