Study On ApxⅡc~-/apxIA~+ Actinobacillus Pleuropneumoniae Serotype-7 And An Indirect TonB2-ELISA To Detect The Antibody Against APP

Porcine contagious pleuropneumoniae caused by Actinobacillus pleuropneumoniae (APP),is a severe,contagious pulmonary disease of pigs that cause important economic losses in industrialized pigs production worldwide.Immunization is the main method to prevent and control this disease.The current commercial vaccines are still primarity killed whole cell bacterins and subunit vaccines, which generally reduce mortality from APP infection but frequently fail to prevent serever morbidity and economic loss due to the chronic effects of the disease on growth rate and feed efficiency.In contrast,natural and experimental infection can induce protection against any heterologous serotype.The attenuated live vaccines could re-present protective antigens and stimulate a lasting immune response that may be more efficacious in preventing the disease.Therefore,studies of A.pleuropneuminiae vaccines tend to focus on live attenuated vaccines constructed by inactivating virulence-associated genes.Based on the APP HB sero-7 strain which was isolated and identified by our lab, Doctor Bei weicheng constructed a genetic mutant strain HBC/GFP⁺.After immunification,the Balb/C mice and piglets obtained immunity against APP sero-7 and 75%protection against heterogenetic sero type 1 strains.This result indicated an excellent application prospect of this genetic mutant strain.However,the protection against heterogenetic sero type strains is 75%,and we aim to improve this protection much higher.An efficient diagnostic kit will contribute to control the disease.Now,the commonly used diagnostic method of APP is HIA which has strong sero-type specificity,and not good in mass survey.The CFT has higher specificity but low sensitivity and some times false positive results.Besides,The CFT method is complex and only applicable in lab. The LAT method is simple and fast,but it has cross-reaction with diferrent sero-types. The ELISA method was adopted by most researchers because the manipulation is simple and the result is easy to judge.Iron acquisition in vivo by Actinobacillus pleuropneumoniae depends upon a functional TonB system,and two kind of this system,TonB1 and TonB2,have been reported in Actinobacillus pleuropneumoniae.TonB2 protein was expressed during the infection of all the sero types of APP and it has species specificity,so it can be used in mass survey of APP.Based on above considerations,The main research was described as follows: 1.Cloning of APP apxâ… A gene,Selection and evaluation of the genetic engineering recombinant strain apxâ…¡C^-/ apxâ… A⁺The GenBank accession number()of the sequence of apxâ… A from Actinobacilluspleuropneumoniae serotype 1 type and the chromosomal DNA of A. pleuropneuminiae serotype 1(SLWO1) was used as template,the target DNA fragment was generated by PCR using the designed primers,The apxâ… A gene was sequenced by Sanger's sequencing technique.Then,the apxâ… A gene was inserted into downstream of the T7 promoter of a shuttle plasmid-pJFF_NX,to yield the recombinant plasmids of pJFF-â… A.Then it was transformed into the genetic engineering mutant strain HBC^-/GFP⁺ by electrotransformation.The chloramphenicol resistant recombinant strain were selected, The genetic engineering recombinant strain which was referred to as apxâ…¡C^- / apxâ… A⁺ was verified and evaluated by PCR.Then,Its some biological activities such as growth rate, the genetic stabily of the insertional mutants were more investigated.2.Characterization of apxâ… A gene expression in A.pleuropneuminiaeThe apxâ… A gene encoding the constitutive protein of Apxâ… ,The Actinobacillus-pleuropneumoniae-RTX-toxins(Apx) was purified as following:By using (NH4)₂SO₄ precipitation,then centrifugation of the supernatant was applied,and the pellet was resuspended in 5 mL PBS and dialyzed against PBS for 3 day and to get the APP toxins.These toxins were characterized by using SDS-PAGE and Western-blot。And the results showed that The apxâ… A.gene could expression stably in The genetic engineering recombinant strain apxâ…¡C^-/ apxâ… A⁺.3.Vaccination and challenge efficacy of the the genetic engineering recombinant strain apxâ…¡C^-/ apxâ… A⁺ in pigletsTo test the protective efficacy of the genetic engineering recombinant strain apxâ…¡C^-/ apxâ… A⁺ for piglets,piglets were vaccinated twice via intranasal inoculation and intramuscular injection with 1×10⁸CFU of the genetic engineering recombinant strain apxâ…¡C^-/ apxâ… A⁺ at 2-week internals.The antibody of each antigen was detected with ELISA.Piglets were challenged intranasally(IN) with 1.0ml containing 1×10⁸CFU of SLWO1(serovar 1) two weeks after second vaccination,The protective efficacy of immunization was evaluated by clinical,bacteriological,serological and post-mortem examination.The results demonstrated that antibody level of Apxâ…¡_ELISA and Apxâ… -ELISA increased obviously after second immunization.Piglets vaccinated via intratracheal injection with the genetic engineering recombinant strain apxâ…¡C^-/ apxâ… A⁺ had 100%(5/5) and vaccinated intramuscularly with the genetic engineering recombinant strain apxâ…¡C^-/ apxâ… A⁺ had 80%(4/5) protection against heterologous serotype(serotype 1) challenge with A.pleuropneumoniae,The results suggested that immunization with the genetic engineering recombinant strain apxâ…¡C^-/ apxâ… A⁺ could alleviate clinical sign and lung lesion,and the genetic engineering recombinant strain apxâ…¡C^- / apxâ… A⁺ is a kind of potential APP vaccine strain.4.Cloning of APP tonB2 gene and its expression in E.colithe 858bp DNA fragment of TonB2 gene of A.pleuroneumoniae was amplified and orderly fused to the downstream of glutathione S-transferase(GST) of pGEX-KG expression vector.The construct was transformed into E.coli BL21(DE3) After induction by(1.0 mmol/mL)IPTG,a recombinant protein about 90kD in size,designed as GST-TonB2,Westem-blot was performed to confirm that the upper purified GST-TonB2fusion proteins could specifically react with antiserum against APP.5.Development and pre-validation of an TonB2-ELISAPlates were coated using purified TonB2,The optimal antigen concentration and the optimal serum dilution were determined by checkerboard titration,we developed this indirect ELISA which was referred to as TonB2-ELISA,use of TonB2-ELISA and Indirect Hmmgglntimnion Assay(IHA) parallel detection and analysis of field sera of pigs that were collected from 17 A.pleuropneumoniae infected pig farms in china,revealed that the positive coincidence rate of two detection method was 92.8%and the negative coincidence rate was 84.2%.The pre-validation study of the TonB2-ELISA revealed that TonB2-ELISA was specific and sensitive.we also evaluated The ELISA with sera from rabbit experimentally infected with 12 different A.pleuropneumoniae serovars of biotype 1,valence of antibody of all these serum are over 1:10240,The findings suggest that all serotypes of A.pleuropneumoniae infections could be detected by means of the TonB2-ELISA...