牛多杀性巴氏杆菌znuA基因的原核表达及其免疫保护作用的研究
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摘要
为研究牛多杀性巴氏杆菌(P.multocida)高亲和力锌吸收蛋白znuA的免疫学活性及其免疫保护作用,本研究利用PCR方法扩增了P.multocida znuA基因,构建表达载体pET-28a-znuA,将其转化E.coli BL21后经诱导表达。表达产物经SDS-PAGE和western blot分析显示,重组蛋白约40 ku;以重组znuA蛋白免疫小鼠后用P.multocida菌株Y-1攻毒,结果显示重组znuA蛋白对免疫组小鼠保护率为60%,表明其具有免疫保护作用。本研究首次在原核系统中表达了P.multocida znuA蛋白,且验证了其免疫保护力,为深入探究znuA基因在P.multocida致病过程中的作用及其亚单位疫苗的开发奠定了基础。
To study the immunoprotection of high-affinity zinc uptake system protein znuA against in mice. The target gene,znuA, was amplified by PCR from Pasteurella multocida of Bovine and cloned into the pET-28a vector. The constructed recombinant plasmid pET-28a-znuA was transformed to E.coli BL21(DE3)for expression under induction of IPTG. SDS-PAGE and western blot analysis showed that the expressed recombinant znuA was about 40 ku. The mice were immunized intramuscularly with purified recombinant znuA, and challenged with the Pasteurella multocida Y-1, result showed that the recombinant znuA provided a 60% protection for the immunized mice. All results indicated that the target protein can be over expressed in E.coli BL21, which would induced protective immune response in mice, indicating its potential as an antigen of Pasteurella multocida protein vaccine.
To study the immunoprotection of high-affinity zinc uptake system protein znuA against in mice. The target gene,znuA, was amplified by PCR from Pasteurella multocida of Bovine and cloned into the pET-28a vector. The constructed recombinant plasmid pET-28a-znuA was transformed to E.coli BL21(DE3)for expression under induction of IPTG. SDS-PAGE and western blot analysis showed that the expressed recombinant znuA was about 40 ku. The mice were immunized intramuscularly with purified recombinant znuA, and challenged with the Pasteurella multocida Y-1, result showed that the recombinant znuA provided a 60% protection for the immunized mice. All results indicated that the target protein can be over expressed in E.coli BL21, which would induced protective immune response in mice, indicating its potential as an antigen of Pasteurella multocida protein vaccine.
引文
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[16]Olsen W A,Follmann F,Erneholm K,et al.Protection against chlamydia trachomatis infection and upper genital tract pathological changes by vaccine-promoted neutralizing antibodies directed to the VD4 of the major outer membrane protein[J].J Infect Dis,2015,212(6):978.
[17]刘丽娜,张洁,周静,等.金黄色葡萄球菌疫苗候选分子StbA主要抗原表位区的表达及重组蛋白抗血清的制备[J].中国兽医科学,2015,(4):375-379.
[18]韩明月,潘秀珍,邵珠卿,等.2型猪链球菌中国强毒株znuA基因的原核表达及免疫学活性分析[J].免疫学杂志,2010,(3):220-223.
[2]Dziva F,Muhairwa A P,Bisgaard M,et al.Diagnostic and typing options for investigating diseases associated with Pasteurella multocida[J].Vet Microbiol,2008,128(1-2):1-22.
[3]Banerjee S,Wei B,Bhattacharyyapakrasi M,et al.Structural determinants of metal specificity in the zinc transport protein ZnuAfrom synechocystis 6803[J].J MBio,2003,333(5):1061-1069.
[4]Yang Xing-hong,Becker T,Walters N,et al.Deletion of znuAvirulence factor attenuates Brucella abortus and confers protection against wild-type challenge[J].Infect Immun,2006,74(7):3874-3879.
[5]Kim S,Watanabe K,Shirahata T,et al.Zinc uptake system(znuA locus)of Brucella abortus is essential for intracellular survival and virulence in mice[J].J Vet Med Sci,2004,66(9):1059-1063.
[6]Lewis D A,Klesneytait J,Lumbley S R,et al.Identification of the znuA-encoded periplasmic zinc transport protein of Haemophilus ducreyi[J].Infect Immun,1999,67(10):5060-5068.
[7]Ammendola S,Pasquali P,Pistoia C,et al.High-Affinity Zn2+uptake system ZnuABC is required for bacterial Zinc homeostasis in intracellular environments and contributes to the virulence of Salmonella enterica[J].Infect Immun,2007,75(12):5867-5876.
[8]Chen C Y,Morse S A.Identification and characterization of a high-affinity zinc uptake system in Neisseria gonorrhoeae[J].FEMS Microbiol Lett,2001,202(1):67-71.
[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]Tang Jia-qi,Wang Chang-jun,Feng You-jun,et al.Streptococcal toxic shock syndrome caused by Streptococcus suis serotype2[J].PLo S Med,2006,3(5):e151.
[11]Janulczyk R,Ricci S,Bj?rck L.MtsABC is important for manganese and iron transport,oxidative stress resistance,and virulence of Streptococcus pyogenes[J].Infect Immun,2003,71(5):2656-2564.
[12]Garmory H S,Titball R W.ATP-binding cassette transporters are targets for the development of antibacterial vaccines and therapies[J].Infect Immun,2004,72(12):6757-6763.
[13]Staats J J,Feder I,Okwumabua O,et al.Streptococcus suis:past and present[J].Vet Res Commun,1997,21(6):381-407.
[14]Bartelink A K,Van K E.Streptococcus suis as threat to pigfarmers and abattoir workers[J].Lancet,1995,346(8991-8992):1707.
[15]Gottschalk M,Segura M,Xu J.Streptococcus suis infections in humans:the Chinese experience and the situation in North America[J].Ani Heal Res Rev,2007,8(1):29-45.
[16]Olsen W A,Follmann F,Erneholm K,et al.Protection against chlamydia trachomatis infection and upper genital tract pathological changes by vaccine-promoted neutralizing antibodies directed to the VD4 of the major outer membrane protein[J].J Infect Dis,2015,212(6):978.
[17]刘丽娜,张洁,周静,等.金黄色葡萄球菌疫苗候选分子StbA主要抗原表位区的表达及重组蛋白抗血清的制备[J].中国兽医科学,2015,(4):375-379.
[18]韩明月,潘秀珍,邵珠卿,等.2型猪链球菌中国强毒株znuA基因的原核表达及免疫学活性分析[J].免疫学杂志,2010,(3):220-223.