迟缓爱德华菌外膜蛋白OmpA原核表达及其免疫原性研究
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摘要
为研究迟缓爱德华菌外膜蛋白OmpA免疫保护性,本研究利用PCR方法扩增迟缓爱德华菌ompA基因,构建重组载体pET-32a-ompA,将其转化大肠杆菌BL21后诱导表达,表达产物经SDS-PAGE和western blot分析显示,重组蛋白大小约58 ku;将纯化的重组蛋白免疫小鼠后,以迟缓爱德华菌强毒株ET-13攻毒,结果显示该重组蛋白对免疫组小鼠具有保护力,保护率为55%。本研究克隆了迟缓爱德华菌ompA基因并表达了相应重组蛋白,免疫小鼠后能够提供一定保护,为重组OmpA蛋白亚单位疫苗的研制奠定基础。
To study the immunoprotection of OmpA against Edwardsiella tarda in mice, the encoding gene, ompA, was amplified by PCR from E.tarda ET-13 and cloned into the pET-32 a vector. The recombinant plasmid was transformed into E.coli BL21(DE3) cells, in which a recombinant OmpA protein(rOmpA) was expressed by inducing with IPTG. SDS-PAGE and western blot analysis showed that the expressed rOmpA was about 60 ku which was recognized by the positive serum against E.tarda. The mice were immunized intramuscularly with purified rOmpA, and challenged with the E.tarda ET-13. Result showed that the rOmpA provided a 55% protection for the immunized mice. The data demonstrated that the OmpA could be used as the antigen for developing the subunit vaccine.
To study the immunoprotection of OmpA against Edwardsiella tarda in mice, the encoding gene, ompA, was amplified by PCR from E.tarda ET-13 and cloned into the pET-32 a vector. The recombinant plasmid was transformed into E.coli BL21(DE3) cells, in which a recombinant OmpA protein(rOmpA) was expressed by inducing with IPTG. SDS-PAGE and western blot analysis showed that the expressed rOmpA was about 60 ku which was recognized by the positive serum against E.tarda. The mice were immunized intramuscularly with purified rOmpA, and challenged with the E.tarda ET-13. Result showed that the rOmpA provided a 55% protection for the immunized mice. The data demonstrated that the OmpA could be used as the antigen for developing the subunit vaccine.
引文
[1]Golub V,Kim A C,Krol V.Surgical wound infection,tuboovarian abscess,and sepsis caused by Edwardsiella tarda:case reports and literature review[J].Infection,2010,38(6):487-489.
[2]Li Jie,Mo Zhao-lan,Li Gui-yang,et al.Generation and evaluation of virulence attenuated mutants of Edwardsiella tarda as vaccine candidates to combat edwardsiellosis in flounder(Paralichthys olivaceus)[J].Fish Shellfish Immunol,2015,43(1):175-180.
[3]Suezawa C,Yasuda M,Negayama K,et al.Identification of genes associated with the penetration activity of the human type of Edwardsiella tarda EdwG II through human colon epithelial cell monolayers[J].Microb Pathog,2016,95:148-156.
[4]Hirai Y,Asahata-Tago S,Ainoda Y,et al.Edwardsiella tarda bacteremia.A rare but fatal water-and foodborne infection:Review of the literature and clinical cases from a single centre[J].Can J Infect Dis Med Microbiol,2015,26(6):313-318.
[5]Katharios P.Virulence regulation during late infection by a fish pathogen;sense and sensibility of bacteria may lead to novel vaccine development strategy[J].Virulence,2017,8(7):1-3.
[6]胡晓娜,朱瑞良,刘红珍,等.禽波氏杆菌外膜蛋白的提取及其免疫原性的检测[J].微生物学报,2007,47(04):714-717.
[7]朱必凤,杨旭夫,彭凌,等.副猪嗜血杆菌外膜蛋白的免疫原性[J].中国兽医学报,2013,33(06):838-842.
[8]田丁,许斌福,林能峰,等.创伤弧菌外膜蛋白免疫刺激复合物对欧洲鳗鲡的免疫保护性分析[J].水生生物学报,2010,34(02):431-435.
[9]李洪涛,张新涛,刘明,等.A型禽流感病毒不同拷贝数基质蛋白胞外区基因的原核表达及免疫保护力研究[J].中国预防兽医学报,2010,32(10):795-799.
[10]徐全圆,石明,杨夷平,等.马链球菌马亚种FNEB蛋白的截短表达及其免疫保护性研究[J].中国预防兽医学报,2015,37(02):132-135.
[11]王宝松,李明义,单虎,等.猪链球菌2型MRP-FBP串联表达蛋白对小鼠的免疫保护力[J].中国兽医科学,2009,39(06):522-526.
[12]张斌,汤承,岳华.副猪嗜血杆菌主要外膜蛋白基因研究进展[J].动物医学进展,2013,34(01):91-96.
