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目的 :利用 Bac- to- Bac表达系统高效表达成熟的胰岛素样生长因子 (IGF- )。方法 :首先构建含 IGF- c DNA的重组供体质粒 p Fast Bac1,然后重组 p Fast Bac1所含的转位元件 mini- Tn7,在感受态细胞 DH10 Bac内转位到粘粒上的连接位点mini- att Tn7构成重组粘粒 ,重组粘粒转染昆虫细胞 Sf9产生重组杆状病毒 ,重组杆状病毒进行感染 Sf9细胞。结果 :琼脂糖凝胶分析显示重组供体质粒 p Fast Bac1的成功构建 ;琼脂糖凝胶分析重组粘粒的 PCR产物表明已获得重组粘粒 ;十二烷基磺酸钠(SDS) - PAGE和 Western Blotting显示 7KD左右蛋白质条带的出现。结论 :利用 Bac- to- Bac表达系统成功表达具有免疫学活性的 IGF- ,可用于临床疾病的诊治。
OBJECTIVE: To express mature insulin-like growth factor (IGF-) efficiently using Bac-to-Bac expression system. METHODS: Firstly, a recombinant donor plasmid p Fast Bac1 containing IGF-c DNA was constructed and then translocation element mini-Tn7 contained in recombinant p Fast Bac1 was transplanted into the cosmid junction site in competent cells DH10 Bac mini-att Tn7 constitute recombinant cosmids, recombinant cosmids transfected insect cells Sf9 recombinant baculovirus, recombinant baculovirus infection of Sf9 cells. Results: The agarose gel analysis showed the successful construction of the recombinant donor plasmid p Fast Bacl; PCR products of the recombinant cosmids by agarose gel indicated that recombinant cosmids were obtained; sodium dodecyl sulfate (SDS) -pAGE and Western Blotting shows the appearance of a protein band around 7KD. Conclusion: The successful expression of immunologically active IGF-1 using Bac-to-Bac expression system can be used to diagnose and treat clinical diseases.