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目的:从人源化噬菌体抗体库中筛选高亲和、特异结合人骨唾液酸蛋白(BSP)的人源可变区单链抗体(scFv)。方法:采用噬菌体表面展示系统,以重组的BSP蛋白为包被抗原,从噬菌体可变区scFv中筛选特异性结合BSP的小分子scFv,经过3轮亲和富集筛选后,再用ELISA方法进一步鉴定与抗原BSP有特异结合活性的scFv阳性克隆,PCR扩增鉴定阳性克隆的插入轻、重链基因片段,并对阳性scFv分子测序和序列分析。结果:筛选得到的scFv片段可以特异地结合BSP蛋白,PCR扩增都得到了长为368 bp、527 bp和935 bp的轻链、重链和轻链-连接片段-重链的基因片段,测序结果分析发现上述scFv片段在轻链有11处的氨基酸组成不同,在重链区域氨基酸组成则有3处不同。基因序列分析结果表明符合人源单链可变区抗体基因序列的结构特征。结论:利用噬菌体抗体库技术成功获得BSP蛋白的特异性人源单链可变区抗体。
OBJECTIVE: To screen human variable region single chain antibody (scFv) with high affinity and specific binding to human bone sialoprotein (BSP) from humanized phage antibody library. Methods: The recombinant BSP protein was used as coating antigen in the bacteriophage surface display system. The small scFv specific for BSP was screened from phage variable region scFv. After 3 rounds of affinity scFv screening, the ELISA method was further used The scFv positive clones with specific binding activity to the antigen BSP were identified. The positive clones were identified by PCR amplification of inserted light and heavy chain gene fragments, and the positive scFv molecules were sequenced and sequenced. Results: The scFv fragment was able to bind specifically to BSP protein. The PCR products of light, heavy chain and light chain - linker fragment - heavy chain with length of 368 bp, 527 bp and 935 bp were sequenced Analysis of the results showed that the above scFv fragments had 11 different amino acid compositions in the light chain and 3 amino acid residues in the heavy chain region. The results of gene sequence analysis showed that the structure of the human single-chain variable region antibody gene was consistent with the structure. Conclusion: The specific human single chain variable region antibody of BSP protein was successfully obtained by phage antibody library.