In silico cloning of partial EST sequence from cDNA library of normal swine peripheral blood lymphocytes and its identification

【Objective】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 bioinformatic 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 contig110) and another unknown (EST contig133) EST of swine were chosen to predict the full-length cDNA annotation. It has been observed that the predicted amplified fragment of obtained EST contig110 and EST contig133 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 contig133, a novel swine protein 60S ribosomal protein L18a subunit was further predicted and it was found that ORF 133 protein was of non secretion type, and consisted of 6 folding and 6 helicalstructures, 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.