| Porcine infectious pleuropneumonia is a respiratory disease that caused by Actinobacillus pieuropnewnoniae (APP). The most important virulent factor of APP is its excreted exotoxin-Apx toxin. In order to better understand the relationship between structure and function of Apx II toxin that be excreted by a field isolate L25-4 of APP serotype 7, and to extend the use of recombinant Apx toxin protein, we expressed the structure gene apxIlA of Apx II toxin in E.coli with prokaryotic expression vector pGEX-6p-l and co-expressed with APP-E.coli shuttle vector pGZRS-38 which is used to express the post-transcriptional activation gene apx IIC. When be expressed in E.coli, both of the expressed products formed inclusions. The inclusion proteins were oxidated, refolded and restored natural protein status after be treated by washing, degeneration, renaturation and purification. Upon detecting the haemolytic activity of the recombinant protein rApxII A that be activated and inactivated, the product of apx IIC is proved to be essential for the haemolytic activity of Apx II toxin. Moreover, in order to detect the immunogenicity of rApx II A, we immunized rabbits with rApx IIA antigen and followed by challenge with homologous strain (serotype 7 L25-4) and heterologous strain (serotype 1 4074). The result showed that rApx IIA is a good antigen, when it was added into inactivated vaccine, the immunity efficacy could be improved. It can provide completely protection against the challenge by homologous strain and partial protection against challenge by heterologous strain.Since the first gene-deletion vaccine was approved to be used in U.S.A in 1986, the technique of gene-deletion has been a very useful tool in the research of the relationship between structure and function of virulent genes of pathogenic microorganisms, it is also a utilitarian method to construction an attenuated mutant and has been applied to many kinds of pathogenic germs. So, it was also developed in the study of APP. Though a great deal of advances has been achieved by many researchers abroad, it is very few or even no research about it within our homeland. A worthy attempt on this will do help to lessen the gap. Our research focus on the apxIIC gene of APP serotype 7 L25-4 strain, a large fragment containing apxIIC gene was amplified and cloned into suicide vector and then apxIIC was deleted by PCR in vitro followed by insertion of promoterless Ampr gene amplified from pUCl9 to form the target vector plasmid pMD-Apx II ACAmp. pMD-Apx II ACAmp was electrotransformed into APP wild strain with the possibility that the target fragment on the plasmid will occur homologous recombination with the homologous region on the chromosome of APP. Thus, apxIIC gene will be completely replaced by Ampr gene. As a result, apxIIC gene will be deleted from chromosome and the mutant will attenuated on molecular level. In order to obtain the highest electrotransformation efficiency and homologous recombination efficiency, we had pre-electrotransformed APP competent cells with APP-E.coli shuttle vector pGZRS-18. Though many kinds of combination of electrotranformation parameters was performed and the optimal electrotransformation condition was determined, we found that pGZRS-18 transformants obtained with serotype 7 L25-4 strain was extremely lower than those of the same serotype reported by others. Thus, we draw the conclusion that APP serotype 7 L25-4 strain is a recalcitrant strain to electrotransformation because of its strong restriction to heterologous DNA. |
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