VP22融合猪细小病毒VP2蛋白在杆状病毒系统中的表达及免疫原性分析
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摘要
旨在利用杆状病毒-昆虫细胞表达系统表达融合VP22短肽的重组猪细小病毒(PPV)衣壳蛋白VP2,分析其免疫原性。以PPV N株为模板,通过重叠延伸PCR技术分别将VP22融合到VP2基因的N端或C端,构建重组杆状病毒rBV-VP2、rBV-VP22-VP2和rBV-VP2-VP22,通过感染昆虫细胞进行重组蛋白的表达,并在小鼠上初步验证其免疫原性。间接免疫荧光和Western blot分析结果表明表达产物均能与鼠抗PPV抗体发生特异性反应;透射电镜观察到rBV-VP2、rBV-VP22-VP2的表达产物可装配形成直径约22～24 nm的病毒样粒子,VP2-VP22没有观察到病毒样粒子。PPV ELISA抗体检测结果显示,3种表达产物均能有效诱导小鼠产生PPV特异性抗体,其中VP2、VP22-VP2组产生抗体水平与PPV灭活苗相当;细胞因子检测结果显示,3种表达产物均能有效刺激细胞因子IL-2和IFN-γ的分泌,其中VP22-VP2组IL-2和IFN-γ的含量要显著高于VP2和PPV灭活苗组及重组蛋白VP2-VP22组,表明VP2 N端融合VP22蛋白的免疫效果要优于其C端,VP22-VP2虽不能提高VP22蛋白的体液免疫效果,但具有增强小鼠机体细胞免疫反应的能力。
The purpose of this study was to express VP22-VP2 fused protein by using the Bac-to-Bac expression system and analyze the immunogenicity of the recombinant protein.Using PPV N strain as template,VP22 were fused to the N-terminal or C-terminal of VP2 gene by SOE-PCR,recombinant baculovirus rBV-VP2,rBV-VP22-VP2 and rBV-VP2-VP22 were constructed,and then the recombinant proteins were expressed in insect cells,also,their immunogenicity were preliminary verified in mice.The results of indirect immunofluorescence and Western blot showed that all the recombinant proteins could react specifically with mouse anti-PPV antibody.The proteins of VP2 and VP22-VP2 could be assembled to form the diameter of about 22-24 nm virus-like particles,but there was not observed virus-like particles of VP2-VP22 fused protein.The results of ELISA showed that the three expressed products could effectively induce the production of PPV specific antibodies in mice,and the antibody levels induced by VP2 or VP22-VP2 groups were comparable to that of PPV inactivated vaccine.The results of cytokine assay showed that,all the three fusion proteins could induced IL-2 and IFN-γ secreted,and the expressions of IL-2 and IFN-γ in VP22-VP2 group were significantly higher than that in VP2,PPV inactivated vaccine,and the VP2-VP22 group,which suggested that the immunogenicity of VP22 fused at the N terminal of VP2 was better than that fused at VP2's C-terminal.Although VP22-VP2 did not enhance the humoral immunity of VP22 protein,but had the ability to enhance cellular immune response in mice.
The purpose of this study was to express VP22-VP2 fused protein by using the Bac-to-Bac expression system and analyze the immunogenicity of the recombinant protein.Using PPV N strain as template,VP22 were fused to the N-terminal or C-terminal of VP2 gene by SOE-PCR,recombinant baculovirus rBV-VP2,rBV-VP22-VP2 and rBV-VP2-VP22 were constructed,and then the recombinant proteins were expressed in insect cells,also,their immunogenicity were preliminary verified in mice.The results of indirect immunofluorescence and Western blot showed that all the recombinant proteins could react specifically with mouse anti-PPV antibody.The proteins of VP2 and VP22-VP2 could be assembled to form the diameter of about 22-24 nm virus-like particles,but there was not observed virus-like particles of VP2-VP22 fused protein.The results of ELISA showed that the three expressed products could effectively induce the production of PPV specific antibodies in mice,and the antibody levels induced by VP2 or VP22-VP2 groups were comparable to that of PPV inactivated vaccine.The results of cytokine assay showed that,all the three fusion proteins could induced IL-2 and IFN-γ secreted,and the expressions of IL-2 and IFN-γ in VP22-VP2 group were significantly higher than that in VP2,PPV inactivated vaccine,and the VP2-VP22 group,which suggested that the immunogenicity of VP22 fused at the N terminal of VP2 was better than that fused at VP2's C-terminal.Although VP22-VP2 did not enhance the humoral immunity of VP22 protein,but had the ability to enhance cellular immune response in mice.
引文
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[26] 徐志文,郭万柱,蒋清蓉,等.猪细小病毒2型ORF2基因插入猪细小病毒VP2基因3’端对病毒样颗粒形成的影响[J].中国兽医科学,2009,39(2):95-100.
[27] WANG J,GUJAR S A,COVA L,et al.Bicistronic woodchuck hepatitis virus core and gamma interferon DNA vaccine can protect from hepatitis but does not elicit sterilizing antiviral immunity[J].J Virol,2007,81(2):903-916.
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[31] 孙亚萍,王英明,乔守怡.干扰素及其最新研究进展[J].中国免疫学杂志,2006,22(7):676-679.
[2] 孙文超,刘立明,赵翠青,等.猪细小病毒6型SYBR Green I real-time PCR检测方法的建立[J].中国兽医学报,2018,38(3):442-445.
[3] 杜毅超,吴健敏,刘金凤.猪细小病毒病毒样颗粒研究进展[J].动物医学进展,2016,37(7):71-75.
