摘要
目的构建人类免疫缺陷病毒1型(HIV-1)tat基因重组慢病毒表达载体。方法将pCDH-GFP和pCDHRFP载体分别用EcoRⅠ和HindⅢ双酶切制备线性化载体;通过设计引物及PCR扩增,获得5′端与3′端分别与15nt线性化载体3′端与5′端互补的tat基因片段;tat基因片段和线性化载体经纯化后,进行重组反应;将重组产物转化感受态细胞DH5α,通过菌落PCR、质粒双酶切、序列测定等方法鉴定重组克隆。结果菌落PCR扩增产物经琼脂糖凝胶电泳,在约354bp位置出现预期的条带;EcoRⅠ和HindⅢ双酶切重组质粒经琼脂糖凝胶电泳,在约7544bp和354bp位置上出现预期条带;通过测序,重组质粒中插入的tat基因序列与GenBank基因库中的HIV-1tat基因序列同源。结论采用本研究方法能成功构建HIV-1tat基因重组慢病毒表达载体。
Objective To construct recombinant lentivirus expression vector for human immunodeficiency virus 1（HIV-1）tat gene. Methods The pCDH-GFP and pCDH-RFP vectors were used to prepare the linear vectors respectively by EcoRⅠand HindⅢ.By designing primers and PCR amplification,15 nt complementary sequences of 3′terminal and 5′terminal of linearized vectors was added to the 5′terminal and 3′terminal of tat gene,and the gene fragments that the 5′terminal and 3′terminal respectively complemented 3′terminal and 5′terminal of 15 nt linearized vectors were obtained.Subsequently,the recombinant reaction was performed after the tat gene fragments and the linearized vectors were purified.The recombinant products were transformed into competent cells DH5αby conventional methods.Finally,the recombinant clones were identified with colony PCR,plasmid double enzyme digestion and sequencing. Results Expected DNA bands occurred at about354 bp after the colony PCR amplification products were separated by agarose gel electrophoresis,two DNA bands with the expected size at about 7500 bp and 350 bp were visible when EcoRⅠand HindⅢ double enzyme digestion recombinant plasmids were separated by agarose gel electrophoresis.The data of the gene sequencing showed that the tat gene inserted in recombinant plasmid was homologous to the HIV1 tat gene sequence of GenBank. Conclusion The HIV1 tat gene lentivirus recombinant expression vector can be successfully constructed by the protocol designed in this study.
出处
《右江民族医学院学报》
2017年第6期444-447,共4页
Journal of Youjiang Medical University for Nationalities
基金
广西自然科学基金2014GXNSFAA118136
