坏死梭杆菌亚单位疫苗候选抗原蛋白的筛选、表达及免疫原性分析

doi:10.16431/j.cnki.1671-7236.2019.01.025

摘要/Abstract

摘要：

试验旨在筛选坏死梭杆菌（Fusobacterium necrophorum，Fn）外膜蛋白（outer membrane protein，Omp）中的候选抗原蛋白，为Fn的亚单位疫苗研究奠定基础。采用Uniprot对Fn Omp序列进行预测，基于预测结果筛选拟表达蛋白，并根据GenBank中登录的Fn Omp相应基因序列设计特异性引物，以QL03株为模板进行PCR扩增、原核表达，以及SDS-PAGE和Western blotting分析，并通过血清杀菌试验和小鼠免疫攻毒保护试验，鉴定重组蛋白的免疫原性，筛选最佳候选蛋白。结果显示，预测得到121个蛋白，顺次命名为1-Fn~121-Fn，根据预测结果筛选出8-Fn、11-Fn、41-Fn、95-Fn和102-Fn 5个候选蛋白，其PCR扩增产物大小分别为672、1 164、570、1 059、729 bp。SDS-PAGE和Western blotting分析显示，除8-Fn未表达外，其余4个蛋白均成功表达，大小分别为60、39、59、44 ku，命名为P11-Fn、P41-Fn、P95-Fn和P102-Fn，均能与Fn阳性血清反应。血清杀菌试验结果显示，4个重组蛋白均有一定杀菌效果，其中抗P102-Fn血清的杀菌效果最佳，杀菌率可达40.49%。小鼠免疫保护试验结果显示，P11-Fn、P41-Fn、P95-Fn和P102-Fn对小鼠均具有一定的保护效果，其中P102-Fn保护效率最高，达60%。结果表明，P11-Fn、P41-Fn、P95-Fn和P102-Fn均具有良好的免疫原性，其中P102-Fn的免疫原性及诱导机体产生保护免疫反应能力最佳，最有望成为Fn亚单位疫苗的候选蛋白。

关键词: 坏死梭杆菌; 原核表达; 外膜蛋白; 免疫原性; 杀菌试验

Abstract:

This study was aimed to screen the sub-unit vaccine candidate antigen protein of outer membrane protein (Omp) of Fusobacterium necrophorum (Fn),and lay the foundation for the development sub-unit vaccine of Fn.Fn Omp sequence was predicted by Uniprot,and the expression protein was screened based on the predicted results.Specific primers were designed according to the corresponding gene sequence of Fn Omp in GenBank,the QL03 strain was used as template for PCR amplification,prokaryotic expression,SDS-PAGE and Western blotting analysis.The proteins immunogenicity were analyzed by bactericidal assays and vaccination efficacy assessment in mice to screen the best candidate protein.The results showed that 121 proteins were obtained through the predicting outcomes,named 1-Fn to 121-Fn in turn,and 8-Fn,11-Fn,41-Fn,95-Fn and 102-Fn were screened to be candidate proteins,the fragments of 672,1 164,570,1 059 and 729 bp were amplified by PCR.SDS-PAGE and Western blotting results showed that 8-Fn expressed unsuccessfully,and the sizes of the others expressed proteins were 60,39,59 and 44 ku,which named P11-Fn,P41-Fn,P95-Fn and P102-Fn,and all proteins could react with Fn positive serum.The bactericidal assays results showed the 4 recombinant proteins had certain bactericidal effects,and the anti-P102-Fn serum was the best,the sterilizing rate reached 40.49%.The vaccination efficacy assessment results in mice showed the 4 recombinant proteins could protect mice from the challenge of QL03,and the P102-Fn was the best,the protection rate reached 60%.These results indicated that P11-Fn,P41-Fn,P95-Fn and P102-Fn were all of effective immunogenicity,P102-Fn could induce an immune response and it might be useful as an antigen for vaccine.

Key words: Fusobacterium necrophorum (Fn); prokaryotic expression; outer membrane protein (Omp); immunogenicity; bactericidal assay

中图分类号:

S852.61⁺6.3

肖璐, 谢晶, 康润敏, 叶勇刚, 于吉锋, 曹冶, 李兴玉, 魏勇, 林毅, 廖党金, 潘梦, 叶健强, 戴卓建. 坏死梭杆菌亚单位疫苗候选抗原蛋白的筛选、表达及免疫原性分析[J]. 《中国畜牧兽医》, 2019, 46(1): 215-222.

