| Interferon-gamma (IFN-γ) is a kind of glycoprotein generated by activated T cells and NK cells and could also be produced and secreted by T cells which were stimulated by mitogen or special antigen. Besides the function of antivirus, anti-intracellular bacteria and parasitic protozoa, anti-tumor, IFN-γhas also the distinct immune regulation function. It can promote expression of MHCⅡantigens, enhance the interaction between antigen present cells (APC) and T cells, accelerate differentiation of Th cells and cytotoxicity T lymphocyte (CTL), and so on. IFN-γposses strict species-specificity and the activity would be depressed or missed if it were in common use in different species. IFN-γneeds to form homodimer through combination of two monomer chains to have the bioactivity since the monomer chain do not have the bioactivity. As a product of host immune response, IFN-γis a necessary component to eliminate intracellular pathogen and is closely related to the nosogenesis and development of disease, on the other hand, if IFN-γwere over secreted, it would cause pathological reaction. Accordingly, IFN-γhas an extensive application in the aspects of diagnosis, therapy and disease prevention, however, IFN-γhas some side effect including fever, shakes, nausea, and so on. Furthermore, some side effect could cause nonreversible damage. All of these limited the clinical application of IFN-γand it is important to develop a new type IFN-γwith lower side effect. IFN-γexerts the biological activities through binding to its receptors which will transform the signal into intracellular. So the receptor play a critical role and the study on IFN-γreceptor (IFNGR) would be benefit to demonstrate the mechanism of IFN-γand find the new methods for treatment and prevention of clinical disease.In this paper, the cloned pIFN-γgene was reconstructed to cut off the signal sequence and fused with GST presented in the pGEX-4T-1 vector. The recombinant gene was expressed in prokaryotic expression system. A series of study was carried out such as induction conditions, recombinant protein purified methods and eventually the recombinant protein was obtained with the purity of up to 95% and the yield of 0.4~0.5 g per litre culture medium. In vitro experiment indicated that the purified protein had a higher bioactivity and could efficiently inhibit the replication of PRV and FMDV. In addition, the recombinant protein had a good stability and lower side effect. The effect of GST-IFN-γanti-toxoplasma gondii was studied and the results showed that pIFN-γcould induce the anti-toxoplasma gondii effect of the porcine abdominal macrophage cell, and the function of anti-toxoplasma goddii presented obviously with the increase of pIFN-γdose. But it could not induce PK15 cell to generate anti- toxoplasma goddii effec. pIFN-γhas the ability of inducing immortalized cells (PK15 cell) apoptosis. At the high concentration of pIFN-γ, the level of PK15 cell telomerase significantly decreased and apoptosis appeared, such as cell rounding and falling off and the"ladder pattern"of the DNA, pyknotic nucleus and apoptotic body appeared by fluorescein stain. However, at the low concentration of pIFN-γ, the level of PK15 cell telomerase significantly increased. This provided the direction of clinical treatment of the tumor with pIFN-γ. The BALB/C mice were immunized with purified GST-pIFN-γand then the spleen cell and SP2/0 were fused. The fusion cell was selected by GST-pIFN-γand pIFN-γwith His tag as antigens. Finally 2 hybridoma cell strains which secreted anti-pIFN-γmonoclonal antibody were obtained. The identification of the monoclonal antibody showed all of them belong to IgG2a subclass withκtype light chain. Analysis of the caryotype, antibody stability and antigen site showed that antibody was stable. After clustal alignment of IFNGR gene sequence from many species, conservative region was selected as primer to amplify porcine IFNGR gene. Porcine peripheral blood lymphocyte was isolated and stimulated in vitro, then total RNA was extracted. Porcine IFNGR two chain genes were cloned by RT-PCR method. Sequence analysis showed that porcine IFNGR1 ORF had 1413 bp, encoding 470 amino acid and porcine IFNGR2 ORF had 1110 bp encoding 369 amino acid. Sequence comparison showed that they were obviously different from those of other species and that is the possible reason that IFN-γpossesed species-specificity. The primers used to amplify extracellular region of IFNGR was designed based on the protein structure prediction. The amplified fragment was expressed in E.coli system. The study of relationship between IFNGR extracellular region and character of pIFN-γantivirus was carried out. The results showed IFNGR1 extracellular region decreased the antivirus activity of pIFN-γ, but IFNGR2 extracellular region increased the antivirus activity of pIFN-γ. These results proved that 2 chains of IFNGR has the function of binding pIFN-γ. In addition, according to the results, it can draw a conclusion that porcine IFNGR1 occupied mainly position in signal transmit, but IFNGR2 had occupied secondary position in signal transmit or not function of signal transmit.
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