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目的构建成纤维生长因子21(FGF21)表达载体。方法将FGF21基因克隆到腺病毒穿梭质粒p AdTrack经DH5α感受态菌扩增,Pme I酶切后热激转化到含腺病毒骨架质粒p Ad Easy-1的BJ5183感受态菌内同源重组,经卡那霉素筛选及Pac I酶切鉴定,并在DH5α菌中扩增得到Ad-FGF21重组腺病毒质粒,再经Pac I酶切线性化后转染到293A细胞包装成重组腺病毒。结果基因测序结果表明成功合成穿梭载体p Ad-Track-FGF21。PCR和酶切鉴定结果证实含FGF21基因的重组腺病毒表达载体构建成功。Ad-FGF21转染293A细胞并制备出高滴度的重组腺病毒。结论利用腺病毒表达载体获得了携带FGF21的重组腺病毒表达载体。
Objective To construct fibroblast growth factor 21 (FGF21) expression vector. Methods The FGF21 gene was cloned into adenovirus shuttle plasmid pAdTrack and amplified by DH5α competent cells. After digestion with Pme I, the FGF21 gene was homologously recombined into BJ5183 competent cells containing adenovirus backbone plasmid pAd Easy-1. Kanamycin screening and Pac I digestion and identification, and amplified in DH5α bacteria Ad-FGF21 recombinant adenovirus plasmid, and then digested with Pac I linearized 293A cells were transfected into recombinant adenovirus. Results The gene sequencing results showed that the shuttle vector p Ad-Track-FGF21 was successfully synthesized. PCR and restriction enzyme digestion confirmed that the recombinant adenovirus containing FGF21 gene was successfully constructed. Ad-FGF21 was transfected into 293A cells and a high titer of recombinant adenovirus was prepared. Conclusion The recombinant adenovirus vector carrying FGF21 was obtained by adenovirus expression vector.