HIV-1 p24单链抗体的构建及其噬菌体文库筛选

Construction and selection from a peptide phage display library of the single-chain Anti-HIV-1 P24

目的通过噬菌体展示技术构建HIV-1 p24单链抗体库,从中筛选出高亲和力的抗体基因,为进一步单链抗体表达打下基础。方法从HIV患者的血清中提取mRNA,RTP-CR扩增出HIV-1 p24基因,扩增的产物用ClaⅠ和BstxⅠ双酶切消化后,连接到同样双酶切的pIRES1表达质粒,把构建的重组pIRES1-p24质粒免疫Balb/c小鼠。1个月后,取鼠脾细胞mRNA构建出单链抗体(ScFv)cDNA文库。该文库以噬菌粒pCANTAB5E cDNA为载体,转化大肠杆菌TG1,最后用p24蛋白对表达的重组噬菌体单链抗体文库进行筛选。结果经过4轮吸附-洗脱-富集筛选,洗脱噬菌体滴度大于1010pfu/ml,ELISA及其测序表明获取了表达抗HIV1p24单链抗体的基因。结论单链抗体和噬菌体展示技术成功的应用简化了获取特异性的单链抗体基因程序。

Objective cDNA library of anti-HIV1 p24 Single Chain Fragment of Variable Region（ScFv） was construct by phage display technology in order to express the ScFv in the future, Methods The template of mRNA was extracted from serum of patients, subsequently the gene of HIV-1 p24 was amplied by RT-PCR. The amplied product was digested by Cla Ⅰ and Bstx Ⅰ , then the digested product was transfered into pIRES1 express vector digested by the same two enzymes. The recomblnant plasmid immuned Balb/c mice, after one month, ScFv cDNA library was constructed from the spleen cell mRNA of mice. ScFv cDNA library of pCANTAB5E plasmid carrier was transformed into E.coli TG1 that of secreting specific anti- HIV1 p24 ScFv was obtained by p24 protein affinity selection. Results After four times absorb-wash^enrich selection, 2 × 10^10 phage form unit（pfu）/ml was got. Both ELISA and sequenced measures proved to succeed in secreting specific anti- HIV1 p24 ScFv gene.Conclusion ScFv and phage display technologies successly applied that simplied the acquiring procedure of specific ScFv gene.