抗人黑色素瘤单链抗体基因的克隆和分泌型表达

应用ＲＴ－ＰＣＲ技术从分泌抗人黑色素瘤单克隆抗体的杂交瘤细胞ＨＢ８７６０中克隆了抗体轻、重链可变区基因，然后用（Ｇｌｙ４Ｓｅｒ）３连接肽基因将ＶＨ、ＶＬ连接成ＳｃＦｖ基因，并进行了序列测定．计算机分析表明ＶＨ，ＶＬ均符合小鼠抗体可变区的特征，为功能性重排的抗体可变区基因．ＶＨ、ＶＬ、ｌｉｎｋｅｒ拼接正确．ＳｃＦｖ基因全长７２９ｂｐ，其中ＶＨ基因长３６０ｂｐ，编码１２０个氨基酸，ＶＬ基因长３２４ｂｐ，编码１０８个氨基酸．在噬菌粒表达载体ｐＣＡＮＴＡＢ５Ｅ中表达了可溶性的ＳｃＦｖ蛋白，表达产物经流式细胞仪检测可特异地与黑色素瘤细胞结合，不与肝癌、胃癌及良性黑痣细胞结合

Cloning and Expression of An Anti human Melanoma Single chain Fv Gene

The VH and VL genes were amplified from a mouse anti human melanoma hybridoma cell line HB8760 by RT PCR.The heavy and light chain variable regions were connected by a flexible linker,(Gly 4Ser) 3.The genes were sequenced and comarped with those published in EMBL genbank.The results showed that the VH and VL genes were homologous with the published mouse antibody variable region gene sequences.The VH and VL genes were 360bp and 324bp respectively and both of them were capable of encoding 120 and 108 amino acids.The deduced amino acid sequences contained four FRs,three CDRs and two cysteine residues necessary for the maintenance of the antibody structure,indicating that they were functionally rearranged.The fusion gene was cloned into a phagemid vector pCANTAB 5E and expressed in E.coli .The results of SDS PAGE and Western blot demonstated that the IPTG induced bacteria expressed an extra band of 30 kD in comparison with the uninduced ones.Immunofluorescence assay showed that the expressed protein had the affinity to a human melanoma cell line LiBr,but not to a liver cancer cell line or a stomach cancer cell line.