摘要
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.
出处
《浙江大学学报(医学版)》
CAS
CSCD
2007年第6期588-591,609,共5页
Journal of Zhejiang University(Medical Sciences)
基金
浙江省科技厅科研基金资助项目(2003C33031)