Preparation And Characterization Of The Monoclonal Antibodies Against Porcine Aminopeptidase N And Analysis Of Blocking Function Of Porcine Aminopeptidase N

Porcine epidemic diarrhea(PED) and Transmissible Gastroenteritis(TGE) causes high fatality rate in seronegative neonatal piglets. the high mortality of infected nursing piglets has resulted in significant economic losses in the China pig industries. This requires us to understand the PEDV,TGEV and theirs related information, especially the disease mechanisms and pathogenesis of PEDV and TGEV. Porcine aminopeptidase(porcine aminopeptidase N papn) is a kind of metal glycoprotein, which widely distributed in the surface of a variety of tissue cells, the main function is the hydrolysis of amide bonds, resulting in the release of different N- neutral amino acids. So it can participate in the reaction of many important processes. It was reported that porcine aminopeptidase N(pAPN) is the receptor for PEDV and TGEV, p APN can bind to PEDV and TGEV specifically, and this binding can be inhibited b y anti p APN antibody.Therefore, the preparation of anti p APN antibody and its biological characteristics of the analysis can provide theoretical basis and experimental data for the effective prevention and treatment of PED and TGE.The natural pAPN protein were used to immunize BALB/c mice, The hybridoma cell lines 3E5 and 2E3 against natural p APN protein that secreted Mc Abs were obtained. The isotype of 3E5 and 2E3 were identified as Ig G1 and I gM. Continuous passages of the hybridomas were detected by indirect ELISA.The result of Western Blot indicated that the monoclonal antibod y could react to SPC protein(the 500aa-963 aa of p APN). The indirect immunofluorescence shows that the binding between PEDV, TGEV and p APN can be inhibited by anti p APN antibody, and the CPE of IEC-2 cells and PK-15 cells after reaction with Mc Ab can be inhibited by it. further more, we got the cellular infection after the adsorption/24 h and 48 h after virus infection, and used the quantitative real time PCR to get its CT, the results shows that, the copy number of virus after interaction with McAb is got reducer. then, we used the different concentration of McAb interact with cells, and got the cellular infection after the adsorption, and used the quantitative real time PCR to get its CT.then we got the results that the less McAb the less blocking action of it. In addition, we infect the different concentration of PEDV and TGEV to know the influence to the blocking action, then, we final discovery that a relationship between the number of viruses and the blocking effectAll these results showed that the Mc Ab against p APN protein were highly specific to detect pathogeny.the Mc Ab against pAPN to make sure the major antibody-binding domains of p APN associated with PEDV and TGEV, and the functional characteristics of the major antibody-binding domains of p APN,also to provide theoretical basis for the pathogenesis of PEDV and TGEV.which is for further design of antiviral drugs, closed virus receptor, prevent virus infection and so on.