Establishment Of Antibody Detection Methods And Determination Of The Antibody Growth And Decline Law Induced By Porcine Contagious Pleuropneumonia Bacterin-toxiod Vacine

Porcine contagious pleuropneumonia is a serious respiratory infectious disease caused by Actinobacillus pleuropneumoniae(APP),mainly manifesting pulmonary hemorrhage,necrosis and fibrinous exudation.Porcine pleuropneumonia has a high mortality rate when acute infection occured,which has caused significant economic losses to the pig industry around the world.Vaccination is an important way of preventing this disease,and has an outstanding effect in decreasing the use of antibiotics and drug resistance.APP can secrete Apx?,Apx? and Apx?,which are fatty acylated proteins,the most important virulence factor and protective antigen of APP.Our laboratory mixed the three recombinant non-fatty acylated Apx toxin proteins(rApx?A,rApx?A and rApx?A)with inactivated serotype 1 APP bacterial antigens to produce the "Porcine contagious pleuropneumonia bacterin-toxiod vaccine".The vaccine immunized pigs produced high multi-serotype cross-immunity protection without obvious side effects.This study aims to establish three ELISA antibody detection methods of rApx?A,rApx?A,rApx?A.Then conducted the immunization test of the Porcine contagious pleuropneumonia bacterin-toxiod vaccine.Finally,the growth and decline laws of antibody after immunization were determined,which can provide scientific basis on the evaluation of the immunity of the vaccine and formulation of immunization procedures.The main research results are as follows: 1.Establishment of rApx?A,rApx?A and rApx?A indirect ELISA methodsThe recombinant plasmids pQE-Apx?A,pQE-Apx?A and pQE-Apx?A were constructed in our laboratory.They were transformed into the competent E.coli M15 cells respectively.And then the cells were induced to express rApx?A,rApx?A and rApx?A recombinant proteins,which were purified to establish three ELISA antibody detections.The conditions of ELISA method were optimized with good repeatability.In the rApx?A indirect ELISA detection method,the rApx?A antigen was coated overnight at 4? with protein concentration of 22.57 ?g/m L.Then 5% BSA was used to block the plates for 2 hours at 37?.The serum was diluted at 40 times,incubated at 37? for 45 min.Enzyme-labeled secondary antibody was diluted to 5000 times,reacted at 37? in the incubator for 30 min.TMB substrate was applied to allow the colour to develop for 30 min in dark.Under the optimal reaction conditions,the cutoff value is 0.267.rApx?A antigen coating concentration was 39.88 ?g/m L.After coating overnight at 4?,1% BSA was used to block the plate for 2h at 37?.Serum was diluted at 40 times and incubated at 37? for 30 min.Enzyme-labeled secondary antibody was diluted at 5000 times,reacted at 37? incubator for 30 min.TMB substrate was applied to allow the colour to develop for 30 min in dark.Under the optimal reaction conditions,the cut-off value is 0.447.rApx?A antigen coating concentration was 11.10 ?g/m L,coated overnight at 4?.Then 5% BSA was used to block the plates for 2 hours at 37?.Serum was diluted at 80 times and incubated at 37? for 30 min.Enzyme-labeled secondary antibody was diluted at 5000 times,reacted at 37? incubator for 30 min.TMB substrate was applied to allow the colour to develop for 30 min in dark.Under the optimal reaction conditions,the cut-off value is 0.283.2.Determination of the antibody levels after immunizationIn the porcine contagious pleuropneumonia bacterin-toxiod vaccine immunization test,9 piglets at the age of 50-days-old piglets were used,in which 3 piglets were injected with PBS as control group,and 6 piglets were injected with bacterin-toxiod vacine as the vaccination group.They were injected behind the ear intramuscular.PBS control and subunit-inactivated vaccine were injected 28 days after the first immunization.Serum was collected at 0,14,21,28,60,68,75,82,89,96 and 110 days after the first immunization.The ELISA test results showed that the trend of antibody growth and decline of rApx?A,rApx?A,and rApx?A in the vaccination group were basically the same after immunization,The change of antibody titer was not obvious from 0 day to 28 days after the first immunization,and increased significantly after the second immunization.The rApx? antibody in the vaccine group reached a peak at 75 days(with titers of 230 times)after the first immunization.The peak of rApxII antibody reached at 60 days(with titers of 483 times)after the first immunization.The rApx? antibody reached the peak value at 68 days(with titers of 73 times)after the first immunization.And then the antibody level gradually decreased.The antibody of rApx?A,r Apx?A and rApx?A remained at a high level at 110 days after the first immunization.These results indicated that the porcine contagious pleuropneumonia bacterin-toxiod vaccine stimulated the body to produce specific antibodies.In summary,three indirect ELISA methods for Apx toxins' detection were established with good repeatability in this study.The growth and decline law of antibody after immunization showed that the porcine contagious pleuropneumonia bacterin-toxiod vaccine could stimulate the production of specific antibodies of rApx?A,rApx?A and rApx?A.It is recommended that the first immunization can be at 50 days age of piglets,and the second immunization would better been taken at 28 days after the first immunization.