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

发布时间：2019-04-25 12:33

【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+30μg 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+50μg 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。
[Abstract]:Porcine (Sus scrofa) infection with pasteurella multocida (Pasteurella multocidain,Pm) can cause swine pulmonary disease, hemorrhagic septicemia and atrophic rhinitis in pigs. At present, the cross-protection effect of inactivated whole bacteria vaccine is poor and the immune protection period is short, so the recombinant vaccine can not provide a high protection rate. In order to study the immune protection of recombinant methionine transporter (Q (recombinant methionine transporter Q, rMetQ) of Pasteurella multocida in pigs, this study was based on the sequence of Pm metQ gene logged in GenBank. Primer pairs were designed according to the corresponding nucleotide sequences to remove the long hydrophobic region of the expression. The metQ gene was amplified by PCR using the genome of Pm as the template, and the bioinformatics analysis was carried out by sequencing. The results showed that the porcine Pm metQ gene contains 768 bp nucleotides and encodes a 255amino acid protein. Its theoretical molecular weight is 28.14 kD, which is an extracellular binding protein related to the toxicity of bacteria. The homologue of the protein in different serotypes was more than 99%. Pm metQ was subcloned into pET-28a () to construct recombinant plasmid pET-metQ and transformed into E. coli (Escherichia coli) BL21 (DE3). Pm rMetQ, was induced to express Pm rMetQ, by isopropyl 尾-D-thiogalactoside (isopropyl 尾-D-thiogalactoside). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE) showed that there was a specific band at 28 kD. The antibody titers of immunoglobulin G (immunoglobulin-G) were determined by enzyme-linked immunosorbent assay (enzyme-linked immuno sorbent assay,ELISA). The animal protection test was carried out with Kunming mouse (Mus musculus). The results showed that on the 14th day after 3 times of immunization, the level of serum IgG antibody in the aluminum adjuvant rMetQ group was significantly higher than that in the control group and the low dose group, and the survival rate of the control group and the low dose group was 20% on the 3rd day after challenge, and the survival rate of the control group and the low dose group was 20%. All died 7 days later. In the aluminum adjuvant 30 渭 g rMetQ-immunized group, 6 mice died after 3 days of immunization, the survival rate was 40% and 7 days later, and the survival rate was 10%. The survival rate of 50 渭 g rMetQ group was 80% after 3 d and 40% after 7 d. The liver of the dead mice was found to be the main pathological organ, showing different degrees of bleeding and necrosis. It can be seen that subcutaneous injection of, Pm rMetQ can induce immune response and high immune protection against aggressive infection in mice. This study provided data for further screening of porcine Pm molecular vaccines.
【作者单位】：湖南农业大学动物科学技术学院;湖南农业大学动物医学院;
【基金】：湖南省自然科学基金项目(No.14JJ4077)
【分类号】：S852.61

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