正常猪外周血淋巴细胞cDNA文库部分EST序列的电子克隆及验证

摘要

[目的]探讨从EST序列电子克隆全长基因序列的有效性,为今后快速克隆和研究免疫相关基因提供新方法.[方法]以正常猪外周血淋巴细胞cDNA文库测序所得的EST序列作为起始材料,应用生物信息库对文库中的部分EST序列进行电子克隆,并用RT-PCR验证其准确性.[结果]电子克隆正常猪外周血淋巴细胞cDNA文库序列,共获得20条片段重叠群( Contigs),经基因注释,证实其均得到了有效延伸,具有完整开放阅读框(ORF),其中已知的猪基因序列14条,猪新基因序列6条；从已知猪基因序列和未知猪新基因序列中各抽取1条感兴趣序列(EST contig110和ESTcontig133)进行全长cDNA预测,发现电子克隆所得序列EST contig110和EST contig133的预期扩增片段(ORF)与实际扩增片段(ORF)基本一致,核苷酸相似性在98.0％以上,氨基酸相似性在93.0％以上.而对EST contig133的ORF序列猪的60S核糖体蛋白L18a亚基)进行基本特性及结构预测,发现ORF133蛋白为非分泌型蛋白,由6个折叠和6个螺旋结构组成,含有8个不同的磷酸化位点.[结论]从EST序列电子克隆全长基因是可行的,为今后快速克隆和研究免疫相关基因提供了参考.%The present experiment was conducted to obtain genetic information from cDNA library of normal swine peripheral blood lymphocytes and to clone whole-length gene sequence from expressed sequence tag (EST) in order to provide new method for rapid cloning and immune-related genetic research. [Method]Partial ESTs from the cDNA library of normal swine peripheral blood lymphocytes were analysed and cloned using in silico cloning techniques and bioin-formatic databases, and the otained sequence was indentified through RT-PCR using specific primers. [Result]By using in silico cloning, the cDNA library sequence of normal swine peripheral blood lymphocytes, 20 contigs were abtained. After assembling and annotating, the contigs were proved to extend effectively and had full length of ORFs. Out of which, 14 gene sequences were consistent with the genes known in the previous studies and 6 genes were new. One known (EST con-tigllO) and another unknown (EST contigl33) EST of swine were chosen to predict the full-length cDNA annotation. It has been observed that the predicted amplified fragment of obtained EST contigl 10 and EST contigl33 were consistent with the in silico amplified fragments, and the similarity of nucleotides and amino acids was over 98.0 and 93.0%. The characteristic and secondary structure of ORF sequence in ET contigl33, a novel swine protein 60S ribosomal protein LI8a subunit was further predicted and it was found that ORF 133 protein was of non secretion type, and consisted of 6 folding and 6 helical-structures, and had 8 phosphorylations. [Conclusion]In silico cloning of full-length genes from ESTs is a feasible method, which provided reference on accelerating immune-related genes cloning and functional study.