The Studies Of The Secretion Of Specific IgA Antibody After Mycoplasma Hyopneumoniae Infection In Pigs

Mycoplasma hyopneumoniae (Mhp) is the causative agent of Swine Enzootic Pneumoniae, a disease which is characterized by highly infective, chronic, high morbidity and low self-mortality.Infected swine has a retarded growth and inefficient food conversion.Mhp destroy the respiratory tract mucosa-cilia barrier, so it can induce the infection of other pathogenic bacteria and increase the mortality.When ppathogens invade the body from the mucous membrane channels,primarily to stimulate the body to produce SIgA mucosal immune antibodies. SIgA antibodies play an important role in mucosal immunity.1 Preparation a lot of standard positive bronchioalveolar lavage fluid (BALF) by utilizing of the preservation of Mhp virulent strain in the lab, and prepared standard negatie BALF. Collected more than 200 portion negative and positive BALF from different areas and different background; this provided powerful references.to the establishment of antibody test methods to M. hyopneumoniae.2 Expressed a variety of immune protein of Mycoplasma hyopneumoniae:P36、P46、 P97R1 in E.coli expression system,making an powfull prepared for ELISA establishing.3 Established single protein indirect ELISA methods, and the methods are optimized. And the optimized program is as follows:Coating protein was diluted to 2μg/mL, coated overnight at 4℃ after incubation for 1 h at37℃ on 96-well microtiter plates. And the plates were subsequently blocked 1h with 1% casein.Then incubated for 2h with samples diluted to 1:20 by PBS containing 0.05%Tween-20 (PBST), the working concentration of HRP-goat anti-swine IgA was 1:10000, the chromogenic time was incubated at 37℃ for 12min. An indirect ELISA methods to detect special IgA in serum are optimized.And the optimized program is as follows:Coating protein was diluted to 4μg/mL, coated overnight at 4℃ after incubation for 1 h at37℃ on 96-well microtiter plates. And the plates were subsequently blocked 1h with 1% casein.Then incubated for 1h with samples diluted to 1:20 by PBS containing 0.05% Tween-20 (PBST), the working concentration of HRP-goat anti-swine IgA was 1:10000, the chromogenic time was incubated at 37℃ for 10min.4 Established SIgA protein indirect ELISA methods, and the methods are optimized. And the optimized program is as follows:mouse anti-pig IgA antibody protein was diluted to 1μg/mL.The protein was coated overnight at 4℃ after incubation for 1 h at37℃ on 96-well microtiter plates. And the plates were subsequently blocked 2h with 1% casein.Then incubated for 30min with BALF diluted to 1:100 by PBS containing 0.05% Tween-20 (PBST), the working concentration of HRP-goat anti-swine IgG was 1:15000, the chromogenic time was incubated at 37℃ for 5min.5 In this study,P97R1,P46,P36-specific SIgA in nasal swabs were respectively measured by indirect ELISA following challenged by Mhp organization virulent and found that the regulation of P97R1, P46, P36 specific SIgA antibody secretion is consistency.To infection pigs,specific SIgA antibody was production six days following challenge.And to the uninfection pigs,there is a falling down untile two days after challenged,and the goup.In a word,all the there antigen protein have the ability to coat for ELISA detecting.As to serum,P97R1 specifical IgA antibody was producted a week after challenged and increasing during the 28 days after challenged.But P46 and P36 special IgA antibody nearly not producted.