Expression Of Apxâ…¡A Gene From Actinobacillus Pleuropneumoniae In Escherichia Coli And Development Of The Monoclonal Antibodies Against Apxâ…¡A And Development Of A Multiplex PCR For Detecting 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. It is difficult to diagnose and control this disease because of 15 serotypes of A. pleuropneumoniae, which produce many virulent factors. Apx toxins are major virulent factors of APP. ApxI is strongly haemolytic and strongly cytotoxic for phagocytic cells, ApxII is weakly haemolytic and weakly cytotoxic, ApxIII is only strongly cytotoxic for macrophages and neutrophils, and its linear cytotoxic and pro-apoptotic epitopes were identified, ApxIV expressed in E.coli is weakly haemolytic. An efficient diagnostic kit will contribute to control the disease. ApxII is species-specific, which is produced by all serotypes of APP except serotype 10. In order to develope an ApxIIA diagnostic method for the detction of APP and provide specific monoclonal antibodies against Apx toxins of APP to study its function and molecular pathogenesis, the following research were explored.1. Cloning of APP apxIIA gene and its expression in E. coli.The GenBank accession number of the sequence of apxIIA from Actinobacillus pleuropneumoniae serotype 1 type strain 4047 is AF232288. To use of Primer Premier, the target DNA fragment was generated by PCR using the designed primers. Then the apxIIA gene was cloned into pCR2.1 vector and recombinant plasmids of pCR-apxIIA were produced. The apxIIA gene was sequenced by Sanger's sequencing technique. Then, the apxIIA gene was inserted into downstream of the T7 promoter of an expression vector, pGEX-6p-1, to yield the recombinant plasmids of pGEX-apxIIA . After induced by IPTG, a high expression of fusion proteins was obtained. SDS-PAGE analysis showed that the fusion protein were about 130kD in size, designed at rApxIIA correspondingly. rApxIIA existed mainly in form of inclusion bodies and was shown to be specific to antisera against APP by Western-blot analysis.2. Preparation and purification of nature App toxins.From A. pleuropneumoniae serotype 1 strain 4047, a mixture of two major toxins namely, ApxI and ApxII was obtained. 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.3. Preparation of monoclonal antibodies against ApxIIA toxins.Two hybridoma clones were generated by fusion of SP2/O myeloma cells and the splenocytes obtained from BALB/c mice immunized with the APP outer toxins. Western-blot and indirect ELISA analysis indicated that both of the monoclonal antibodies(Mabs) were specific to the rApxIIA antigen and could not react with the proteins from GST, E.coli, Pasteurella multocide(PM) or Haemophilu parasuis(HPs). And MAb 3A11 was of IgG1 isotype and MAb 8B3 was of IgG2a。4. Development of a Multiplex PCR for Detecting APP.Specific primers for cpx and apxIV genes of APP were synthesized and used for development of a multiplex PCR for detecting APP. A 389bp fragment and a 498bp fragment can be simultaneously amplified in one reaction. 97 suspicious samples from cases of PCP collected from Jiangsu area were detected for APP by the multiplex PCR. The results showed that the multiplex PCR seemed to meet its potential application for diagnostic purpose.