猪圆环病毒2型病毒样颗粒的纯化及鉴定
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摘要
为获得较高纯度的猪圆环病毒2型(porcine circovirus type 2,PCV2)Cap蛋白的病毒样颗粒(virus-like particles,VLPs)并对其进行结构和功能研究,本试验以大肠杆菌表达系统表达了PCV2重组Cap(PCV2 rCap)蛋白,通过硫酸铵分级沉淀、Sephacryl S-300 HR凝胶过滤层析、Capto Q离子交换层析、CsCl密度梯度离心等方法对其进行了纯化。通过Western blotting对纯化所得蛋白进行免疫反应性鉴定,透射电镜(TEM)观察纯化蛋白的粒径及形态,高效液相尺寸排阻色谱(HPSEC)检测其组分分布。结果显示,纯化后PCV2 rCap蛋白经SDS-PAGE检测,可见28 ku的目的蛋白带,灰度扫描法检测其纯度为97.53%。Western blotting结果显示,该处的特异性蛋白条带有明显的免疫反应性;TEM检测其为粒径17.35～19.24 nm的规则球形颗粒,表明所获得的PCV2 rCap蛋白在表达时能在菌体内自我装配成VLPs,且在纯化过程中没有被破坏而解聚,仍保持天然状态;HPSEC检测纯化样品中VLPs含量为92.67%。本试验结果为进一步研究PCV2 VLPs的空间结构及其免疫保护机制提供了参考依据。
To obtain and analyze highly purified porcine circovirus virus type 2(PCV2) virus-like particles(VLPs),the PCV2 recombinant Cap protein(PCV2 rCap) was expressed by Escherichia coli expression system,and was purified by the means of ammonium sulfate precipitation,gel filtration chromatography,ion exchange chromatography and CsCl density gradient centrifugation.The immunoreactivity of purified VLPs were identified by Western blotting,the diameter and morphology of purified VLPs were observed by transmission electron microscopy(TEM).Its components were detected and analyzed by high performance liquid size exclusion chromatography(HPSEC).After purification,the PCV2 rCap protein was detected by SDS-PAGE,the target protein band of 28 ku was detected,and its purity was 97.53% by gray scale scanning.The results of Western blotting showed that the specific protein strips had obvious immunoreactivity.The TEM detected the regular spherical particles with the particle size of 17.35 to 19.24 nm,indicating that the obtained PCV2 rCap protein could express itself in the bacteria.They were assembled into VLPs and deagglomerated without being destroyed during the purification process,and remained in a natural state;In addition,HPSEC detected that the proportion of normally assembled VLPs was 92.67%.The results of this study provided a reference for further study of the spatial structure of PCV2 VLPs and their immune protection mechanisms.
To obtain and analyze highly purified porcine circovirus virus type 2(PCV2) virus-like particles(VLPs),the PCV2 recombinant Cap protein(PCV2 rCap) was expressed by Escherichia coli expression system,and was purified by the means of ammonium sulfate precipitation,gel filtration chromatography,ion exchange chromatography and CsCl density gradient centrifugation.The immunoreactivity of purified VLPs were identified by Western blotting,the diameter and morphology of purified VLPs were observed by transmission electron microscopy(TEM).Its components were detected and analyzed by high performance liquid size exclusion chromatography(HPSEC).After purification,the PCV2 rCap protein was detected by SDS-PAGE,the target protein band of 28 ku was detected,and its purity was 97.53% by gray scale scanning.The results of Western blotting showed that the specific protein strips had obvious immunoreactivity.The TEM detected the regular spherical particles with the particle size of 17.35 to 19.24 nm,indicating that the obtained PCV2 rCap protein could express itself in the bacteria.They were assembled into VLPs and deagglomerated without being destroyed during the purification process,and remained in a natural state;In addition,HPSEC detected that the proportion of normally assembled VLPs was 92.67%.The results of this study provided a reference for further study of the spatial structure of PCV2 VLPs and their immune protection mechanisms.
引文
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[18] ZHOU J Y,SHANG S B,GONG H,et al.In vitro expression,monoclonal antibody and bioactivity for capsid protein of porcine circovirus type Ⅱ without nuclear localization signal[J].Journal of Biotechnology,2005,118(2):201-211.
