Research On Signaling Pathway Mediated By GP5 Protein Contains The ITIM Motif

Porcine reproductive and respiratory syndrome(PRRS) is viral infectious diseases caused by porcine reproductive and respiratory syndrome virus(PRRSV) which causes serious damage to the pig industry.when the body infected PRRSV,The body only induced delay, low levels of neutralizing antibodies, and virus persistent infection in the body cause immunosuppression.PRRSV structure protein and the nonstructural proteins play an important role in the immune suppression. GP5 is the main structural protein induced neutralizing antibodies which are encoded by PRRSV ORF5,contains four glycosylation and six antigenic determinant.GP5 varies the largest in PRRSV structural protein, which encods 201 amino acids, a total of 603 bp, research and analysis of the sequence found that the 229-246 amino acid residues contains ITIM inhibitory motif.The process of the body’s immune response to the external environment can maintain the proper strength depends on the adjustment of the negative feedback system, and immune receptor tyrosine motif inhibition(ITIM) play a decisive role in the negative regulation. Tyrosine phosphorylation of ITIM motif is the most critical step in the process of immune suppression signal transduction,the activation of ITIM can be the potential binding sites of tyrosine phosphatase(SHP-l,SHP-2, SHIP) with SH2 domain. When the inhibition pathway was activated, ITIM can combine with the SH2 domain of SHP-l/SHP-2 and make the SHP- l/SHP- 2 activate. In order to reveal the mechanisms of PRRSV escape innate immune, this paper carried on the preliminary exploration and research on GP5 protein of including ITIM motif mediated signaling pathway.Specific content as follows:In experiment one,c DNA sequences of SHP-1 domain and SHP-2 domain were cloned from the Marc145 cells by RT-PCR.The sequences were then subcloned into the prokaryotic expression vector p ET-32 a to construct recombinant expression plasmid p ET-SHP-1 and p ET-SHP-2 and expressed in BL21. After induction with IPTG, we obtained the fusion proteins, which molecular mass were about 37.5,40.1KDa respectively. The polyclonal antibodies were obtained by immunizing mouse with the two purified fusion proteins and analyzed by indirect ELISA and Western blotting.The results of ELISA showed that the antibody titers were both 1:12800. Western blotting results indicated that the prepared polyclonal antibodies could bind to the recombinant proteins specifically,suggesting the recombinant proteins had good immunogenicity. The two-step method of ammonium sulfate precipitation method and the DE-52 ion exchange chromatography technology was used to extract and purify to obtain two specific antibodies of high purity. The results verified the presence of SHP-1and SHP-2 from Marc145 cells,and the structure domains of SHP-1、SHP-2 were cloned expression. Foundation was laid for following experiment and further study.In experiment two, through research and analysis of the GP5 gene sequences, combining the need for subsequent experiments, RT-PCRtechnique to clone GP5 gene c DNA sequence and sequencing analysis comparison, the GP5 gene cloning and expression, through gene recombination technology and overlap extension PCR technology successfully build the GP5 gene eukaryotic expression vector pc DNA-GP5、pc DNA-GP5△229-246.In experiment three, by PRRSV infection Marc145 cells and by liposome transfection method will successfully build pc DNA-GP5、pc DNA-GP5△229-246 eukaryotic expression plasmid transfection to Marc145 cell, in accordance with the real-time PCR method of oneself Established to detect Marc145 cells the m RNA transcription level of SHP-1、SHP-2、IFN-β、NF-κB, Using RT-PCR and Western blotting technology from m RNA and protein level Mutual authentication GP5 and GP5△229-246 effectively expressed in Marc145 cell. With our laboratory set up real-time PCR method to detec pc DNA-GP5、pc DNA-GP5△229-246 transfection Marc145 cells,SHP-1、SHP-2、IFN-β、NF-κBm RNA transcription level results show that the influence of the ITIM motif can significantly reduce the transcription level of SHP-1, explain ITIM motif can combined with SHP-1 reduceits transcription level to inhibit the expression of SHP-1, ITIM motif in general can significantly increase transcription level of SHP- 2 and significantly increased at early stage, Show ITIM motif can combined with SHP- 2 to raised its transcription level to activated the SHP-2, make its downstream signals mediated phosphorylation to play a role of function; Relative to the IFN-β, Experimental results show that ITIM motif can reduce IFN-β transcription level significantly in 24 h, but, TIM motifcan can rise IFN-βtranscription level significantly in36 h,48h tends to be stable and have lower trend; Showed the ITIM motif to its inhibition has a certain time limit. However, Research for the NF-κB, ITIM motif can reduce the NF-κB transcription level significantly in different period of time and had obvious inhibitory effect in NF-κB. Therefore, containing ITIM motifof GP5 protein may depend on the ITIM phosphorylation manner interactedwith the host intracellular tyrosine phosphatase SHP-1/SHP-2, Promote SHP-1/SHP-2interacted with TRAF6 protein, Thus inhibiting TRAF6 ubiquitin, thereby inhibiting the NF-κB and the activation of MAP kinase. the ITIM motif combined with SHP-1 reduce its transcription level, the ITIM motif combined with SHP-2 raised its transcription leveland activate the SHP-2, The ITIM motif had obvious inhibitory effect in NF-κB in different periods, showed that the SHP-2 may play a major role in negative control in Marc145 cells.