XIAO Lu, XIE Jing, KANG Runmin, YE Yonggang, YU Jifeng, CAO Ye, LI Xingyu, WEI Yong, LIN Yi, LIAO Dangjin, PAN Meng, YE Jianqiang, DAI Zhuojian. Screening,Expression and Immunogenicity Analysis of Sub-unit Vaccine Candidate Antigen Protein of Fusobacterium necrophorum[J]. , 2019, 46(1): 215-222.

参考文献

[1] 陆承平.兽医微生物学[M].北京:中国农业出版社,2001. LU C P.Veterinary Microbiology[M].Beijing:China Agriculture Press,2001.(in Chinese)
[2] 孟祥玉,佟盼盼,冯二凯,等.坏死梭杆菌研究进展[J].动物医学进展,2017,5:86-89. MENG X Y,TONG P P,FENG E K,et al.Progress on Fusobacterium necrophorum[J].Progress in Veterinary Medicine,2017,5:86-89.(in Chinese)
[3] TERRY R.Human infection with Fusobacterium necrophorum (necrobacillosis),with a focus on Lemierre's syndrome[J].Clinical Microbiology Reviews,2007,20(4):622-625.
[4] ROGERS M B,BROWER-SINNING R,FIREK B,et al.Acute appendicitis in children is associated with a local expansion of Fusobacteria[J].Clinical Infectious Diseases an Official Publication of the InfectiousDiseases Society of America,2016,63(1):71-78.
[5] 李世宏,杨亚军,孔晓军,等.奶牛腐蹄病综合防治措施[J].中国草食动物科学,2017,37(5):48-50. LI S H,YANG Y J,KONG X J,et al.Comprehensive prevention and control measures for cow foot rot[J].Chinese Herbivorous Animal Science,2017,37(5):48-50.(in Chinese)
[6] 谢晶,廖党金,叶勇刚,等.坏死梭杆菌QL03株绵羊感染模型的建立及免疫保护性试验[J].中国草食动物科学,2012,32(5):50-53. XIE J,LIAO D J,YE Y G,et al.Study on development and immunity of an infection model in sheep induced with Fusobacterium necrophorum isolate QL03 from dairy cattle[J].Chinese Herbivorous Animal Science,2012,32(5):50-53.(in Chinese)
[7] KUPPALLI K,LIVORSI D,TALATI N J,et al.Lemierre's syndrome due to Fusobacterium necrophorum:The lancet infectious diseases[J].Lancet Infectious Diseases,2012,12(10):808-815.
[8] 胡玲玲,汤德元,曾智勇,等.贵州某动物园羊驼坏死梭杆菌感染的诊治[J].中国兽医杂志,2017,5:49-50. HU L L,TANG D Y,ZENG Z Y,et al.Diagnosis and treatment of Fusobacterium necrophorum infection of alpaca in a Guizhou zoo[J].Chinese Journal of Veterinary Medicine,2017,5:49-50.(in Chinese)
[9] 王启菊,李稳欣,金花,等.海西地区绵羊羔羊"肝脓肿病"病因的分子生物学鉴定[J].青海畜牧兽医杂志,2017,47(4):21-23. WANG Q J,LI W X,JIN H,et al.Diagnose of pathogens on liver abscess from lamb in Haixi area of Qinghai province[J].Qinghai Journal of Animal Husbandry and Veterinary Medicine,2017,47(4):21-23.(in Chinese)
[10] 鱼艳荣,刘希成,张彦明,等.革兰氏阴性菌外膜蛋白研究进展[J].动物医学进展,2000,2:35-39. YU Y R,LIU X C,ZHANG Y M,et al.Progress ofsutdy on membrane protein of gram-negative bacteria[J].Progress in Veterinary Medicine,2000,2:35-39.(in Chinese)
[11] 代先祝,邵娜娜,罗峰.革兰氏阴性细菌β-桶状结构外膜蛋白体外折叠的研究进展[J].微生物学报,2014,54(11):1241-1247. DAI X Z,SHAO N N,LUO F,et al.The in vitro refolding of β-barrel outer membrane protein of gram-negative bacteria[J].Acta Microbiologica Sinica,2014,54(11):1241-1247.(in Chinese)
