NYD-SP5基因发夹结构小干扰RNA真核表达载体的构建

Construction of eukaryotic vector of small hairpin interfering RNA against NYD-SP5

目的:NYD-SP5基因是在人睾丸cDNA微阵列差异杂交的基础上,克隆的一个新的成人睾丸高度表达的基因。它由3598个核苷酸组成,包括一个编码1027个氨基酸的开放阅读框架。NYD-SP5基因是一条人-小鼠同源基因。结构域分析推测NYD-SP5蛋白是一种跨膜蛋白。本研究构建NYD-SP5基因发夹结构小干扰RNA(shRNA)表达质粒,拟为下一步建立基因NYD-SP5的转基因小鼠模型及研究该基因的功能奠定基础。方法:根据NYD-SP5基因mRNA序列,设计有小发夹结构的4条寡核苷酸序列,克隆到空载体pGPU6/GFP/Neo中,构建重组质粒,同时设计构建分别针对人GAPDH的干扰质粒作为阳性对照,不针对任何特异基因的质粒作为阴性对照。通过酶切鉴定和测序验证阳性克隆。结果:经重组质粒筛选及测序鉴定证实,重组质粒与设计的shRNA转录模板序列相同,提示重组质粒构建成功。结论:利用RNA干扰技术路线可成功构建NYD-SP5的发夹结构小干扰RNA表达载体。

Objective：NYD-SP5 is a newly cloned gene highly expressed in human testes,which consists of 3 598 nucleotides including a 1 027-amino acid open reading frame.It is a human-mouse homologous gene.The domain analysis indicated that the NYD-SP5 protein is a transmembrane protein.This study aimed to design and establish recombinant plasmids of small hairpin interfering RNA （shRNA） against NYD-SP5,and to pave the way for the analysis of the function of NYD-SP5 in the testis using the transgenic mouse model.Methods：Four sequences of oligonucleotides with the small hairpin structure were designed based on the NYD-SP5 mRNA sequence.Recombinant plasmids were constructed by cloning these oligonucleotides into pGPU6/GFP/Neo vectors.Interfering plasmids against GAPDH were established as positive controls and those targeting non-specific genes used as negative controls.The positive constructs were verified by enzyme digestion and sequencing.Results：Plasmid screening and sequencing showed the sequences of the recombinant plasmids to be the same as the shRNA transcribed sequences,which indicated the successful establishment of the recombinant vectors.Conclusion：The shRNA expression vector targeting NYD-SP5 could be established successfully.