利用毕赤酵母表达系统表达兔出血症病毒样颗粒及其免疫原性研究
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摘要
为研究毕赤酵母表达的兔病毒性出血症病毒(RHDV)病毒样颗粒(VLP)的免疫保护性,本研究将RHDV VP60的基因克隆到pPIC3.5K载体中,并导入毕赤酵母KM71菌株中,重组菌株经发酵和甲醇诱导,通过western blot检测目的蛋白的表达;透射电镜观察表达产物是否组装形成VLP;并通过动物试验评估VLP的免疫原性和攻毒保护效果。Western blot结果显示VP60蛋白能够在酵母内表达,电镜下观察到表达的VP60能够自发装配成大小均一的颗粒,与天然RHDV大小相当,约为30 nm～40 nm,表明表达的VP60可以自发组装成VLP。动物实验结果表明:表达的VLP与佐剂MONTANIDE GEL 01 PR混合后免疫实验兔,能够刺激实验兔产生保护性抗体,免疫后21 d攻毒,对实验兔的保护率达100%,且保护性抗体至少可以持续6个月。本研究利用毕赤酵母表达了RHDV的VLP,为研制RHDV亚单位疫苗奠定了基础。
To study the immunoprotecive effect of rabbit hemorrhagic disease virus(RHDV) virus-like particles(VLPs)expressed by the yeast of Pichia pastoris, the gene of RHDV VP60 was cloned into the pPIC3.5 k vector, then the resulting recombinant plasmid was transformed into P.pastoris KM71 strain to construct the recombinant yeasts. After fermentation and methanol induction of the recombinant yeasts, the expression of target protein was detected by western blot, and the VLPs was identfied by observation under transmission electron microscopy(TEM), then the immunogenicity and protection effect of the expressed VLPs were evaluated by animal experiments. Western blot detection showed that the VP60 proteins were efficiently expressed in yeast cells which was able to spontaneously assemble into 30 nm to 40 nm VLPs observed with TEM. Animal experiments showed that the expressed VLPs were capable to effectively stimulate the rabbits to produce protective antibody after emulsifying with MONTANIDE GEL 01 PR adjuvant, which lasted at least six months. Moveover, the immune protection rate of the VLPs for the immunized rabbits against RHDV challenge was 100% on the twenty-first day post the vaccination. This study provided a new way to development of RHDV subunit vaccine.
To study the immunoprotecive effect of rabbit hemorrhagic disease virus(RHDV) virus-like particles(VLPs)expressed by the yeast of Pichia pastoris, the gene of RHDV VP60 was cloned into the pPIC3.5 k vector, then the resulting recombinant plasmid was transformed into P.pastoris KM71 strain to construct the recombinant yeasts. After fermentation and methanol induction of the recombinant yeasts, the expression of target protein was detected by western blot, and the VLPs was identfied by observation under transmission electron microscopy(TEM), then the immunogenicity and protection effect of the expressed VLPs were evaluated by animal experiments. Western blot detection showed that the VP60 proteins were efficiently expressed in yeast cells which was able to spontaneously assemble into 30 nm to 40 nm VLPs observed with TEM. Animal experiments showed that the expressed VLPs were capable to effectively stimulate the rabbits to produce protective antibody after emulsifying with MONTANIDE GEL 01 PR adjuvant, which lasted at least six months. Moveover, the immune protection rate of the VLPs for the immunized rabbits against RHDV challenge was 100% on the twenty-first day post the vaccination. This study provided a new way to development of RHDV subunit vaccine.
引文
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[10]Bachmann A S,Corpuz G,Hareld W P,et al.A simple method for the rapid purification of copia virus-like particles from Drosophila Schneider 2 cells[J].J Virol Methods,2004,(115):159-165.
[11]Braeden D,Farah A B,Simon J P,et al.Multi-target chimaeric VLP as a therapeutic vaccine in a model of colorectal cancer[J].J Immunother Cancer,2017,5(69):1-13.
[12]World Health Organization.Human papillomavirus vaccines:WHO position paper[J].Wkly Epidemiol Rec,2014,89(43):465-492.
[13]Wood V,Gwilliam R,rejandream M A,et al.The genome sequence of Schizosaccharomyces pombe[J].Nature,2002,415:871-880.
[14]李超美,王芳,蔡少平,等.检测兔出血症病毒抗体间接ELISA方法的建立[J].江苏农业学报,2010,26(3):546-550.
[2]杜念兴,许为燕,刘胜江.一种新病毒-兔出血症病毒的鉴定初报[J].病毒学报,1989,2(2):146-152.
[3]Abrantes J,van der Loo W,Le Pendu J,et al.Esteves rabbit haemorrhagic disease(RHD)and rabbit haemorrhagic disease virus(RHDV):A review[J].Vet Res,2012,43(1):2879-2981.
[4]Zheng Xue-xing,Wang Shu-kun,Zhang Wei-jiao,et al.Development of a VLP-based vaccine in silkworm pupae against rabbit hemorrhagic disease virus[J].Int Immunopharmacol,2016,11(40):164-169.
[5]Hukowska-Szematowicz B,Tokarz-Deptula B,Deptula W.Genetic variation and phylogenetic analysis of rabbit haemorrhagic disease virus(RHDV)strains[J].Acta Biochim Pol,2012,59(4):459-465.
[6]Jiang Qian,Yu Zuo,Liu Jia-sen,et al.Recombinant canine adenovirus type 2 expressing rabbit hemorrhagic disease virus VP60protein provided protection against RHD in rabbits[J].Vet Microbiol,2018,(213):15-20.
[7]Song Yan-hua,Fan Zhi-yu,Zuo Yuan-yuan,et al.Binding of rabbit hemorrhagic disease virus-like particles to host his to-blood group antigens is blocked by antisera from experimentally vaccinated rabbits[J].Arch Virol,2017,162(11):3425-3430.
[8]范志宇,魏宏军,胡波,等.兔出血症病毒杆状病毒载体灭活疫苗安全性及效力验[J].江苏农业科学,2015,43(11):272-275.
[9]严维巍,崔治中,王永坤.兔出血症病毒衣壳蛋白基因在毕赤酵母中的表达[J].生物工程学报,2005,21(1):135-138.
[10]Bachmann A S,Corpuz G,Hareld W P,et al.A simple method for the rapid purification of copia virus-like particles from Drosophila Schneider 2 cells[J].J Virol Methods,2004,(115):159-165.
[11]Braeden D,Farah A B,Simon J P,et al.Multi-target chimaeric VLP as a therapeutic vaccine in a model of colorectal cancer[J].J Immunother Cancer,2017,5(69):1-13.
[12]World Health Organization.Human papillomavirus vaccines:WHO position paper[J].Wkly Epidemiol Rec,2014,89(43):465-492.
[13]Wood V,Gwilliam R,rejandream M A,et al.The genome sequence of Schizosaccharomyces pombe[J].Nature,2002,415:871-880.
[14]李超美,王芳,蔡少平,等.检测兔出血症病毒抗体间接ELISA方法的建立[J].江苏农业学报,2010,26(3):546-550.