应用RACE法分离和克隆猪GPX2基因研究

Study on the Cloning and Isolation of sus scrofa GPX2 Gene by RACE Method

[目的]运用分子生物学技术克隆猪GPX2基因。[方法]以猪十二指肠总RNA为模板，根据人、大鼠、小鼠、牛、狗GPX2基因同源序列分析，设计兼并引物对，采用RT—PCR技术扩增获得了l段330bp的猪GPX2基因序列。根据获得的已知序列分别设计引物，采用3＇-RACE和5＇-RACE技术手段分离、克隆猪GPX2基因。并对所得基因进行基因序列分析。[结果]该试验成功克隆分离了1段长924bp的mRNA序列，该序列包含完整3’-末端，与人、鼠、牛、狗GPX2基因具有较高的序列同源性，并在基因第114～116位置具有编码硒代半胱氨酸残基（Sec）的密码子TGA。[结论]序列分析比对的结果表明克隆的基因就是猪GPX2基因（NCBI GeneBank数据库序列号为DQ98982）。

[Objective]Using molecular biotechnology to clone the sus scrofa GPX2 gene. [Method] Using total RNA of sus scrofa duodenum as template, degenerated primer pairs were designed according to the homology alignment analysis of GPX2 gene of human, rat, mouse, dog and cattle. A sus scrofa GPX2 gene sequence of 330 bp was obtained by RT-PCR application method. Primes were designed respectively according to the known sequence, sus scrofa GPX2 gene was isolated and cloned by 3-RACE and 5-RACE method and analyzed the gene sequence. [Result] A mRNA sequence of 924 bp was successfully cloned and isolated in this research. This sequence contained complete 3＇ end and had higher sequence homology with human, mouse, cattle and dog GPX2 gene, and there was codon called TGA which encoding Sec on the position of No. 114- 116 gene. [Conclusion] Sequence alignment analysis showed that the cloned gene was sus scrofa GPX2 gene （ NCBI GenBank database, the sequence number was DQ98982）.