表达NDV HN和IBDV VP2融合蛋白的重组减毒沙门菌的构建及其生物学特性分析
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摘要
为构建表达鸡新城疫病毒(NDV)HN和传染性法氏囊病病毒(IBDV) VP2融合蛋白的重组减毒沙门菌,并分析其生物学特性。本研究扩增了NDV HN和IBDV VP2主要抗原基因片段,通过重叠延伸PCR扩增获得HN-VP2融合基因,构建重组质粒pYA-HN-VP2,将该重组质粒电转化至减毒鼠伤寒沙门菌SL1344ΔcrpΔcyaΔasd中,获得表达HN-VP2融合蛋白的减毒重组菌SL1344ΔcrpΔcyaΔasd (pYA-HN-VP2),并分析其生化特性、遗传稳定性和毒力等生物学特性。Western blot结果显示,重组减毒沙门菌可分泌表达HN-VP2融合蛋白,大小为84 ku,且该融合蛋白分别能与相应特异性阳性血清发生反应;生物学特性结果显示,重组菌株失去了利用麦芽糖、山梨醇及乳糖等碳源的能力,其生长速度显著慢于野生菌株SL1344;毒力较野生菌株至少下降5个对数单位,且能够稳定遗传重组质粒pYA-HN-VP2。本研究为鸡新城疫-传染性法氏囊病-沙门菌病的三联口服疫苗研制奠定重要基础。
In order to construct the recombinant attenuated Salmonella which expresses Newcastle disease virus(NDV) HN and Infectious bursal disease virus(IBDV) VP2 fusion protein and analyze its biological characteristics, the recombinant vector pYA-HN-VP2 was constructed by inserting the fusion gene fragment HN-VP2 into prokaryotic expression vector pYA3493, and SL1344ΔcrpΔcyaΔasd(pYA-HN-VP2) was constructed by transform the recombinant vector pYA-HN-VP2 into attenuated Salmonella typhimurium SL1344ΔcrpΔcyaΔasd. The results of western blot showed that SL1344ΔcrpΔcyaΔasd(pYA-HN-VP2)could express and secrete 84 ku HN-VP2 fusion protein, which could react with specific positive sera against HN or VP2 protein,respectively. Biological characteristics results showed that recombination incapacitates the strain digesting ferment maltose, sorbitol and lactose, and the growth rate of the recombinant strain was slower than wt SL1344. LD_(50) of recombinant strain reduced at least 5 Logarithmic units compared with SL1344 and recombinant plasmid pYA-HN-VP2 could be stably inherited in the recombinant salmonella strain. These results provide new insights for triple oral vaccine candidate of ND, IBD and salmonellosis investigation.
In order to construct the recombinant attenuated Salmonella which expresses Newcastle disease virus(NDV) HN and Infectious bursal disease virus(IBDV) VP2 fusion protein and analyze its biological characteristics, the recombinant vector pYA-HN-VP2 was constructed by inserting the fusion gene fragment HN-VP2 into prokaryotic expression vector pYA3493, and SL1344ΔcrpΔcyaΔasd(pYA-HN-VP2) was constructed by transform the recombinant vector pYA-HN-VP2 into attenuated Salmonella typhimurium SL1344ΔcrpΔcyaΔasd. The results of western blot showed that SL1344ΔcrpΔcyaΔasd(pYA-HN-VP2)could express and secrete 84 ku HN-VP2 fusion protein, which could react with specific positive sera against HN or VP2 protein,respectively. Biological characteristics results showed that recombination incapacitates the strain digesting ferment maltose, sorbitol and lactose, and the growth rate of the recombinant strain was slower than wt SL1344. LD_(50) of recombinant strain reduced at least 5 Logarithmic units compared with SL1344 and recombinant plasmid pYA-HN-VP2 could be stably inherited in the recombinant salmonella strain. These results provide new insights for triple oral vaccine candidate of ND, IBD and salmonellosis investigation.
引文
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[2]Si Wei,Yu Shen-ye,Liu Heng-gui,et al.A bacterial ghost improves the immunological efficacy of a Newcastle disease virus inactivated vaccine[J].Vet Microbiol,2017,203:189-195.
[3]Dey S,Chellappa M M,Pathak D C,et al.Newcastle disease virus vectored bivalent vaccine against virulent infectious bursal disease and Newcastle disease of chickens[J].Vaccines,2017,5(4):pii:E31.
[4]Yang Xin,Zhou Ying-shun,Li Jia-nan,et al.Recombinant infectious bronchitis virus(IBV)H120 vaccine strain expressing the hemagglutinin-neuraminidase(HN)protein of Newcastle disease virus(NDV)protects chickens against IBV and NDV challenge[J].Arch Virol,2016,161(5):1209-1216.
