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目的探讨基因转运载体中甲基化XbaⅠ位点在基因克隆中的效用。方法以pIRES2-EGFP为母本,PCR扩增获得携附加XhoⅠ位点和满足甲基化序列组成的XbaⅠ位点、同时缺失IRES和EGFP区段的载体骨架pΔSG,其PCR产物与携带对等酶切位点的EGFP PCR产物经酶切、连接后,构建重组质粒rpΔSG-EGFPV-Xho、rpΔSG-EGFPV-Xba。另将pΔSG和EGFP的PCR产物分别与pMD 18-T Simple T载体连接,构建重组质粒psT-ΔSG和psT-EGFP,并亚克隆获得重组质粒rpΔSG-sT-EGFPV-Xho。以DsRed2基因置换rpΔSG-sT-EGFPV-Xho中的EGFP基因,获得重组质粒rpΔSG-sT-DsRed2V-Xho。各重组质粒经脂质体介导转染CHO细胞。结果各重组质粒转染的细胞中明显可见EGFP或DsRed2的高效表达。结论 PCR产物中满足甲基化序列组成的XbaⅠ位点,在体外酶切与连接的克隆效果良好;该序列经体外重组并导入Dam+和/或Dcm+宿主菌中扩增时被重新甲基化而受保护,且对重组质粒在宿主细胞内的稳定性和生物活性没有明显影响。
Objective To investigate the utility of methylated Xba Ⅰ locus in gene transfer in gene cloning. Methods The XbaⅠ locus carrying XhoⅠ site and methylation sequence was obtained by PCR amplification with pIRES2-EGFP as the parent. The pESS vector carrying IRES and EGFP was deleted. The EGFP PCR product of the cut site was digested and ligated, and then the recombinant plasmids rpΔSG-EGFPV-Xho and rpΔSG-EGFPV-Xba were constructed. In addition, the PCR products of pΔSG and EGFP were ligated to pMD 18-T Simple T vector respectively to construct recombinant plasmids psT-ΔSG and psT-EGFP, and subcloned to obtain recombinant plasmid rpΔSG-sT-EGFPV-Xho. The EGFP gene in rpΔSG-sT-EGFPV-Xho was replaced by DsRed2 gene to obtain recombinant plasmid rpΔSG-sT-DsRed2V-Xho. Each recombinant plasmid was transfected into CHO cells by liposome. Results High expression of EGFP or DsRed2 was observed in all the transfected cells. Conclusion The XbaⅠ locus in the PCR product which satisfies the methylation sequence is well cloned in vitro and cloned. The sequence was recombined and introduced into Dam + and / or Dcm + host bacteria for remethylation Protected, and had no significant effect on the stability and biological activity of the recombinant plasmid in host cells.