Construction Of CDNA Library Of Porcine Cytokine And Screening, Cloning Of Important Functional Genes

Constructing and screening cDNA library is a reliability method to obtain new genes. There is no report about construction of the cDNA library of porcin cytokine at the present time. So the cDNA library of porcin cytokine was constructed and some important cytokine is screened in our study.1. The mononuclear cells were isolated from the peripheral blood and lymph nodes of healthy porcine and were stimulated by PHA+LPS at different time and different dose. Then , abundant cytokine were transcripted, expressed and secreted. Total RNA was extracted and mRNA was isolated from the mixed sample. The cDNA library was constructed according to the instruction of kit. The tilter of the library , recombinant efficiency and the length of cDNA fragment were assayed. The results demonstrated that a cDNA library of porcine cytokine has been constructed successfully. The tilter of the primary library was 8×l0~5pfu/mL and the recombinant efficiency was 93.3%. Data showed that the length of inserts was about 300bp~2000bp.2, In order to screen and clone genes from the library and establish a new membrane in-situ hybridization technique , a random gene was selected and labled with DIG. In order to identify whether the system is reliable, it was hybridated with itself phage, the control was set at same time. The result showed that this system is highly sensitive. The target clone of 0.4pg/μL, could be tested. By contrast with control, the chance of false positive result is low. It can be used as a effective nucleic acid hybridization method to screening the new genes.3, The porcine IL-2 and IL-4 genes were amplified from the library by special primer, the PCR product was cloned into the pMD18-T vector and sequenced. The sequenced result analyzed using DNAstar software showed that the ORF of IL-2 is 465 bp, encoding a pre-protein of 154 amino acids with MW 27.4ku. it has 99.1% homology with the IL-2 gene sequence obtained from GenBank. The ORF of IL-4 is 402bp, encoding a protein of 133 amino acids with MW 15ku, it has 99.3% homology with the IL-4 gene sequence obtained from GenBank. The result showed that the full-length IL-2 and IL-4 genes have been cloned.