基因转运载体中甲基化XbaⅠ位点的体外克隆与应用

In vitro recombination and use of methylated XbaⅠ in gene transmitting vectors

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 This study sought to explore the use of methylated XbaⅠ in gene transmitting vectors.Methods A pair of primers to amplify the vector backbone pΔSG was designed according to pIRES2-EGFP.pΔSG with potentially methylating XbaⅠ and deletion of IRES and EGFP segments and individual EGFP were obtained by PCR amplification.Recombinants of rpΔSG-EGFPV-Xho,rpΔSG-EGFPV-Xbawere screened with colony PCR and enzyme digestion after the two PCR products were digested and linked with T4 ligase.The two PCR products were also separately cloned into a pMD 18-T simple vector,and two recombinants of psT-ΔSG and psT-EGFP were identified.The EGFP in psT-EGFP was subcloned into the vector backbone of pΔSG cut out of psT-ΔSG to obtain the recombinant rpΔSG-sT-EGFPV-Xho.With the replacement of EGFP by DsRed2 in rpΔSG-sT-EGFPV-Xho,recombinant rpΔSG-sT-DsRed2V-Xho was obtained.The four final recombinants were transfected into CHO cells with Lipofectamine2000,and EGFP or DsRed2 was detected under a fluorescence microscope.Results The expression of EGFP or DsRed2 was obvious in cells transfected with the different recombinants.Conclusion The potentially methylating sequence of XbaⅠ in PCR products remained active during enzyme digestion and ligation in vitro while being protected by methylation in prokaryotic cells of genotype Dam＋or/and Dcm＋after transformation.The sequence had no effect on the stability and biological activity of recombinants in host cells.