重组猪多杀性巴氏杆菌MetQ诱导小鼠抗攻毒感染的免疫保护力研究

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摘要猪多杀性巴氏杆菌(Pasteurella multocidain, Pm)感染可引起猪(Sus scrofa)产生猪肺疫、猪出血性败血症和猪萎缩性鼻炎等。目前应用的灭活全菌苗交叉保护效果差且免疫保护期短，重组疫苗尚不能提供高的保护率。为研究猪多杀性巴氏杆菌重组甲硫氨酸转运体Q(recombinant methionine transporter Q, rMetQ)的免疫保护性，本研究根据GenBank中登录的Pm metQ基因序列，根据去除影响表达的长片段疏水区的相应核苷酸序列设计引物对，以Pm的基因组为模板PCR扩增metQ基因，测序进行生物信息学分析。结果表明，猪Pm metQ基因片段含768 bp核苷酸，编码255个氨基酸的蛋白质，其理论分子量为28.14 kD，是与细菌的毒性有关的胞外结合蛋白，不同血清型的该蛋白同源型达99%以上。将Pm metQ亚克隆到pET-28a(+)构建重组质粒pET-metQ并转化至大肠杆菌(Escherichia coli) BL21 (DE3)，用异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1- thiogalactopyranoside, IPTG)诱导表达Pm rMetQ，经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)检测，结果表明，在28 kD处出现特异性条带。动物保护实验采用昆明小鼠 (Mus musculus)进行，免疫球蛋白G(immunoglobulin G, IgG)抗体滴度采用酶联免疫吸附测定法(enzyme-linked immuno sorbent assay, ELISA)检测。结果表明，3次免疫后第14天，铝佐剂+rMetQ组小鼠血清中的IgG抗体水平显著升高，明显高于对照组和低剂量组，对照组和低剂量组小鼠攻毒后3 d存活率为20%，7 d后全部死亡。铝佐剂+30 μg rMetQ免疫组，10只免疫组小鼠3 d后，6只死亡，存活率为40%；7 d后，又有3只死亡，存活率为10%。铝佐剂+50 μg rMetQ组 3 d后存活率达80%，7 d后存活率40%。死亡小鼠剖检发现肝脏为主要病变器官，呈现有不同程度的出血点甚至坏死。可见，Pm rMetQ皮下免疫注射可诱导小鼠产生免疫应答和较高的抗攻击感染的免疫保护力。该研究为进一步筛选猪Pm分子疫苗积累了资料。

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关键词 ：多杀性巴氏杆菌, rMetQ, 免疫原性, 免疫保护力, 小鼠

Abstract：Porcine (Sus scrofa) Pasteurella multocidain (Pm) infection caused serious diseases including swine plague, hemorrhagic septicaemia and infectious atrophic rhinitis. The present inactivated vaccine had poor cross protection effect and short immune protection period, and the recombinant vaccine still did not provide high protection rate. Based on Pm methionine transporter Q (metQ) sequences in GenBank, the primers were designed after removing the corresponding nucleotide sequence of expressing hydrophobic amino acid regions to express metQ in Escherichia coli. The partial sequence of Pm metQ was amplified by PCR from Pm genome. Bioinformatics analysis showed that the Pm metQ gene was composed of 768 bp, encoding a protein of 255 amino acids. This protein was an extracellular toxic protein and high homology among different Pm serotypes. Pm metQ gene was subcloned into pET32a (+) to construct pET-metQ. After induced by isopropylβ-D-1- thiogalactopyranoside (IPTG) rMetQ expressed in E.coli BL21, and determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that rMetQ with 28 kD was detected. Animal experiment was conducted by Kunming mice (Mus musculus). The serum immunoglobulin G (IgG) level was determined by enzyme-linked immuno sorbent assay (ELISA). The results showed that serum IgG level significantly increased in immuned group (aluminium adjuvant+rMetQ) at 14 d after third immunization. The infected mice in control groups were 20% survival rate at 3 d and all died at 7 d. The mice in the immunized group (aluminium adjuvant+30 μg rMetQ) showed 40% survival rate at 3 d and 10% survival rate at 7 d. But the mice in the immunized group (aluminium adjuvant+50 μg rMetQ) showed 80% survival rate at 3 days and 40% survival rate at 7 d. Autopsy of dead mice showed that the liver was the main diseased organ with haemorrhagic spot and even necrosis.Mice subcutaneously vaccinated with rMetQ revealed a partial protection against the challenge infection of Pm. This study provides data for screening new molecular vaccines against Pm infection.

Key words： Pasteurella multocida rMetQ antigenicity immune protective efficacy mice

收稿日期: 2017-03-15 出版日期: 2017-10-30

通讯作者: 伍小松 E-mail: wuxiaosong529@126.com

引用本文:

伍小松罗艳辉余兴龙贺建华赵墩. 重组猪多杀性巴氏杆菌MetQ诱导小鼠抗攻毒感染的免疫保护力研究[J]. , 2017, 25(10): 1682-1688.

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http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2017/V25/I10/1682

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