Cloning And Sequence Analysis And Expression Of Nanyang Bovine BoLA-DRA

The prevention and therapy of Bovine disease play a very important role in the healthy development of cattle industry. It was confirmed that a close relationship exist between the cattle disease particularly infectious diseases occurrance and the body's own immune function. Recent researches on modern immunology demonstrate that there is a causal relationship between individual genetic type and the level of immune function.In other words, individual genes are controlled by the immune function. The gene which Determine individual immunity was known as the major histocompatibility complex(MHC). As MHC was closely associated with the disease, It was attracted the most attention. At present, the composition of MHC has been basically clarified on the human and mouse genes, however ,it is far from clear that the basic composition of MHC in animals, especially economic species, which had an effect on the development of animal disease control and genetic breeding areas. Therefore, MHC composition analysis will play a significant role in the future.In this paper, using molecular biology techniques and bioinformatics, Nanyang cattle BoLA-DRA gene was cloned by RT-PCR and structured eprokaryotic and eukaryotic expression vector respectively,which bulid a foundation for breeding and disease resistance research in the future.(1) BoLA-DRA gene's coding region was amplified by RT-PCR from the spleen of Nanyang cattle ,purifiing PCR product, then connect to the pGEM-T vector, transform into E. coli DH5α, to select pGEM-DRA positive clones, meanwhile, identifying by restriction endonuclease and sequencing. The results showed that the cloned genes were 775bp and the sequence homology of 99% through comparing the genes in the GenBank. the corresponding derivation amino acid results were compared with 83%. the nucleotide sequence homology comparison showed that sheep of 96%, 86% of horses, wild boar 85%, 84% human. the corresponding derivation amino acid results were 82%, 83%, 83%, 83%.And the encoded 253 amino acid residues of polypeptide chains, 1-24 amino acids are signal peptide sequence, following by the mature peptide sequence.(2)Using restriction endonuclease Xho I / BamHI , the recombinant T vector plasmid by sequencing and prokaryotic expression plasmid PGEX-4T-1 were digested respectively,linked by T4 ligase, transformed into E. coli BL21 (DE3) Competent cell. Through DNA sequencing, recombinant plasmids contain the target gene. Inducing by IPTG(isopropyl-β-D-thiogalactopyranoside) of recombinant gene in Escherichia coli, The results of SDS-PAGE (polyacrylamide gel electrophoresis) analysis ,showed recombinant protein were more when OD value 0.4, IPTG concentration 1mmol / L, time of 4h. Molecular weight 54.4ku. the same size as the expected.Western-blotting analysis also indicateed that fusion protein retained the antigenicity of the GST tag. It showed that the gene could be expressed in E. coli.(3)Double digestion with the same method ,the cloned fragment was conected into the eukaryotic expression vector pEGFP-C1, the recombinant plasmid was extracted after positive clones were screened. And the enzyme digestion and sequencing results revealed the successful construction of eukaryotic expression vector. bovine fibroblasts were transfected by liposome-mediated . Positive cells were selected by G418. The results through reverse transfection microscope are showed green fluorescence distributed throughout the target cell uniformly. Detectting by RT-PCR, genes were successfully transfected into fibroblast cells.In summary, this research cloned bovine BoLA-DRA gene in Nanyang sucessfully, aqucired the prokaryotic expression product of the BoLA-DRA gene. This gene can be expressed in cattle fibroblasts through the reseach at the molecular level.It provide the foundation for the study of function on BoLA-DRA gene .