Construction of cDNA Expression Library of Porcine Cytokine and Cloning of Related Genes

In order to clone and study the cytokine and related genes of porcine,a cDNA library of porcine cytokine was constructed.The mononuclear cells were isolated from the peripheral blood and lymph nodes of healthy porcine and were stimulated by PHA+LPS at different time.The total RNA were extracted and the mRNA were isolated.Single-strand cDNA and double-strand cDNA were synthesized from mRNA,then ligated to directional EcoRI and HindⅢ linkers.cDNA were ligated into λScreen vector after size fractionating by gel filtration and packaged in vitro.The cloning efficiency was evaluated and the length of the cDNA fragment was assayed by PCR.Using the amplified library as template DNA,2 pair primers were designed according to the sequence of the porcine IL2 and IL4,then the gene were amplified by PCR.The results demonstrated that a cDNA library of porcine cytokine has been constructed and the IL2 and IL4 gene were amplified successfully.The tilter of the cDNA library was 8×10~5 pfu/mL and the length of inserts was about 300~2 000 bp.It is helpful in the further study on screening novel cytokine and related genes.