[4] 丁佳欣,钱晶,徐小洪,等.新城疫病毒样颗粒对树突状细胞成熟的影响[J].中国兽医学报,2017,37(8):1485-1489.
[5] BOSHUIZEN R S,CASAL J I,HOJRUP P,et al.Mapping the antigenic structure of porcine parvovirus at the level of peptides[J].Virus Res,1998,53(2):163-173.
[6] RYMERSON R T,BABIUK L,MENASSA R,et al.Immunogenicity of the capsid protein VP2 from porcine parvovirus expressed in low alkaloid transgenic tobacco[J].Mol Breeding,2003,11(4):267-276.
[7] 司艳红,方明刚,王汉中.猪细小病毒vp2基因的表达和类病毒颗粒的构建[J].中国病毒学,2006,21(2):148-152.
[8] JI P,LIU Y,CHEN Y,et al.Porcine parvovirus capsid protein expressed in Escherichia coli self-assembles into virus-like particles with high immunogenicity in mice and guinea pigs[J].Antiviral Res,2017,139:146-152.
[9] QI T,CUI S.Expression of porcine parvovirus VP2 gene requires codon optimized E.coli cells[J].Virus Genes,2009,39(2):217-222.
[10] GUO C,ZHONG Z,HUANG Y.Production and immunogenicity of VP2 protein of porcine parvovirus expressed in Pichia pastoris[J].Arch Virol,2014,159(5):963-970.
[11] PAN Q,HE K,HUANG K.Development of recombinant porcine parvovirus-like particles as an antigen carrier formed by the hybrid VP2 protein carrying immunoreactive epitope of porcine circovirus type 2[J].Vaccine,2008,26(17):2119-2126.
[12] MARCEKOVA Z,PSIKAL I,KOSINOVA E,et al.Heterologous expression of full-length capsid protein of porcine circovirus 2 in Escherichia coli and its potential use for detection of antibodies[J].J Virol Methods,2009,162(1):133-141.
[13] FENG H,LIANG M,WANG H L,et al.Recombinant canine parvovirus-like particles express foreign epitopes in silkworm pupae[J].Vet Microbiol,2011,154(1):49-57.
[14] 潘群兴何孔旺,王永山,等.重组外源基因的猪细小病毒VP2蛋白病毒样颗粒的构建与鉴定[J].畜牧兽医学报,2008,39(9):1245-1250.
[15] ELLIOTT G,O'HARE P.Intercellular trafficking and protein delivery by a herpesvirus structural protein[J].Cell,1997,88(2):223-233.
[16] HARMS J S,REN X,OLIVEIRA S C,et al.Distinctions between bovine herpesvirus 1 and herpes simplex virus type 1 VP22 tegument protein subcellular associations[J].J Virol,2000,74(7):3301-3312.
[17] BOLHASSANI A,GHOLAMI E,ZAHEDIFARD F,et al.Leishmania major:protective capacity of DNA vaccine using amastin fused to HSV-1 VP22 and EGFP in BALB/c mice model[J].Exp Parasitol,2011,128(1):9-17.
[18] 范兴琼,吴双林,刘成倩,等.穿膜肽在疫苗中应用的研究进展[J].中国兽药杂志,2012,46(1):51-53.
[19] KUSHNIR N,STREATFIELD S J,YUSIBO V.Virus-like particles as a highly efficient vaccine platform:diversity of targets and production systems and advances in clinical development[J].Vaccine,2012,31(1):58-83.
[20] COX M M,HASHIMOTO Y.A fast track influenza virus vaccine produced in insect cells[J].J Invertebr Pathol,2011,107(Suppl):31-41.
[21] FELBERBAUM R S.The baculovirus expression vector system:A commercial manufacturing platform for viral vaccines and gene therapy vectors[J].Biotechnol J,2015,10(5):702-714.
[22] CHEN H,SHAFFER P L,HUANG X,et al.Rapid screening of membrane protein expression in transiently transfected insect cells[J].Protein Expr Purif,2013,88(1):134-142.
[23] WU Y,REN C,GAO Y,et al.A novel method for promoting heterologous protein expression in Escherichia coli by fusion with the HIV-1 TAT core domain[J].Amino Acids,2010,39(3):811-820.
[24] CASAL J I,RUEDA P,HURTADO A.Parvovirus-like particles as vaccine vectors[J].Methods,1999,19(1):174-186.
[25] 蒋春英,魏建超,史子学,等.展示日本乙型脑炎病毒B细胞表位和T细胞表位的猪细小病毒病毒样颗粒的制备[J].中国兽医科学,2013,43(4):358-363.
[26] 徐志文,郭万柱,蒋清蓉,等.猪细小病毒2型ORF2基因插入猪细小病毒VP2基因3’端对病毒样颗粒形成的影响[J].中国兽医科学,2009,39(2):95-100.
[27] WANG J,GUJAR S A,COVA L,et al.Bicistronic woodchuck hepatitis virus core and gamma interferon DNA vaccine can protect from hepatitis but does not elicit sterilizing antiviral immunity[J].J Virol,2007,81(2):903-916.
[28] Malek T R.The Biology of interleukin-2[J].Annual Rev Immunol,2008,26(1):453-479.
[29] BOYMAN O,SPRENT J.The role of interleukin-2 during homeostasis and activation of the immune system[J].Nat Rev Immunol,2012,12(3):180-190.
[30] FERRIS R L,WHITESIDE T L,FERRONE S.Immune escape associated with functional defects in antigen-processing machinery in head and neck cancer[J].Clin Cancer Res,2006,12(13):3890-3895.
[31] 孙亚萍,王英明,乔守怡.干扰素及其最新研究进展[J].中国免疫学杂志,2006,22(7):676-679.