胸膜肺炎放线杆菌复合减毒突变株WF83ΔapxⅡC/apxⅠAN/apxⅠAC的构建  

Construction of a Composite Attenuated Mutant Actinobacillus pleuropneumoniae Strain WF83ΔapxⅡC/apxⅠAN/apxⅠAC

作　　者：张倩[1] 陈广力[1] 文心田[1] 黄小波[1] 伍锐[1] 文翼平[1] 曹三杰[1] 

机构地区：[1]四川农业大学动物医学院猪病研究中心,成都611130

出　　处：《农业生物技术学报》2015年第7期841-850,共10页Journal of Agricultural Biotechnology

基　　金：公益性行业(农业)科研专项(No.201303034);四川省科技支撑计划(No.2012NZ0001)

摘　　要：猪传染性胸膜肺炎(porcine contagious pleuropneumonia,PCP)是猪(Sus scrofa)的一种接触性呼吸道疾病,引起该病的致病菌是胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP),目前该病已成为世界养猪业的五大疫病之一,造成了重大的经济损失。为了提供一种能够保护多种血清型的安全性弱毒疫苗,本研究以APP血清7型菌株WF83(APP-7-WF83)为基本材料,将卡那霉素抗性基因(Kan)作为抗性筛选基因置换完整的溶血外毒素(Actinobacillus pleuropneumoniae RTX-toxin,Apx)家族中的的毒素Ⅱ激活基因C(apxⅡC),同时引入APP血清1型菌株(shope4074)的毒素Ⅰ基因的氮端(apxⅠAN)和碳端(apxⅠAC)片段序列,构建重组转移载体p BSLNKAR,电转化将该载体导入亲本菌株APP-7-WF83,获得突变株WF83ΔapxⅡC/apxⅠAN/apxⅠAC。与亲本株(APP-7-WF83)相比,溶血活性检测显示,突变株WF83ΔapxⅡC/apxⅠAN/apxⅠAC无溶血性;PCR鉴定显示,WF83ΔapxⅡC/apxⅠAN/apxⅠAC缺失了377 bp的apxⅡC,同时插入1 188 bp的apxⅠAN序列和377 bp的apxⅠAC序列;以0.5 m L的胰蛋白胨大豆肉汤(trypticase soy broth,TSB)为空白对照,亲本株APP-7-WF83组和突变株WF83ΔapxⅡC/apxⅠAN/apxⅠAC组均以1.1×109cfu/m L的量接种小鼠(Mus musculus)后,亲本株组小鼠全部死亡,突变株组小鼠没有死亡情况,证实突变株的毒性显著减弱。本研究成功构建了基因缺失复合减毒株WF83ΔapxⅡC/apxⅠAN/apxⅠAC,为进一步研究基因缺失弱毒疫苗提供了基础资料。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 7isolates WF83 strain（APP- 7- WF83） based recombinant transfer vector p BSLNKAR was constructed, which contained the kanamycin resistance gene（Kan） as a resisitance screening gene replacing complete toxin Ⅱactivate gene C（apxⅡC）, with the N-terminal and C-terminal fragment squence of toxinⅠ structural gene A（apx Ⅰ A） of Actinobacillus pleuropneumoniae RTX- toxin（Apx） gene family from APP serotype Ⅰ isolates shope4074 strain inserted. The vector p BSLNKAR was electroporated into the parent strain APP- 7-WF83 to build mutant strain WF83Δapx Ⅱ C/apx Ⅰ AN/apx Ⅰ AC. The antibodies expressed by N- terminal fragment squence of toxin Ⅰstructural gene A（apxⅠAN） neutralized the toxinⅠ so that the mutant had no hemolysis compared with APP- 7- WF83 by hemolytic detection. PCR identification showed that WF83Δapx Ⅱ C/apxⅠAN/apxⅠ AC lacked about 377 bp of apxⅡ C and was inserted about 377 bp of apxⅠ AC and 1 188 bp of apxⅠ AN compared with APP-7-WF83. With 0.5 m L of trypticase soy broth（TSB）（blank control）, the parent strain APP-7-WF83 group and the mutant WF83ΔapxⅡC/apxⅠAN/apxⅠ AC group inoculated mice（Mus musculus）with 1.1×10^9cfu/m L, 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/apxⅠ AC was successfully constructed, which supplies basic data for further research on gene deleted attenuated vaccine.

关 键 词：胸膜肺炎放线杆菌 基因缺失 毒素Ⅱ激活基因C(apxⅡC) 毒素Ⅰ结构基因A氮端序列(apxⅠAN) 毒素Ⅰ结构基因A碳端序列(apxⅠAC) 

分 类 号：S858.28[农业科学—临床兽医学]