Construction Of Grunniens PBMC CDNA SSH Libraries And Sequence Analysis Of Major Functinal Moleculars

Functional molecules of peripheral blood immune-related cells play an important role in immune regulation, antimicrobial, antitumor and signalling,et.al. Peripheral Blood Mononuclear Cells (PBMC) were stimulated by concanavalin A(ConA) and lipopolysaccharide(LPS) as mitogen in order to secrete a number of cytokines.These cytokines are regulated by a complex signaling network,which is composed of many molecules. In this study,we have undertaken suppression subtractive hybridization (SSH) and constructed differentially expressed cDNA library of Peripheral Blood Mononuclear Cells (PBMC) to screen and clone differentially expressed genes in respose to LPS and ConA in Bos grunniens.The identified differentially expressed genes will give insight into immune regulatory mechanisms in bos grunniens.In the present experiment,we used Peripheral Blood Mononuclear Cells (PBMC) of bos grunniens as experiment material: cDNA from the treated with LPS and ConA(as tester) was hybridized twice with untreated cDNA of control (as driver); after double hybridizations the products were used for suppression PCR twice; the secondary PCR products were inserted into pGEM-T Easy vector and transformed into E. coli JM109 competent cells; identification of the inserted cDNA fragments in subtractive library was done using PCR. The results showed that there were inserted fragments of 200～1000 bp in 16 randomly selected positive clones. Meanwhile, in order to confirm the subtraction efficiency of the SSH cDNA library, 6 non-repeatedly sequence were selected randomly to semi-quantitative RT-PCR. It was showed that there were 5 inducible differential expression molecules and 1 inducible specific expression genes. Positive clones were sequenced and BLASTn analyzed, and we obtained 27 differential expression genes ,of which there were 3 novel ESTs,and others were involved in protein biosynthesis,signaling,apoptosis,cell proliferation and differentiation,mRNA transcription regulation,metabolism and cell and organism denfense and so on.The full-length genes CXCR4 and ProTαwere amplified by specific primers. The PCR products were cloned into pMD18-T vector and sequenced. The sequenced result analyzed using DNAstar software showed that the ORF of CXCR4 was 1062 bp, encoding a pre-protein of 353 amino acids. It shared 99.6% homology with the CXCR4 gene retrieved from GenBank. The ORF of ProTαwas 333 bp, encoding a protein of 110 amino acids, It shared 98.2% homology with the ProTαgene retrieved from GenBank. It was showed that the full-length CXCR4 and ProTαgenes were cloned successfully, which may be helpful to further study their structures and function and to illuminate molecular mechanisms of immunity and signaling.