猪胸膜炎放线杆菌重组外膜蛋白LOLA诱导小鼠产生免疫保护力的研究  

Study of the immunoprotective efficacy of recombinant outer membrane carry protein LOLA of Actinobacillus pleuropneumonia in mice

作　　者：张宇航[1] 张雨龙 杨婷婷[1] 黄复深[1,2] ZHANG Yu-hang;ZHANG Yu-long;YANG Ting-ting;HUANG Fu-shen(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China;Hunan Engineering Technology Research Center of Veterinary Drug,Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]湖南农业大学湖南省兽药工程技术研究中心,湖南长沙410128

出　　处：《中国预防兽医学报》2020年第6期607-611,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：湖南省教育厅重点项目(16A101);湖南省自然基金(2017JJ2116)。

摘　　要：为筛选出具有广谱抗猪传染性胸膜肺炎作用的疫苗候选分子,本研究利用PCR技术从血清1型猪传染性胸膜肺炎放线杆菌(App)基因组克隆外膜蛋白(Omp)lola基因。经生物信息学分析显示,App lola基因由633 bp组成,编码210个氨基酸,不同的血清型之间具有很高的同源性。将App lola克隆至pET-32a(+),转化大肠杆菌,表达重组蛋白rLOLA。取4周龄小鼠60只,随机分为6组,分别于0、2、4周经皮下免疫:弗氏不完全佐剂(IFA)+10μg rLOLA(低剂量组)、IFA+30μg rLOLA(中剂量组)、IFA+50μg rLOLA(高剂量组)、30μg rLOLA(rLOLA组)、IFA(对照组)和生理盐水(对照组)。ELISA检测结果显示,第3次免疫接种后相较于对照组(IFA组和生理盐水组),实验组小鼠血清IgG水平均明显升高,尤以高剂量组小鼠(IFA+50μg rLOLA)更显著(p<0.01)。于第3次免疫后2周,利用10×LD50 App对小鼠腹腔注射攻毒,5 d后对照组小鼠全部死亡,30μg rLOLA、IFA+50μg rLOLA、IFA+30μg rLOLA、IFA+10μg rLOLA免疫组小鼠存活率分别为20%、40%、20%和10%。上述结果表明,IFA+rLOLA可以诱导部分抗App攻击感染的免疫保护作用。本研究为进一步研究广谱抗猪传染性胸膜肺炎疫苗积累了基础资料。In order to screen candidate vaccines with broad-spectrum and disease-resistant effects,the outer membrane protein(Omp)lolagene was amplified by PCR from genome of Actinobacillus pleuropneumoniae(App)sera type I.Bioinformatics analysis revealed that the OmpLolA consists of 633 bp,encoding 210 amino acids.Homology analysis showed that the sequence shared high similarity between different serotypes of App.In this study,the App lola was inserted into pET-32a(+)vector and then transformed into Escherichia coli BL21 to express the recombinant protein LOLA(rLOLA).Sixty 4-week old mice were randomly divided into 6 groups and 10 mice in each group.Mice in each groups were immunized with Freund's incomplete adjuvants(IFA)+10μg rLOLA(low-dose group),IFA+30μg rLOLA(medium-dose group),IFA+50μg rLOLA(high-dose group),30μg rLOLA(rLOLA group),IFA(control group)or normal saline(control group)by subcutaneous injection.After 3 times immunization,the IgG levels in serum were detected by enzyme-linked immunosorbent assay(ELISA)which showed that the serum IgG levels of mice in the experimental group were significantly higher than those of in the control group(IFA and saline immunization group),especially significant difference were observed in the high-dose group(IFA+50μg rLOLA)(p<0.01).Two weeks after the third immunization,mice were injected intraperitoneally with 10×LD50 App for infection.Animal experiment results showed that survival rate in group IFA+50μg rLOLA,IFA+30μg rLOLA,30μg rLOLA and 10μg IFA+rLOLA was 40%,20%,20%and 10%.No mice survived in adjuvant group and control group.The above results indicate that IFA+rLOLA induce a partial immune protection in mice against App challenged infection.This study accumulated data for exploring new vaccines of porcine contagious pleuropneumonia.

关 键 词：猪胸膜炎放线杆菌 重组LOLA蛋白 小鼠 免疫保护效果 

分 类 号：S852.61[农业科学—基础兽医学]