胸膜肺炎放线杆菌apxIC基因插入失活突变株构建及免疫原性分析

Construction and immunogenicity of an attenuated mutant of Actinobacillus pleuropneumoniae by insertional inactivation of apxIC

胸膜肺炎放线杆菌是引起猪传染性胸膜肺炎(APP)的呼吸道病原菌,其分泌的Apx毒素是最重要的毒力因子之一。为构建APP突变弱毒菌株,在apxIC基因下游XhoI酶切位点处插入氯霉素抗性基因(Chlr)制备转移载体,通过电转化导入APP血清10型参考菌株(D13039)进行同源重组,筛选获得apxIC基因插入突变菌株D13039C-Chlr。该突变菌株特性鉴定结果表明其溶血活性完全丧失,可正常增殖和分泌ApxI毒素,连续10次传代后基因组中插入的Chlr基因可稳定遗传,利用5个剂量(2×108CFU^2×106CFU)对每组3只小鼠腹腔攻毒结果显示突变菌株毒力较母源菌株降低至少100倍以上,将突变菌株作为弱毒活疫苗经滴鼻途径免疫仔猪后利用APP血清1型(4074)和血清10型(D13039)菌株攻毒进行免疫原性鉴定,结果显示血清1型攻毒后非免疫组4头仔猪全部死亡而免疫组4头中死亡2头,非免疫组肺损伤指数(34.4)显著高于免疫组(17.5),血清10型攻毒后非免疫组肺损伤指数(17.5)也高于免疫组(10.5),同时鼻拭子和肺组织样品的细菌重分离数及PCR检测阳性数非免疫组也明显高于免疫组,表明突变菌株作为弱毒活疫苗对仔猪具有一定的交叉免疫保护力。该突变菌株的构建为鉴定ApxI毒素活性及研制具有交叉保护活性的APP弱毒活疫苗奠定了基础。

Actinobacillus pleuropneumoniae is the aetiological agent of porcine pleuropneumonia. Apx toxin, an exotoxin secreted by A. pleuropneumoniae, is one of the most important virulence factors. To construct an avirulent mutant strain by inactivation of ApxI toxin, the apxIC gene of A. pleuropneumoniae serovar 10 was inactivated by inserting a chloramphenicol resistance gene cassette into the downstream XhoI site of the apxIC gene for constructing the transfer plasmid. The transfer plasmid was introduced into the electrocompetent A. pleuropneumoniae serovar 10 for homologous recombination by electroporation. The mutant strain was obtained and identified by PCR and Southern blotting. The mutant strain was phenotypically identical to the parent strain except that it showed no haemolytic activity. The mutant strain was also able to secret the same ApxI toxin as the parent strain. In the intra-peritoneal mouse model, the virulence of the mutant strain decreased at least 100 fold compared with the parent strain. The mutant was evaluated as a potential vaccine using a vaccination-challenge trial in which pigs were given two intra-nasal doses of the mutant with 14 days＇ interval and then challenged 14 days after the last vaccination with A. pleuropneumoniae serovar 1 and serovar 10 reference strains respectively. The death number and lung lesion score in the vaccinated pigs given the serovar 1 challenge were obviously lower than those in the unvaccinated pigs. And the lower lung lesion score was also observed in the vaccinated pigs challenged with serovar 10. And the positive numbers of A. pleuropneumoniae re-isolation and PCR detection showed the same consistency. The vaccination-challenge trial suggested that the mutant strain could offer partial cross-protection as a live attenuated vaccine against A. pleuropneumoniae infection.