Cloning, Prokaryotic Expression And Construction Of Eukaryotic Expression Vector Of IFN-γ Gene In Ailuropoda Melanoleuca

Abstract: Interferon-gamma(IFN-γ), produced mainly by Th₁ cells, CD₈⁺T cells and natural killer cells, plays a critical role in anti-virus, anti-tumor, and immune modulation. Its immune modulation includes activating macrophages, up-regulating the expression of MHC classâ… and classâ…¡molecules, prompting the presentation of antigens, and inducing Th₁ cell growth and differentiation. The level of IFN-γis an important index to the cellular immunity.Giant panda is a characteristic and precious wildlife in our country,it is named "national treasure" in China.The investigation from Nation Forestry Head Office indicates that the number of wild Giant panda is about 1590, captive Giant panda is only 189, they are still faced to extinction. The long-term captive giant pandas ,especially baby or aging Giant panda with hypoimmunity, are easily infected by virus, germ and parasite.The way of preventing or curing disease is mainly by drug,no special vaccine,but the drug would benoxious for giant panda with adverse reaction and the pathogen would become drug-resistant. All of these are bad for disease prevention and breeding of Giant panda,so we need new technologies to breed giant panda and control diseases, the way of improving autoimmunity by cytokines may be one effective rescueing measure relatively.1. By reverse transcription polymerrase chain reaction(RT-PCR), Giant panda IFN-γgene was cloned from total RNA which was extracted from ConA-stimulated-cultured peripheral blood lymphocytes of giant panda, the amplified products of PCR were cloned into PGEMT vector.Alter identification and sequencing,the sequence analysis revealed that the open reading frame of giant panda IFN-γgene contained 498 nucleotides which encoding 166 amino acids,the signal peptide contained 19 amino acids and the mature IFN-γgene was composed of 147 amino acids. Compared with other mammals in Genbank, including dog, cat, human, pig, cow, horse, goat, rabbit, rat and so on, the homology of nucleotide sequence was between 57.7 % (rat) and 93.2%(dog), the homology of amino acid sequence was between 46.8%(rat) and 92.8 %(dog). Phylogenetic analysis based on the nucleotide sequence from the IFN-γgene showed that Giant panda has the most closed relationgship genetic distance with dog.2. In this study, pET32alFN-γ, the prokaryotic expression plasmid with the complete sequence of IFN-γgene ,was constructed and expressed the corresponding protein. The complete sequence of IFN-γgene from PGEMT was subcloned into prokaryotic expression vetor pET32a,then a recombinant plasmid pET32aIFN-γwas obtained. The plasmid pET32aIFN-γwas transformed into E.coli.BL21 (expression E.coli ).The target protein was detected by SDS-PAGE analysis after induction by IPTG. Then the special band of fusion expressed protein was roughly obtained,and it was approximately 34.6KDa ( target protein and E.coli protein were 17.4 KDa and 17.2 KDa respectively ).3. The complete sequence of IFN-γgene from PGEMT was double cleaved by EcoRI and XhoI, the cleaved product was reclaimed by DNA UNIQ-10 spin column purification kit after gel electrophoresis,then ligated with pcDNA3.1(+) plasmid double cleaved with the same enzyme and transformed into E.coli, pcDNA3.1(+)IFN-γwas identitied by cleaving analysis and sequencing,the result indicated that the eukaryotic expression plasmid was constructed successfully.IFN-γgene was cloned and expressed in prokaryocyte successfully, and the eukaryotic expression plasmid was constructed. It would provide some evidence for the evolutional relationship of different species, Meanwhile, it provided a basis for further research on immunological and molecular biologic activity and its amplification of recombinant IFN-γprotein.