[12] PAUTSCH A,SCHULZ G E.Structure of the outer membrane protein A transmembrane domain[J].Nature Structural Biology,1998,5(11):1013.
[13] MENON S,PILLAI D K,NARAYANAN S.Characterization of Fusobacterium necrophorum subsp.necrophorum outer membrane proteins[J].Anaerobe,2018,50:101-105.
[14] KUMAR A,PETERSON G,NAGARAJA T G,et al.Outer membrane proteins of Fusobacterium necrophorum subsp.necrophorum and subsp.funduliforme[J].Journal of Basic Microbiology,2015,54(8):812-817.
[15] KUMAR A,MENON S,NAGARAJA T G,et al.Identification of an outer membrane protein of Fusobacterium necrophorum subsp.necrophorum that binds with high affinity to bovine endothelial cells[J].Veterinary Microbiology,2015,176(1-2):196-201.
[16] 谢晶,廖党金,汪明,等.四川奶牛腐蹄病主要病原菌调查[J].中国兽医杂志,2013,49(4):35-37. XIE J,LIAO D J,WANG M,et al.The main pathogen of the foot rot of milk cow in Sichuan province[J].Chinese Journal of Veterinary Medicine,2013,49(4):35-37.(in Chinese)
[17] 张春梅,施传信,韩进诚,等.豫西北地区奶牛蹄病的调查分析和防治措施[J].黑龙江畜牧兽医,2018,6:86-88. ZHANG C M,SHI C Y,HAN J C,et al.Investigation and analysis of hoof disease in cows in Northwest Henan province and its prevention and control measures[J].Heilongjiang Animal Science and Veterinary Medicine,2018,6:86-88.(in Chinese)
[18] 李稳欣,薛慧文,张学勇,等.羊场环境中绵羊腐蹄病病原的检测[J].中国兽医学报,2018,38(4):662-664. LI W X,XUE H W,ZHANG X Y,et al.Detection of the pathogens from footrot disease in sheep farm environment[J].Chinese Journal of Veterinary Science,2018,38(4):662-664.(in Chinese)
[19] ROMEROSTEINER S,SPEAR W,BROWN N,et al.Measurement of serum bactericidal activity specific for Haemophilus influenzae type b by using a chromogenic and fluorescent metabolic indicator[J].Clinical & Diagnostic Laboratory Immunology,2004,11(1):89-93.
[20] FRASCH C E,BORROW R,DONNELLY J.Bactericidal antibody is the immunologic surrogate of protection against meningococcal disease[J].Vaccine,2009,27(2):112-116.
[21] KANG J G,LEE H W,KO S,et al.Comparative proteomic analysis of outer membrane protein 43(omp43)-deficient Bartonella henselae[J].Journal of Veterinary Science,2018,19(1):59-70.
[22] SONG X,LI C,QI K,et al.The role of the outer membrane protein gene ybjX in the pathogenicity of Avian pathogenic Escherichia coli[J].Avian Pathology,2018,47(3):294-299.
[23] 吴洪超,刘晓颖,冯二凯,等.坏死梭杆菌120 ku血凝素相关外膜蛋白的原核表达及免疫原性分析[J].中国畜牧兽医,2015,42(11):2843-2849. WU H C,LIU X Y,FENG E K,et al.Prokaryotic expression and immunogenicity analysis of hemagglutinin-related outer membrane protein from F.necrophorum[J].China Animal Husbandry & Veterinary Medicine,2015,42(11):2843-2849.(in Chinese)
[24] 徐晶,陈立志,李淑艳,等.坏死梭杆菌44.5 ku外膜蛋白的免疫原性分析[J].中国兽医科学,2016,4:512-515. XU J,CHEN L Z,LI S Y,et al.Antigenicity analysis of 44.5 ku outer membrane proteins of Fusobacteriumnecrophorum[J].Chinese Veterinary Science,2016,4:512-515.(in Chinese)