Zhao Zhanqin, Wang Chen, Xue Yun, Ding Ke, Zhang Chunjie, Cheng Xiangchao, Li Yinju, Liu Yichen, Wu Tingcai
Lab of Veterinary Microbiology, College of Animal Science and Technology, Henan University of Science and Techology, China.
Wei Sheng Wu Xue Bao. 2010 Sep;50(9):1239-45.
In this study we showed the immunogenicity and protective efficacy of five pertactin recombinants against Bordetella bronchiseptica (Bb) challenge.
The complete coding sequence (2040 bp) of the prn gene (PRN) and its fragments,5'-terminal 1173 bp fragment (PN),3'-terminal 867 bp fragment (PC), two copies of region I (654 bp; PR I) in PN, and 2 copies of region II (678 bp; PR II) in PC, were separately cloned into the prokaryotic expression vector pGEX-KG, and expressed in the Eschierichia coli BL21 (DE3) using induction by isopropyl-beta-D-thiogalactopyranoside. The recombinant proteins were named GST-PRN, GST-PN, GST-PC, GST-2PR I and GST-2PR II. All five recombinant proteins showed immunological reactivity in the Western-blot analysis. Mice, immunized subcutaneously with two doses of the purified proteins mixed with an equal volume of Freund's adjuvant,produced robust PRN-specific IgG antibody levels. When challenged, 6 of 9 mice in GST-2PR I group and all 9 mice in the other groups survived intranasal challenge with three times the 50% lethal dose (LD50) of virulent Bb HH0809. After challenge with 10 LD50 7/9,3/9,6/9,1/10 and 6/10 of the mice survived. Furthermore, complete protection against intraperitoneal (i.p.) challenge with 10 LD50 of HH0809 was observed in mice that were injected i.p. with 0.5 ml rabbit anti-GST-PRN, GST-PN,GST-PC or GST-2PR II serum. Only 1 of 10 mice survived in the group of mice that received anti-GST-2PR I, and no survivors were noted in the group of mice that received PRN-absorbed rabbit antiserum (0/5).
In this study,we showed that all of five pertactin recombinants had differential immunogenicity and protective efficacy against Bb challenge. Mice immunized with GST-PC had better survival against fatal Bb challenge than did those immunized with GST-PN. In addition, GST-2PR II and GST-2PR I provided the similar results These data may have implications for the development of safe and efficacious subunit vaccines for the prevention of bordetellosis on the basis of these five pertactin recombinants.
在本研究中，我们展示了五种百日咳杆菌黏附素重组体针对支气管败血波氏杆菌（Bb）攻击的免疫原性和保护效力。
将百日咳杆菌黏附素基因（prn，编码序列全长2040 bp）及其片段，即5'端1173 bp片段（PN）、3'端867 bp片段（PC）、PN中的两个区域I拷贝（654 bp；PR I）以及PC中的两个区域II拷贝（678 bp；PR II），分别克隆至原核表达载体pGEX-KG，并通过异丙基-β-D-硫代半乳糖苷诱导在大肠杆菌BL21（DE3）中表达。重组蛋白分别命名为GST-PRN、GST-PN、GST-PC、GST-2PR I和GST-2PR II。所有五种重组蛋白在蛋白质免疫印迹分析中均显示出免疫反应性。用等体积弗氏佐剂混合的纯化蛋白分两剂皮下免疫小鼠，可产生较强的PRN特异性IgG抗体水平。在攻毒时，GST-2PR I组的9只小鼠中有6只以及其他组的所有9只小鼠在经三倍50%致死剂量（LD50）的强毒Bb HH0809滴鼻攻毒后存活。在用10 LD50攻毒后，GST-2PR I组7/9、GST-PN组3/9、GST-PC组6/9、GST-2PR II组1/10以及GST-PRN组6/10的小鼠存活。此外，腹腔注射0.5 ml兔抗GST-PRN、GST-PN、GST-PC或GST-2PR II血清的小鼠在经10 LD50的HH0809腹腔攻毒后获得了完全保护。接受抗GST-2PR I血清的小鼠组中10只小鼠仅有1只存活，而接受PRN吸附兔抗血清的小鼠组（0/5）无存活者。
在本研究中，我们表明所有五种百日咳杆菌黏附素重组体针对Bb攻击具有不同的免疫原性和保护效力。用GST-PC免疫的小鼠在抵抗致死性Bb攻击时比用GST-PN免疫的小鼠存活率更高。此外，GST-2PR II和GST-2PR I产生了相似的结果。这些数据可能对基于这五种百日咳杆菌黏附素重组体开发安全有效的亚单位疫苗以预防博德特氏菌病具有启示意义。
