巴氏杆菌糖酵解酶的免疫保护效应研究

Study on Immunoprotective Efficacy of Pasteurella Glycolytic Enzymes

本研究旨在评估巴氏杆菌糖酵解酶全部9个成员作为抗原对巴氏杆菌的免疫保护效应。通过PCR扩增出多杀性巴氏杆菌C51-17株糖酵解酶的基因并将其连接到pET-28a(+)质粒上。重组质粒转化大肠杆菌BL21(DE3)工程菌后进行过夜诱导表达,收集菌体,超声破碎后取上清,纯化出巴氏杆菌糖酵解酶的重组蛋白。将巴氏杆菌糖酵解酶进行SDS-PAGE后转入PVDF膜上,孵育C51-17株感染康复血清,孵育二抗后进行ECL显色。将糖酵解酶免疫C57BL/6小鼠,待免疫组均产生高水平特异性抗体后使用C51-17株攻毒,比较各组存活率。测序结果显示,本研究成功将巴氏杆菌糖酵解酶全部9个成员的基因克隆到pET-28a(+)质粒上,经诱导表达和纯化成功获得了巴氏杆菌糖酵解酶的重组蛋白。Western blotting结果显示,磷酸葡萄糖异构酶(PGI)、丙糖磷酸异构酶(TPI)、甘油醛-3-磷酸脱氢酶(GPD)、磷酸甘油激酶(PGK)、丙酮酸激酶(PYK)能与巴氏杆菌C51-17株感染康复血清反应。免疫保护力试验显示,PGI、醛缩酶(ALD)、GPD、PGK、磷酸甘油酸变位酶(PGM)、烯醇化酶(ENO)具有一定的免疫保护作用。本研究结果表明,巴氏杆菌糖酵解酶部分成员具有一定程度的免疫保护作用,为巴氏杆菌病防控研究提供了新的依据。

The purpose of this study was to evaluate the immunoprotective efficacy of all 9 members of Pasteurella glycolytic enzymes as an antigen.Firstly,the glycolytic enzyme genes of Pasteurella multocida C51-17 strain were amplified by PCR and linked to pET-28a(+)plasmid.The recombinant plasmid was transferred into Escherichia coli BL21(DE3)strain and induced to express overnight.The recombinant proteins of Pasteurella glycolytic enzymes were purified from the supernatant of the collected strain after ultrasonic crushing.After SDS-PAGE,the Pasteurella glycolytic enzymes were transferred to PVDF membranes,and the recovered sera from C51-17 strain infection was incubated.ECL staining was performed after incubating the second antibody.At last,C57BL/6 mice were immunized with glycolytic enzymes.After high level of specific antibody was produced in the immunized groups,C51-17 strain was used to challenge.Sequencing results showed that the genes of all 9 members of the Pasteurella glycolytic enzymes were successfully cloned into pET-28a(+)plasmid,and the recombinant protein was obtained after induced expression and purification.Western blotting results showed that phosophoglucose isomerase(PGI),triose phosphate isomerase(TPI),glyceraldehyde-3-phosphate dehydrogenase(GPD),phosphoglycerate kinase(PGK),and pyruvate kinase(PYK)could react with the recovered sera from Pasteurella multocida C51-17 strain.The results of immunoprotection test showed that PGI,aldolase(ALD),GPD,PGK,phosphoglycerate mutase(PGM)and enolase(ENO)had some protective effects.In conclusion,the results of this study showed that some members of the Pasteurella glycolytic enzymes had a certain degree of immune protection,which provided a new basis for the prevention and control of pasteurellosis.