[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 anal...[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 D098982）.展开更多
目的克隆信号转导与转录活化子1(signal transducer and activators of transcription,STAT)基因片段并构建其真核重组表达质粒。通过研究Stat1 mRNA序列各组成部分与Stat1基因翻译调控的关系,探讨了Stat1蛋白翻译调控机制。方法采用DN...目的克隆信号转导与转录活化子1(signal transducer and activators of transcription,STAT)基因片段并构建其真核重组表达质粒。通过研究Stat1 mRNA序列各组成部分与Stat1基因翻译调控的关系,探讨了Stat1蛋白翻译调控机制。方法采用DNA重组技术,将用SMART RACE法筛选出的Stat1基因片段克隆到pT-Adv载体,然后将其亚克隆到pFLAG-CMV-2真核表达载体上,构建Stat1 cDNA重组质粒。分别采用磷酸钙转染法、DEAE-葡聚糖转染法和脂质体介导转染法将其转染Hela细胞,并对3种方法的转染效率及各重组Stat1质粒mRNA和蛋白质表达的差异进行比较,以研究Stat1 mRNA结构对Stat1表达的影响。结果构建了CS,C3S,CS/3S,5SFC3S,5SFCS和CL/3L共6个重组Stat1质粒。通过比较发现,磷酸钙转染法、DEAE-葡聚糖转染法的转染效率明显低于脂质体介导转染法,转染的质粒均得到清晰的mRNA和蛋白质表达信号。用INF-α刺激转染5SFC3S重组Stat1质粒的Hela细胞,发现刺激组与空白组Stat1 mRNA转录水平和蛋白质表达量无明显差异,蛋白质合成效率无降低,此结果与野生型Stat1对INF-α刺激不同。结论将重组Stat1质粒转染Hela细胞后,在短暂表达条件下,重组Stat1质粒与野生型Stat1对INF-α刺激反应不同,其原因尚有待进一步研究。展开更多
文摘[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 D098982）.
