人乳铁蛋白基因的克隆及序列分析与重组逆转录病毒质粒载体PLNCX2-hLF的构建

Cloning and sequence analysis of human lactoferrin and construction of the recombinant retroviral vector of PLNCX2-hLF

从正常人外周血中性粒白细胞中提取总RNA,用RT-PCR的方法扩增人乳铁蛋白(hLF)cDNA,将其克隆到pMD18-T Simple载体上并测序.用限制性内切酶XhoⅠ和HindⅢ将目的片段与逆转录病毒载体PLNCX2进行双酶切,并用T4连接酶进行连接;然后转化到top10菌中,再次进行DNA序列测定.前后两次序列测定结果一致,没有突变,全长为2,136bp,说明所克隆的为hLF基因的cDNA序列;与GeneBank中登录的序列相比,同源性达99%以上.

White blood cells are isolated from normal human peripheral bloods and total RNA is extracted. The cDNA are amplified by RT-PCR and then cloned into pMD18-T simple vector. After being sequenced, digest the fragment and retroviral vector by restriction enzymes Xho Ⅰ and Hind Ⅲ. The conjugate is transferred into E, coli cell line Top 10 and sequenced again Both sequencings show that the cloned gene sequence hLF consistent, no mutation, 99 % homology with other sequences in GeneBank.