转录抑制因子ZHX2功能片段的克隆及表达

Cloning of the human ZHX2 functional domain

目的构建人ZHX2功能片段的真核融合表达载体。方法以含有全长ZHX2基因的pcDNA3.0-ZHX2-HA质粒为模板,PCR扩增人ZHX2基因功能片段(242-501AA),经EcoRI,KpnI双酶切后克隆入pcDNA3.0,构建真核表达载体pZHX2(242-501)-HA,通过酶切及测序鉴定其正确性。利用脂质体将pcDNA3.0和含有ZHX2功能片段的真核表达载体分别转染肝癌细胞系HepG2.2.15和非洲绿猴肾细胞COS-7,Western blot检测人ZHX2功能片段融合蛋白的表达。结果电泳结果显示:PCR扩增的ZHX2基因242-501AA片段大小与预期相符,重组质粒经双酶切和DNA测序,证明其与GenBank报道序列一致。Western blot结果显示,ZHX2功能片段的融合蛋白在肝癌细胞系HepG2.2.15和非洲绿猴肾细胞COS-7中均可有效表达。结论成功构建含有ZHX2功能片段的真核融合表达载体,为进一步探讨ZHX2基因的功能机制及其应用奠定了基础。

Objective To construct a plasmid containing the functional domain of human Zinc-fingers and homeoboxs 2（ZHX2）. Methods A truncated fragment of human ZHX2（242-501AA） was amplified with pcDNA-ZHX2-HA and cloned into pcDNA3.0 to construct expression vector pZXH2 （242-501AA）. The recombinant plasmids were identified with enzyme digestion and sequencing. Expression vector pZXH2 （242-501AA） was transfected into HepG2.2.15 and COS-7cells by Lipofectamine 2000, and expression of fusion proteins was assayed by western blot. Results Recombinant plasmids containing the functional domain （242-501AA） of the human ZHX2 gene were constructed. Enzyme digestion and sequencing analysis showed successful construction of the plasmids. Western blot showed protein expressions of the functional domain of human ZHX2 in HepG2.2.15 and COS- 7cells lines. Condusion The functional domain of the human ZHX2 gene was successfully cloned and expressed, which sets a good basis for further study on the functional mechanism of ZHX2.