NYD-SP5基因发夹结构小干扰RNA真核表达载体的构建

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。

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

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.