SD大鼠和BALB/c小鼠PRND基因的克隆和序列分析

Cloning and sequence analysis of PRND genes of SD rat and BALB/c mice

以睾丸总RNA为模板,运用RT-PCR方法扩增了SD大鼠和BALB/c小鼠的叠朊蛋白(PRND)基因,并将其克隆到pMD18-T载体中进行测序,运用生物信息学软件对这些序列进行了分析。结果表明获得的SD大鼠和BALB/c小鼠PRND基因的完整ORF片段,基因无内含子,分别编码178和179个氨基酸的前体蛋白。与GenBank登录的其他同品种鼠相应序列进行比较,SD大鼠发生了G187C点突变,相应地引起了G63R的氨基酸改变;BALB/c小鼠发生了G12T、C13G和C528T点突变,但只发生了L5V的氨基酸改变,其余均为同义置换。氨基酸一级结构分析显示2种PRND编码的Dopple蛋白均由氨基端的信号肽、中间的成熟蛋白和羧基端的GPI锚定结合区组成。二级结构预测表明,Dopple蛋白由3个α-螺旋和2个β-折叠片层组成。研究结果为进一步研究Dopple蛋白的结构、功能及其在传染性海绵状脑病发生发展中的作用提供了基础数据。

Prion like-doppel （PRND） genes of SD rat and BALB/c mouse were amplified respectively by RT-PCR from the total RNA extracted from testicles of the two species of animals as templates. The genes were sequenced after cloned respectively to the vector pMD-18T and analyzed by bioinformatics softwares. The results showed that the whole open reading frames （ORFs） of PRND genes of SD rat and BALB/c mouse encode precursor proteins with 178 and 179 amino acids, respectively and without intron. Compared the sequence with other corresponding genes of the same breed in Genebank, the mutation site G187C was found in SD rat, and accordingly, the G63R amino acids changed. Whereas mutation sites G12T, C13G and C528T were found in BALB/c mouse, but only the LSV amino acids changed, and the rest were all isosemantic substitution. The primary structure analysis of amino acids showed that both doppel proteins were made up of N-terminal signal peptide, ms-maturation protein and carboxyl terminus GPI anchor combined region. The secondary structure prediction indicated that both doppel proteins had three α-hehx and two β-sheet. The results provide the basic data for the further study of structure and function of the doppel protein and its role in transmissible spongiform encephalopathies.