表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌对小鼠的免疫原性

Immunogenicity of attenuated Salmonella choleraesuis vaccine strain expressing immunogenic genes of Mycoplasma hyopneumoniae in mice

摘要:

摘要: 【目的】构建表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌，对重组菌株的生物学特性以及对小鼠的免疫原性进行研究。【方法】分别将猪肺炎支原体的免疫原性基因p36、p46、p65 和p97R1-Nrdf 克隆到pYA3493，得到重组质粒pYA-36、pYA-46、pYA-65 和pYA-97R1-Nrdf。重组质粒和空质粒pYA3493 分别电转asd 基因缺失株C500ˉ，获得重组菌株C36(pYA-36)、C46(pYA-46)、C65(pYA-65) 、C97R1-Nrdf( pYA-97R1-Nrdf) 和空质粒菌株CpYA( pYA3493) 。研究重组菌株的生物学特性，并以小鼠为动物模型评价重组菌株在口服、肌注两种不同免疫途径下的免疫原性。【结果】成功构建表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌，重组菌株能表达外源蛋白，生化和生长特性未发生改变，插入的外源基因亦稳定存在。小鼠的免疫原性结果显示: 口服C36 + C46 + C65 + C97R1-Nrdf 组的猪肺炎支原体抗体极显著高于口服C36 + C46 + C65 组和肌注商品疫苗组(P＜0.01) ，但与肌注C36 + C46 + C65 组无显著性差异(P＞ 0.05);IFN-γ为肌注C36 + C46 + C65 组显著高于肌注商品疫苗组( P ＜ 0. 05) ，而与口服C36 + C46 + C65 或C36 + C46 + C65+ C97R1-Nrdf 组差异均不显著(P＞ 0.05); IL-4 水平为口服C36 + C46 + C65 组＞ 口服C36 + C46 + C65 + C97R1-Nrdf 组＞ 肌注商品疫苗组＞ 肌注C36 + C46 + C65 组，但各组之间差异均不显著(P＞0.05)。对照组的猪肺炎支原体抗体、IFN-γ 以及IL-4 均与试验组差异极显著(P＜0.01)。【结论】构建的表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌，采用肌注免疫时具有较好的免疫原性，有望发展为猪肺炎支原体的基因工程疫苗。

关键词:关键词: 猪肺炎支原体，重组猪霍乱沙门氏菌，免疫原性，抗体，IFN-γ，IL-4

Abstract:

Abstract: ［Objective］The study was carried out to construct and characterize Salmonella choleraesuis vaccine strain expressing immunogenic genes of Mycoplasma hyopneumoniae and to test its immunogenicity in mice． ［Method］We made p36，p46，p65 and p97R1-Nrdf，the main immunogenic genes of Mycoplasma hyopneumoniae，to insert into the prokaryotic expression plasmid pYA3493. Then these recombinant plasmids and pYA3493 were electroporated into C500 asd- mutant，resulting in the recombinant Salmonella choleraesuis vaccine strains C36( pYA-36) ，C46( pYA-46) ，C65( pYA-65) ，C97R1-Nrdf( pYA-97R1-Nrdf) and CpYA( pYA3493) ． We characterized these recombinant Salmonella choleraesuis vaccine strains and tested the immunogenicity in mice by intramuscular injection or orally immunized． ［Result］ The results of the immunogenicity in mice indicated that the group orally immunized with C36，C46，C65，C97R1-Nrdf showed significantly higher Mycoplasma pneumoniae antibody than both the group orally immunized with C36，C46，C65 and the group intramuscular injected with the Mycoplasma hyopneumoniae bacterin ( M + PAC) ( P ＜ 0. 01 ) ． The group intramuscular injected with C36，C46，C65 showed higher IFN-γ production than the group injected with the Mycoplasma hyopneumoniae bacterin ( M + PAC) ( P ＜ 0. 05) ，but there was no significant difference between the group orally immunized with C36，C46，C65 and the group orally immunized with C36，C46，C65，C97R1-Nrdf ( P ＞ 0. 05) ． The highest level of IL-4 was found in the group orally immunized with C36，C46，C65; higher levels of IL-4 was observed in the group orally immunized with C36，C46，C65，C97R1-Nrdf than the group injected with the Mycoplasma hyopneumoniae bacterin ( M + PAC) ; and the lowest IL-4 level was found in the group injected with C36，C46，C65. There were no significant differences among them ( P ＞ 0. 05) ． The Mycoplasma pneumoniae antibody，IFN-γ or IL-4 production of the each group was obviously higher than the control group ( P ＜ 0. 01) ． ［Conclusion］The attenuated Salmonella choleraesuis vaccine strain expressing immunogenic genes of Mycoplasma hyopneumoniae which has immunogenicity in mice especially by intramuscular injection could probably serve as a vaccine against mycoplasmal pneumonia of swine．

Key words:Keywords: Mycoplasma hyopneumoniae，attenuated Salmonella choleraesuis，Immunogenicity，Antibody，IFN-γ，IL-4