前列腺癌患者抗体库构建中抗体基因的扩增

Amplification of human antibody genes in the construction of antibody library

目的:探讨如何扩增出种类更多、产量更丰的免疫球蛋白基因,从而增加抗体库的多样性,为抗体库的构建及筛选奠定基础。方法:分离前列腺癌患者外周血淋巴细胞,提取其总RNA,逆转录为cDNA,通过改变PCR反应条件扩增出全部κ、λ轻链及大部分重链Fd段;改变PCR反应条件扩增未成功的重链片段改用半套式PCR进行扩增。结果:不仅所有引物都扩增出产物,而且其产量也较高。结论:这表明改变PCR反应条件与半套式PCR联合应用可扩增出种类更多、产量更丰的免疫球蛋白基因,从而增加抗体库的多样性,为抗体库的构建及筛选奠定基础。

Objective:To research how to enlarge the content and types of the human immunoglobulin' s diversity of variable region genes in the antibody library.Methods:Total RNA was extracted from human peripheral blood lymphocytes of prostate cancer patients. Through reverse transcription and routine Polymerase chain reaction(PCR) , the genes of light chain and most Fd of immunoglobulin were amplified.The remainder genes of Fd which could not be directly gained by routine PCR were amplified by semi-nested PCR.Results:We got all the gene of light chain and heavy chain of antibody. All of semi-nested PCR produced much stronger bands. The molecular weight of products amplified by routine PCR and semi-nested PCR was about 700 bp.Conclusion:The results suggested that the diversity of variable region genes of human Ig and the antibody library can be maximized by semi-nested PCR.