欢迎访问《中国农学通报》,

中国农学通报 ›› 2010, Vol. 26 ›› Issue (23): 22-25.

所属专题: 生物技术 畜牧兽医

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

Touchdown PCR扩增多样性小鼠Fab抗体基因

王乃东 何浪 薛立群   

  • 收稿日期:2010-04-29 修回日期:2010-05-26 出版日期:2010-12-05 发布日期:2010-12-05
  • 基金资助:

    国家自然科学基金;湖南省教育厅科学研究优秀青年项目;湖南省研究生创新基金项目资助

Amplify mouse Fab antibody gene with diversity by Touchdown PCR

  • Received:2010-04-29 Revised:2010-05-26 Online:2010-12-05 Published:2010-12-05

摘要:

摘 要:【研究目的】为了扩增多样性Fab抗体基因产物,保证噬菌体抗体库的足够库容。【方法】分离C57BL\6鼠的脾脏,并制备脾细胞悬液,提取其总RNA,逆转录为cDNA,利用普通PCR和Touch down(TD)PCR扩增鼠Fab基因产物,通过琼脂糖凝胶电泳验证PCR产物,比较分析两种方法的扩增效果。【结果】轻链和重链Fd基因的PCR产物大小约为680bp,与理论值相符。电泳结果显示,TD-PCR获得8个重链Fd基因和6个轻链基因条带,普通PCR获得7个重链Fd基因和5个轻链基因条带。在普通PCR产物中,轻链基因引物VkB未能扩增出相应基因,而TD-PCR扩增出该基因。TD-PCR扩增出引物IIIA和IIIC的对应基因,而普通PCR却未见对应结果,普通PCR扩增出IIIB引物的对应基因,TD-PCR未见对应结果。【结论】比较分析认为,TD-PCR可以增加抗体基因多样性,有潜力成为保证噬菌体Fab抗体库库容的途径,为Fab抗体基因的扩增及相关研究提供实验参考。关键词:小鼠;噬菌体展示技术;抗体库

关键词: 有色稻, 有色稻, 栽插密度, 产量, 群体质量

Abstract:

Abstract:【OBJECTIVE】To amplify various products of Fab antibody genes with diversity, guarantee enough reservoir capacity of phage antibody library.【METHOD】The spleen of C57BL\6 mouse was isolated, spleen cell suspension was prepared, total RNA was extracted, cDNA synthesis was accomplished by reverse transcription. The product of Fab antibody was amplified by PCR and Touch down PCR, the products were analyzed by agarose gel electrophoresis, we performed a comparative analysis of two PCR methods. 【RESULTS】the products of light chain and high chain gene was about 680bp, and the result corresponds with the theoretical value. The results of electrophoresis analysis showed that TD-PCR had 8 high chain gene and 6 light chain gene, PCR had 7 high chain gene and 5 light chain gene. The amplified gene product with VkB primer was not appeared in the products of PCR, and appeared in the products of TD-PCR. The gene product with IIIA and IIIC primer were obtained by TD-PCR, and not successfully amplified by PCR. The amplified gene product with IIIB primer was appeared in the products of PCR, and not appeared in the products of TD-PCR. 【 CONCLUSION 】TD-PCR may increase diversity of Fab antibody genes, is an approach to ensure reservoir capacity of phage antibody library,which would be a reference for amplification and other research of Fab antibody gene.