[13]Chu Chun-yen,Liu Chia-hui.Development of a subunit vaccine containing recombinant Riemerella anatipestifer outer membrane protein A and Cp G OD7N adjuvant[J].Vaccine,2015,33(1):92-99.
[14]Zhang Xiao-jiao,Yang Tian-xiang,Cao Ju,et al.Mucosal immunization with purified OmpA elicited protective immunity against infections caused by multidrug-resistant Acinetobacter baumannii[J].Microb Pathog,2016,96:20-25.
[15]Simborio H L,Reyes A W,Hop H T,et al.Immune modulation of recombinant OmpA against Brucella abortus 544 infection in mice[J].J Microbiol Biotechnol,2016,26(3):603-609.
[16]Ou Chang-bo,Tian De-yu,Ling Yong,et al.Evaluation of an omp A-based phage-mediated DNA vaccine against Chlamydia abortus in piglets[J].International Immunopharmacol,2013,16(4):505-510.
[17]Zhu Fu-jie,Liu Xiao,Sun Zhen-hong,et al.Immune-enhancing effects of taishan pinus massoniana pollen polysaccharides on DNA vaccine expressing bordetella avium omp A[J].Front Microbiol,2016,7(66):1-8.
[2]Li Jie,Mo Zhao-lan,Li Gui-yang,et al.Generation and evaluation of virulence attenuated mutants of Edwardsiella tarda as vaccine candidates to combat edwardsiellosis in flounder(Paralichthys olivaceus)[J].Fish Shellfish Immunol,2015,43(1):175-180.
[3]Suezawa C,Yasuda M,Negayama K,et al.Identification of genes associated with the penetration activity of the human type of Edwardsiella tarda EdwG II through human colon epithelial cell monolayers[J].Microb Pathog,2016,95:148-156.
[4]Hirai Y,Asahata-Tago S,Ainoda Y,et al.Edwardsiella tarda bacteremia.A rare but fatal water-and foodborne infection:Review of the literature and clinical cases from a single centre[J].Can J Infect Dis Med Microbiol,2015,26(6):313-318.
[5]Katharios P.Virulence regulation during late infection by a fish pathogen;sense and sensibility of bacteria may lead to novel vaccine development strategy[J].Virulence,2017,8(7):1-3.
[6]胡晓娜,朱瑞良,刘红珍,等.禽波氏杆菌外膜蛋白的提取及其免疫原性的检测[J].微生物学报,2007,47(04):714-717.
[7]朱必凤,杨旭夫,彭凌,等.副猪嗜血杆菌外膜蛋白的免疫原性[J].中国兽医学报,2013,33(06):838-842.
[8]田丁,许斌福,林能峰,等.创伤弧菌外膜蛋白免疫刺激复合物对欧洲鳗鲡的免疫保护性分析[J].水生生物学报,2010,34(02):431-435.
[9]李洪涛,张新涛,刘明,等.A型禽流感病毒不同拷贝数基质蛋白胞外区基因的原核表达及免疫保护力研究[J].中国预防兽医学报,2010,32(10):795-799.
[10]徐全圆,石明,杨夷平,等.马链球菌马亚种FNEB蛋白的截短表达及其免疫保护性研究[J].中国预防兽医学报,2015,37(02):132-135.
[11]王宝松,李明义,单虎,等.猪链球菌2型MRP-FBP串联表达蛋白对小鼠的免疫保护力[J].中国兽医科学,2009,39(06):522-526.
[12]张斌,汤承,岳华.副猪嗜血杆菌主要外膜蛋白基因研究进展[J].动物医学进展,2013,34(01):91-96.
[13]Chu Chun-yen,Liu Chia-hui.Development of a subunit vaccine containing recombinant Riemerella anatipestifer outer membrane protein A and Cp G OD7N adjuvant[J].Vaccine,2015,33(1):92-99.
[14]Zhang Xiao-jiao,Yang Tian-xiang,Cao Ju,et al.Mucosal immunization with purified OmpA elicited protective immunity against infections caused by multidrug-resistant Acinetobacter baumannii[J].Microb Pathog,2016,96:20-25.
[15]Simborio H L,Reyes A W,Hop H T,et al.Immune modulation of recombinant OmpA against Brucella abortus 544 infection in mice[J].J Microbiol Biotechnol,2016,26(3):603-609.
[16]Ou Chang-bo,Tian De-yu,Ling Yong,et al.Evaluation of an omp A-based phage-mediated DNA vaccine against Chlamydia abortus in piglets[J].International Immunopharmacol,2013,16(4):505-510.
[17]Zhu Fu-jie,Liu Xiao,Sun Zhen-hong,et al.Immune-enhancing effects of taishan pinus massoniana pollen polysaccharides on DNA vaccine expressing bordetella avium omp A[J].Front Microbiol,2016,7(66):1-8.