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目的:构建并鉴定含有人骨形成蛋白2(hBMP2)及神经生长因子(hNGF)目的基因的慢病毒载体。方法:使用限制性内切酶,将新霉素抗性基因(Neo)从载体pLentiTrident1-EGFP-Neo切下,插入pLentiTrident1空载体的相应多克隆位点上,构建出含新霉素抗性的表达载体。采用PCR扩增得到hBMP2与hNGF的目的片段,通过多克隆位点将2个目的基因插入表达载体。对该重组载体进行酶切鉴定、PCR鉴定以及DNA序列测序分析。结果:通过酶切鉴定及测序分析,慢病毒表达载体(plentiTrident 1-hBMP2-Neo-hNGF)构建成功。结论:成功构建了hBMP2与hNGF的共表达慢病毒载体,为研究神经因素在骨组织再生中的作用奠定了基础。
Objective: To construct and identify a lentiviral vector containing human bone morphogenetic protein 2 (hBMP2) and nerve growth factor (hNGF). Methods: The neomycin resistance gene (Neo) was excised from the vector pLentiTrident1-EGFP-Neo using restriction endonuclease and inserted into the corresponding multiple cloning site of pLentiTrident1 empty vector to construct neomycin-resistant Expression vector. The target fragment of hBMP2 and hNGF was amplified by PCR and inserted into the expression vector through multiple cloning sites. The recombinant vector was identified by restriction enzyme digestion, PCR identification and DNA sequencing analysis. Results: The lentiviral expression vector (plentiTrident 1-hBMP2-Neo-hNGF) was successfully constructed by restriction analysis and sequencing analysis. Conclusion: The co-expression of hBMP2 and hNGF lentiviral vector was successfully constructed, which laid the foundation for the study of the role of neural factors in the regeneration of bone tissue.