BMP2基因重组慢病毒载体质粒的构建及鉴定

摘要

目的 构建重组慢病毒载体质粒pLV.EX2d.P/neo-EF1A>BMP-2/T2A/EGFP并进行鉴定.方法 从Genbank获得BMP2基因序列,结合载体上的酶切位点需要,设计上下游引物,通过PCR方法扩增目的 基因片段,利用Gateway技术 BP反应构建pDown-BMP2-T2A-EGFP,并进行阳性克隆测序,应用LR反应把pDown-BMP2-T2A-EGFP重组入慢病毒目的 载体质粒pLV.Des2d.P/neo,进行阳性克隆测序.结果 获得长度为1 191 bp的BMP2目的 基因片段,质粒pLV.EX2d.P/neo-EF1A>BMP2/T2A/EGFP经双酶切后凝胶电泳鉴定正确,测序结果与Genbank报道序列一致.结论 成功构建重组慢病毒载体质粒pLV.EX2d.P/neo-EF1A>BMP2/T2A/EGFP.%Objective To construct and identify a lentiviral vector carrying human BMP2 gene. Methods BMP2 gene was obtained from Genbank directly. Primers were designed according to the BMP2 gene sequences reported in Genbank and the restriction sites of the vector. The BMP2 gene was amplified by polymerase chain reaction(PCR). pDown-BMP2-T2A-EGFP was constructed with the BP reaction of Gateway technology, then positive cloning was sequenced. pDown-BMP2-T2A-EGFP was inserted into destination vector plasmid pLV. Des2d. P/neo with the LR reaction of Gateway technology. The positive cloning was sequenced. Results 1 191 bp BMP2 gene fragment was obtained,pLV. EX2d. P/neo-EFlA>BMP2/T2A/EGFP was identified with double digestion and sequencing, the result was completely in accordance with the BMP2 gene sequences reported in Genbank. Conclusion Recombination lentiviral vector plasmid pLV. EX2d. P/neo-EFlA>BMP2/T2A/EGFP is constructed successfully.