南方农业学报2011，Vol.42Issue(9)：1136-1143,8.
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
In silico cloning of partial EST sequence from cDNA library of normal swine peripheral blood lymphocytes and its identification
摘要
Abstract
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.关键词
猪/外周血淋巴细胞/电子克隆/表达序列标签( EST)Key words
swine peripheral blood lymphocytes/ in silico cloning/ expression sequence tag (EST)分类
农业科技引用本文复制引用
李晓宁,罗廷荣,苏丽娟,尹珊,李晓泉,章民,李延生,孙石开,蔡新斌,杨坚..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[J].南方农业学报,2011,42(9):1136-1143,8.基金项目
国家“863”计划项目(2007AA10Z160) （2007AA10Z160）
国家自然科学基金项目(31060336) （31060336）
广西科学研究与技术开发计划项目(桂科攻0480001) （桂科攻0480001）
广西大学科学技术研究重点基金项目(2003ZD01) （2003ZD01）
