兔支气管败血波氏杆菌DNT蛋白的分段表达及其免疫保护性研究  

The expression and immunoprotection of the truncated dermonecrotic toxin of rabbit Bordetella bronchiseptica

作　　者：赵宁[1] 王芳[1] 恽时锋[2] 范志宇[1] 胡波[1] 

机构地区：[1]江苏省农业科学院兽医研究所农业部动物疫病免疫与诊断重点开放实验室/国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京军区南京总医院比较医学科,江苏南京210002

出　　处：《中国预防兽医学报》2011年第8期648-652,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：现代农业产业技术体系建设专项资金(nycytx-44);江苏省农业科技自主创新资金[CX(10)214]

摘　　要：皮肤坏死毒素(DNT)是支气管败血波氏杆菌(Bb)的主要毒力因子之一。为研究DNT结合位点和催化位点的免疫保护性,本研究分别将其核苷酸序列N-端的1 612 bp片段(DNT1)和C-端的973 bp片段(DNT3)克隆到原核表达载体pET-28a(+)中,经IPTG诱导后在E.coli BL21(DE3)中获得表达。SDS-PAGE和western blot检测表明重组蛋白DNT1、DNT3均具有良好的反应原性。在主动免疫保护试验中,重组蛋白免疫组小鼠均能够产生较高的DNT抗体水平;当使用2 LD50的Bb强毒株BJL0504进行腹腔攻毒后,其存活率为100%,而阴性对照组则为40%;当使用1.07×108 cfu/mL Bb强毒株BJL0504进行滴鼻攻毒后第9 d时,重组蛋白免疫组小鼠已基本清除肺脏内的Bb菌,而阴性对照组小鼠肺脏内的Bb菌落数仍高达1.2×105 cfu/肺。本实验结果表明,DNT的结合位点和催化位点均具有良好的免疫保护性,有希望作为疫苗添加成分,为新型疫苗的研制提供实验依据。Dermonecrotic toxin （DNT） is identified as one of the main virulence factor of Bordetella bronchiseptica. To study the immunoprotection of the binding and catalytic regions of DNT, the N-terminal 1,612 bp （DNT1） and the C-terminal 973 bp （DNT3） fragments of the DNT were cloned into the pET-28a（＋） and expressed in E. coli BL21 （DE3）, respectively. The expression of recombinant protein DNT1 and DNT3 were identified by SDS-PAGE and western blot analysis. High level antibody against DNT was induced in the mice immunized with the recombinant proteins, and all the mice in immunized groups were survived when challenged intraperitoneally with virulent B. bronchiseptic strain BJL0504 at 2 dosages of LDso, while the mice mortility were 60% in control group. Furthermore, the bacteria in lung was eliminated completely from the immunned mice post challenging intranasally with B. bronchiseptic （1.07 x 10^8 cfu/mL） at 9 days, on the contrary, the bactera counts in mice lung were up to 1.2 x 10^5 cfu in control group. These results shown that the DNT1 and DNT3 had strong immunoprotection against 13. bronchiseptic infection and could be used as a subunit vaccine against B. bronchiseptic.

关 键 词：支气管败血波氏杆菌 坏死毒素 原核表达 免疫保护 

分 类 号：S852.614[农业科学—基础兽医学]