Abstract：Porcine contagious pleuropneumonia (PCP), caused by Actinobacillus pleuropneumoniae (APP), is an infectious porcine (Sus scrofa) respiratory tract disease causing severe economic losses worldwide in the swine industry. Aiming at developing a safe attenuated vaccine against multiple serotypes, the APP serotype 7 isolates WF83 strain (APP-7-WF83) based recombinant transfer vector pBSLNKAR was constructed, which contained the kanamycin resistance gene (Kan) as a resisitance screening gene replacing complete toxin Ⅱ activate gene C (apxⅡC), with the N-terminal and C-terminal fragment squence of toxinⅠstructural gene A (apxⅠA) of Actinobacillus pleuropneumoniae RTX-toxin (Apx) gene family from APP serotype Ⅰ isolates shope4074 strain inserted. The vector pBSLNKAR was electroporated into the parent strain APP-7-WF83 to build mutant strain WF83ΔapxⅡC/apxⅠAN/apxⅠAC. The antibodies expressed by N-terminal fragment squence of toxin Ⅰstructural gene A (apxⅠAN) neutralized the toxinⅠso that the mutant had no hemolysis compared with APP-7-WF83 by hemolytic detection. PCR identification showed that WF83ΔapxⅡC/apxⅠAN/apxⅠAC lacked about 377 bp of apxⅡC and was inserted about 377 bp of apxⅠAC and 1 188 bp of apxⅠAN compared with APP-7-WF83. With 0.5 mL of trypticase soy broth (TSB) (blank control), the parent strain APP-7-WF83 group and the mutant WF83ΔapxⅡC/apxⅠAN/apxⅠAC group inoculated mice (Mus musculus) with 1.1×109 cfu/mL, respectively. The results showed that the mice of parent strain groups died of 100%, the mice of mutant group had no death. The results confirmed that the gene deletion composite attenuated strain WF83ΔapxⅡC/apxⅠAN/apxⅠAC was successfully constructed, which supplies basic data for further research on gene deleted attenuated vaccine.
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