Cloning And Expression Of Tumor Necrosis Factor ? Gene Of Macaca Mulatta Lasiotis

Tumor necrosis factor alpha (TNF-a), as a potent cytokine, plays different roles in immune response, inflammation, injury, cell proliferation, cell differentiation and apoptosis. TNF-a and its inhibitors are essential in early diagnosis, prognosis and treatment of diseases. Macaca mulatta lasiotis, one of the most valuable experimental animals in researches of the effects of TNF-a biologicals on many kinds of diseases, has been used to construct primate model for a variety of critical human illnesses. So far, there has been many researches about TNF-a in terms of human and other animals, but little in macaques, and also there is no relevant report on the expression of macaca mulatta lasiotis TFN-a (MmTFN-a) in domestic. In this study, we get the MmTNF-a gene by gene cloning first, then construct MmTNF-a expression strain in prokaryote and eukaryote, and finally express the protein. This research lays a foundation for further study about the function of the protein activity and structure, monoclonal antibody production and development of kit. In addition, it provides information for the protection of the subspecies of macaques in Western Sichuan. 1.Cloning and expression of macaca mulatta lasiotis TNF-a in Escherichia coliDesign and biosynthesize a pair of primer based on the sequence of macaque TNF-a mRNA in NCBI/GenBank. The MmTNF-a gene is amplified by using specific primers and insert it to pMD-19T simple vector, then generate recombinant strain DH5a/pMD-19T-MmTNF?. the positive clones will identified by PCR and sequence analysis. The amplified MmTNF-a gene is about 702 bp, and the sequence in BLAST shows that MmTNF-a gene has the homology 99% compared with macaques TNF-a gene which posted on GenBank (NCBI Reference Sequence:001047149.1 NM).Design and biosythesize expression primers based on successfully sequenced MmTNF-a gene, use subclone to construct the prokaryotic expression vector (pGEX-4T-1-MmTNF-a). which was screened by PCR and identified by double enzyme, and express it in E. coli BL21 (DE3).The fusion protein has been obtained, which is 52 kDa and mainly expressed in the form of inclusion body after 4h inducible expression the prokaryotic expression strains E.coliBL21(DE3)/pGEX-4T- 1-MmTNF-? in 0.1mmol/L IPTG at 37?, consistents with the expected size, and WB analysis confirms the recombinant protein possesses good immunoreactivity. 2. Construction and expression of macaca mulatta lasiotis TNF-a. in eukaryoteInsert MmTNF-a cloning gene into the pPIC9K polyclonal loci, use Sal I restriction enzymes to linearize recombinant plasmid pPIC9K-MmTNF-?, and put it into GS115 by electro-transformation. After transformation, the positive clones were screened by PCR, the transformants phenotypes were identified by PCR> MM and MD plate, and the high copy recombinant strains were screened by G418 gradient plate. Use methanol to induct recombinant strain pPIC9K-MmTNF-a/GS115, and analyse the results by SDS-PAGE and Western-blot. The phenotype of eukaryotic expression strains GS115/pPIC9k-MmTNF-a is Muts. After 72h continuously inducing by 1%methanol at 30?, the interest protein, which is about 26 kDa, is detected from supernatant. And WB shows it has a good immunoreactivity.