受控型蜕皮激素诱导表达截短型hIGF-Ⅰ转基因载体的构建及其在细胞水平上的有效性检测

Construction and availability test in eukaryocyte of ecdysone-inducible controlled transgene expressing truncated hIGF-Ⅰ

目的:构建一种可以在哺乳动物细胞中蜕皮激素诱导表达截短型hIGF-Ⅰ的受控型转基因载体,为制备受控型蜕皮激素诱导表达截短型hIGF-Ⅰ的转基因小鼠奠定基础。方法:利用分子克隆技术构建受控型蜕皮激素诱导表达截短型hIGF-Ⅰ的转基因载体;将其电转至AM1菌中,利用其中的Cre重组酶将载体上两个同向LoxP序列锚定的新霉素(neomycin)基因删除,解除其对蜕皮激素诱导表达系统的阻断作用,利用PCR、酶切和测序鉴定删除情况;将重组后的载体转染至COS7细胞中进行瞬间表达,蜕皮激素诱导后回收培养上清和细胞裂解物进行W est-ern印迹分析,检测hIGF-Ⅰ的表达情况。结果和结论:成功构建大小为13.6 kb的转基因载体pCE-IGF-Ⅰ;转化至AM1中后,PCR、酶切和测序的结果都证明其中的Cre酶能够将载体上neomycin基因删除;重组后的载体转染至COS7细胞中进行诱导表达,W estern印迹实验证明截短型hIGF-Ⅰ能够在COS7细胞中顺利表达。上述结果证明该蜕皮激素诱导表达截短型hIGF-Ⅰ的受控型转基因载体能够用于转基因小鼠的制备。

Objective： To construct one kind of controlled transgene and lay a foundation of preparing transgenic mice in which truncated hIGF-Ⅰ can be induced to express by ecdysone in mammalian cells. Methods：The transgene was constructed according to molecular cloning technique,then it was transformed by electric shocking into AM1 bacteria, in which Cre recombinase could delete neomycin gene between two syntropic LoxP sequences, and its blocking effect on ecdysone-inducible expression system was removed. PCR, enzyme-cutting and sequencing were used to check the deletion of blocking sequence during the process. Transgenes in AM 1 were recovered and transfected into COS7 cells, after ecdysone induction, medium supematant and cell lysate were recovered, Western blotting were run to detect hIGF-Ⅰ expression. Results and Conclusion： 13.6 kb Transgene pCE-IGF-Ⅰ was constructed successfully, after transforming into AM1, PCR, enzymecutting and sequencing results showed that Cre recombinase in AM1 could delet blocking sequence between LoxP sites, Transgenes after recombination were transfected into COS7 and induced by ecdysone, Western blotting results showed that truncated hIGF-Ⅰ could be expressed successfully in COS7. All results showed that the transgene can be used for preparing transgenic mice.