人抵抗素基因全长的体外拼接及真核表达载体的构建

Assembly of full-length human resistin gene in vitro and construction of its eukaryotic expression vector

目的:利用合成的寡核苷酸片段,在体外拼接人抵抗素(Resistin,Retn)基因的全长,并构建其真核表达载体。方法:根据已知抵抗素基因(GenBank:AF323081),设计并合成10条寡核苷酸片段,采用降落PCR方法进行拼接,获得预期大小的PCR产物后将其克隆入pSecTag2B载体进行测序确认。结果:降落PCR法扩增的特异条带与目的基因的大小一致。克隆载体测序结果与GenBank中已知序列完全符合。结论:降落PCR成功地拼接和扩增了人抵抗素全长基因,并构建了含有该基因的真核表达载体,为进一步研究该基因的功能奠定了实验基础。

Objective： To assemble the full-length of human resistin gene in vitro by using oligonucleotides and to construct its eukaryotic expression vector. Methods： According to the gene sequence of resistin （GenBank： AF323081）, 10 oligonucleotides were designed and synthesized,followed by a touch down PCR to assemble the full-length gene. The PCR products were cloned into pSecTag2B vector and confirmed by sequencing. Results： The band of PCR products and gene sequencing showed the insert fragment in pSecTag2B vector was identical to that as designed. Conclusion： The full-length of human resistin coding sequence was successfully assembled and amplified by touch down PCR, and a resistin-expressing eukaryotic vector was constructed.