Study On Functional Domains Of Interacting Proteins Between PMGA1.2and ApoA-I

It is considered that Mycoplasma gallisepticum (MG) is the primary cause of Chronic Respiratory Disease (CRD) in chickens. This kind of disease is widespread all over the world and has resulted in severe economic losses in poultry industry. After entering the chicken in vivo, MG infected host by connecting its adhesive protein (protein Mycoplasma gallisepticum adhesion, pMGA) with surface receptors of respiratory tract mucous membrane. It was found that Apolipoprotein ApoA-I was the interacting protein of pMGA1.2. In this research, The interaction functional domains between pMGA1.2and ApoA-I was studied which will be the foundation of interaction loci study between pMGA1.2and ApoA-I, The main results were as follows.1. It was predicted that pMGA1.2contains four functional domains by bioinformatics. Two FIVAR (Uncharacterised Sugar-binding Domain) domains are located respectively in25-77amino acids and105-166amino acids. Myco-haema (Mycoplasma haemagglutinin Domain) domain is located in171-596amino acids and Galactose-binding domain-like domain is located in491-595amino acids.2. pMGA1.2was divided into four sections according to the predicted functional domains, which are section1:25-77aaâ†'>73-231bp,159bp; section2:105-166aaâ†'313-498bp,186bp; section3:171-491aaâ†'511-1473bp,963bp; section4:491-595aaâ†'1471-1785bp,315bp. Four fragments pMGA1.2-N (N=1,2,3,4)were successfully cloned which contain functional domains of pMGA1.2.3. Prokaryotic expression plasmid pGEX-KG-pMGA1.2-1and pGEX-KG-pMGA1.2-2were successfully constructed. Prokaryotic expression plasmids were transformed into E.coli BL21(DE3), fusion protein of pMGA1.2-1and pMGA1.2-2were taken by1mmol/L IPTG and inducted8h at22℃; After affinity chromatography purification of GST, purified fusion protein of pMGA1.2-1and pMGA1.2-2were95%. Western blot experiment showed that fusion protein of pMGAl.2-1and pMGAl.2-2have obvious ribbon; the empty vector controls of PGEX-KG has no ribbon. It means fusion protein of pMGA1.2-1and pMGA1.2-2have a good immune activity. Virus overlay骗人 otein blot assay (VOPBA) experiment showed that fusion protein of pMGA1.2-1and pMGA1.2-2have obvious ribbon; the empty vector controls of PGEX-KG has no ribbon. It means fusion protein of pMGA1.2-1and pMGA1.2-2can react with purified ApoA-I fusion protein specifically. The protein fragment of pMGA1.2-1and pMGA1.2-2contain the functional domain what is binding with ApoA-I, and it’s consistent with the result of bioinformatics prediction.4. Eukaryotic expression plasmid PG-3(pCDNA3.1-GFP-pMGA1.2-3), PR-3(pCDNA3.1-RFP-pMGA1.2-3), PG-4(pCDNA3.1-GFP-pMGA1.2-4), PR-4(pCDNA3.1-RFP-pMGA1.2-4) were successfully constructed. After transfected into Hela cells, these Eukaryotic expression plasmids produced corresponding red/green fluorescence,and the transfection efficiency of PR-3, PR-4is better than PG-3, PG-4, so PR-3, PR-4and PG-AI (pCDNA3.1-GFP-ApoA-I) were transfected into Hela cells separately or together, the expression of proteins of ApoA-I, pMGA1.2-3, pMGAl.2-4were detected by flow cytometry. The results demonstrated that: compared with separate transfection, the two co-transfected group expression of pMGA1.2-3,pMGA1.2-4and ApoA-I were significantly lower, the difference was significant (P<0.01), that means pMGA1.2-3, pMGA1.2-4between ApoA-I protein have interaction in the cell.The protein fragment of pMGA1.2-3and pMGAl.2-4contain the functional domain which is binding with ApoA-I, and it’s consistent with the result of bioinformatics prediction.