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[21] WU P C,CHEN T Y,CHI J N,et al.Efficient expression and purification of porcine circovirus type 2 virus-like particles in Escherichia coli[J].Journal of Biotechnology,2016,220:78-85.
[22] ANGELO J M,LENHOFF M.Determinants of protein elution rates from preparative ion exchange adsorbents[J].Journal of Chromatography A,2016,1440(1):94-104.
[23] 杨延丽.灭活病毒及病毒样颗粒的结构表征和稳定性研究[D].北京:中国科学院,2015.YANG Y L.Characterization and stabilization of inactivated virus and virus like particles[D].Beijing:Chinese Academy of Sciences,2015.(in Chinese)
[24] CHEN Y,ZHANG Y,QUAN C,et al.Aggregation and antigenicity of virus like particle in salt solution-A case study with hepatitis B surface antigen[J].Vaccine,2015,33(35):4300-4306.
[25] HUANG Y,BI J,ZHANG Y,et al.A highly efficient integrated chromatographic procedure for the purification of recombinant hepatitis B surface antigen from Hansenula polymorpha[J].Protein Expression and Purification,2007,56(2):301-310.
[26] SANCHEZ-RODRIGUEZ S P,MUNCH-ANGUIANO L,ECHEVERRIA O,et al.Human parvovirus B19 virus-like particles:In vitro assembly and stability[J].Biochimie,2012,94(3):870-878.
[2] 于静,陈彦永,何小江,等.猪圆环病毒2型巢式PCR检测方法的建立与初步应用[J].中国畜牧兽医,2015,42(12):3173-3178.YU J,CHEN Y Y,HE X J,et al.Establishment and preliminary application of nested PCR for the detection of porcine circovirus type 2[J].China Animal Husbandry & Veterinary Medicine,2015,42(12):3173-3178.(in Chinese)
[3] NEIRA V,RAMOS N,TAPIA R,et al.Genetic analysis of porcine circovirus type 2 from pigs affected with PMWS in Chile reveals intergenotypic recombination[J].Journal of Virology,2017,14(1):191-197.
[4] LI P C,QIAO X W,ZHENG Q S,et al.Immunogenicity and immunoprotection of porcine circovirus type 2 (PCV2) Cap protein displayed by Lactococcus lactis[J].Vaccine,2016,34(5):696-702.
[5] 李富强,王利丽,李秀丽,等.猪圆环病毒2型分子生物学研究进展[J].天津农业科学,2015,21(9):57-60.LI F Q,WANG L L,LI X L,et al.Progress on molecular biology of PCV2[J].Tianjin Agricultural Sciences,2015,21(9):57-60.(in Chinese)
[6] 高田,张丽琳,黄金海,等.PCV2病毒样颗粒疫苗的研究进展[J].生命的化学,2017,37(2):233-238.GAO T,ZHANG L L,HUANG J H,et al.Progress on porcine circovirus type 2 virus-like particle[J].Chemisty of Life,2017,37(2):233-238.(in Chinese)
[7] 侯强,侯绍华,贾红,等.猪圆环病毒2d亚型病毒样颗粒的制备与鉴定[J].中国兽医科学,2017,47(11):1385-1391.HOU Q,HOU S H,JIA H,et al.Preparation and identification of porcine circovirus type 2d Cap protein as virus-like particles in insect cells[J].Chinese Veterinary Science,2017,47(11):1385-1391.(in Chinese)
[8] GARG H,SEDANO M,PLATA G,et al.Development of virus-like particle vaccine and reporter assay for Zika virus[J].Journal of Virology,2017,91(20):e00834-17.
[9] 赵晓云,乔绪稳,陈瑾,等.利用E.coli表达猪圆环病毒2型Cap蛋白生产病毒样颗粒疫苗[J].中国农业科学,2015,48(5):976-986.ZHAO X Y,QIAO X W,CHEN J,et al.PCV2 virus like particles vaccine produced with recombinant Cap protein expressed in E.coli[J].Scientia Agricultura Sinica,2015,48(5):976-986.(in Chinese)
[10] NAINYS J,LASICKIENE R,PETRAITYTE-BURNEIKIENE R,et al.Generation in yeast of recombinant virus-like particles of porcine circovirus type 2 capsid protein and their use for a serologic assay and development of monoclonal antibodies[J].BioMed Central Biotechnology,2014,14(1):100-110.