[5]Liu Lin-lin,Zhang Wang,Song Yu-xin,et al.Recombinant Lactococcus lactis co-expressing OmpH of an M cell-targeting ligand and IBDV-VP2 protein provide immunological protection in chickens[J].Vaccine,2018,36(5):729-735.
[6]Liu Jing-jing,Zhu Jie,Xu Hai-xu,et al.Effects of the HN antigenic difference between the vaccine strain and the challenge strain of Newcastle disease virus on virus shedding and transmission[J].Viruses,2017,9(8):225.
[7]Hajam I A,Lee J H.An influenza HA and M2e based vaccine delivered by a novel attenuated Salmonella mutant protects mice against homologous H1N1 infection[J].Front Microbiol,2017,15:872.
[8]Ding Ke,Shang Ke,Yu Zu-hua,Recombinant-attenuated Salmonella Pullorum strain expressing the hemagglutinin-neuraminidase protein of Newcastle disease virus(NDV)protects chickens against NDV and Salmonella Pullorum challenge[J].JVet Sci,2018,19(2):232-241.
[9]Zhang Dan,Huang Xiao-bo,Zhang Xiao-hui,et al.Construction of an oral vaccine for transmissible gastroenteritis virus based on the TGEV N gene expressed in an attenuated Salmonella typhimurium vector[J].J Virol Methods,2016,227:6-13.
[10]Lalsiamthara J,Won G,Lee J H.Effect of immunization routes and protective efficacy of Brucella antigens delivered via Salmonella vector vaccine[J].J Vet Sci,2018,19(3):416-425.
[11]Chin'ombe N,Lebeko M,Kgatle M.Overexpression of recombinant HIV-1 subtype C Tat and Nef in a Salmonella vaccine vector[J].Pan Afr Med J,2013,16(19):19.
[12]El Mouali Y,Gaviria-cantin T,Sanchez-romero M A,et al.CRP-cA MP mediates silencing of Salmonella virulence at the post-transcriptional level[J].PLoS Genet,2018,14(6):e1007401.
[13]Zhao Xin-xin,Dai Qin-long,Jia Ren-yong,et al.Two novel Salmonella bivalent vaccines confer dual protection against two Salmonella serovars in mice[J].Front Cell Infect Microbiol,2017,7:391.
[14]Liu Xue-yan,Liu Qing,Xiao Kang-peng,et al.Attenuated Salmonella Typhimurium delivery of a novel DNA vaccine induces immune responses and provides protection against duck enteritis virus[J].Vet Microbiol,2016,186:189-198.
[15]李正,程相朝,张春杰,等.鼠伤寒沙门氏菌SL1344株ΔcrpΔcya缺失株的构建及其生物学特性初步研究[J].中国预防兽医学报,2012,34(7):534-538.
[16]Deutscher J.The mechanisms of carbon catabolite repression in bacteria[J].Curr Opin Microbiol,2008,11(2):87-93.
[17]杨亚东,张俊峰,张春杰,等.稳定表达PRRSV ORF5-ORF6融合基因重组减毒猪霍乱沙门菌的构建及其生物学特性[J].中国兽医科学,2017,47(01):47-53.
[18]Chen Song-biao,Liao Cheng-shui,Zhang Chun-jie,et al.Roles of the crp and sipB genes of Salmonella enterica serovar Typhimurium in protective efficacy and immune responses to vaccination in mice[J].Can J Vet Res,2018,82(2):102-105.
[19]Pradhan S N,Prince P R,Madhumathi J,et al.Protective immune responses of recombinant VP2 subunit antigen of infectious bursal disease virus in chickens[J].Vet Immunol Immunopathol,2012,148(3-4):293-301.
[20]Pradhan S N,Prince P R,Madhumathi J,et al.DNA vaccination with VP2 gene fragment confers protection against infectious bursal disease virus in chickens[J].Vet Microbiol,2014,171(1-2):13-22.
[21]Zhao Xin-xin,Dai Qin-long,Zhu De-kang,et al.Recombinant attenuated Salmonella Typhimurium with heterologous expression of the Salmonella Choleraesuis O-polysaccharide:high immunogenicityand protection[J].Sci Rep,2017,7(1):7127.
[22]Lalsiamthara J,Kamble N M,Lee J H.A live attenuated Salmonella Enteritidis secreting detoxified heat labile toxin enhances mucosal immunity and confers protection against wild-type challenge in chickens[J].Vet Res,2016,47(1):60.
[23]Chen Hua-you,Wu Bang-guo,Zhang Tian-xi,et al.Effect of linker length and flexibility on the Clostridium thermocellum esterase displayed on Bacillus subtilis spores[J].Appl Biochem Biotechnol,2017,182(1):168-180.
[24]Chen Rong,Zhou Jing-jing,Qin Ling-yun,et al.A fusion protein of the p53 transaction domain and the p53-binding domain of the oncoprotein MdmX as anefficient system for high-throughput screening of MdmX inhibitors[J].Biochemistry,2017,56(25):3273-3282.