[11] HUANG X,WANG X,ZHANG J,et al.Escherichia coli-derived virus-like particles in vaccine develop-ment[J].NPJ Vaccines,2017,2(1):3-11.
[12] WU P C,LIN W L,WU C M,et al.Characterization of porcine circovirus type 2 (PCV2) capsid particle assembly and its application to virus-like particle vaccine development[J].Applied Microbiology and Biotechnology,2012,95(6):1501-1507.
[13] 于孟冉.超大孔色谱纯化类病毒颗粒的过程研究[D].北京:中国科学院,2015.YU M R.Study on gigaporous chromatographic process of virus-like particles purification[D].Beijing:Chinese Academy of Sciences,2015.(in Chinese)
[14] QUAN F S,LEE Y T,KIM K H,et al.Progress in developing virus-like particle influenza vaccines[J].Expert Review of Vaccines,2016,15(10):1281-1293.
[15] 杨剑,尹丹,罗文涓,等.猪圆环病毒2型诱导小鼠体内免疫细胞氧化胁迫模型的建立[J].中国畜牧兽医,2017,44(2):402-410.YANG J,YIN D,LUO W J,et al.Establishment of model for oxidative stress in mice immune cells induced by PCV2 in vivo[J].China Animal Husbandry & Veterinary Medicine,2017,44(2):402-410.(in Chinese)
[16] LóPEZ-VIDAL J,GóMEZ-SEBASTIáN S,BáRCENA J,et al.Improved production efficiency of virus-like particles by the baculovirus expression vector system[J].Public Library of Science One,2015,10(10):39-51.
[17] 余婉婷,湛洋,雷昕诺,等.猪圆环病毒2型病毒样颗粒的研究及应用[J].中国畜牧兽医文摘,2015,31(5):57-67.YU W T,ZHAN Y,LEI X N,et al.Study and application of porcine circovirus type 2 virus-like particles[J].Chinese Abstracts of Animal Husbandry and Veterinary Medicine,2015,31(5):57-67.(in Chinese)
[18] ZHOU J Y,SHANG S B,GONG H,et al.In vitro expression,monoclonal antibody and bioactivity for capsid protein of porcine circovirus type Ⅱ without nuclear localization signal[J].Journal of Biotechnology,2005,118(2):201-211.
[19] KONG W T,KONG J,HU S M,et al.Enhanced expression of PCV2 capsid protein in Escherichia coli and Lactococcus lactis by codon optimization[J].World Journal of Microbiology Biotechnology,2011,27(3):651-657.
[20] MOHSEN M O,ZHA L,CABRAL-MIRANDA G,et al.Major findings and recent advances in virus-like particle (VLP)-based vaccines[J].Seminars in Immunology,2017,34(1):123-132.
[21] WU P C,CHEN T Y,CHI J N,et al.Efficient expression and purification of porcine circovirus type 2 virus-like particles in Escherichia coli[J].Journal of Biotechnology,2016,220:78-85.
[22] ANGELO J M,LENHOFF M.Determinants of protein elution rates from preparative ion exchange adsorbents[J].Journal of Chromatography A,2016,1440(1):94-104.
[23] 杨延丽.灭活病毒及病毒样颗粒的结构表征和稳定性研究[D].北京:中国科学院,2015.YANG Y L.Characterization and stabilization of inactivated virus and virus like particles[D].Beijing:Chinese Academy of Sciences,2015.(in Chinese)
[24] CHEN Y,ZHANG Y,QUAN C,et al.Aggregation and antigenicity of virus like particle in salt solution-A case study with hepatitis B surface antigen[J].Vaccine,2015,33(35):4300-4306.
[25] HUANG Y,BI J,ZHANG Y,et al.A highly efficient integrated chromatographic procedure for the purification of recombinant hepatitis B surface antigen from Hansenula polymorpha[J].Protein Expression and Purification,2007,56(2):301-310.
[26] SANCHEZ-RODRIGUEZ S P,MUNCH-ANGUIANO L,ECHEVERRIA O,et al.Human parvovirus B19 virus-like particles:In vitro assembly and stability[J].Biochimie,2012,94(3):870-878.