人乳头瘤病毒52型病毒样颗粒制备及其生物活性
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摘要
[目的]利用大肠杆菌系统表达人乳头瘤病毒52型(HPV52) L1蛋白,并获得HPV52病毒样颗粒(VLPs)。[方法]优化并合成HPV52L1基因,构建p ET-30a-52L1表达载体粒,转化大肠杆菌E. coli BL21 StarTM(DE3)。经IPTG诱导表达,阳离子交换层析纯化,SDS-PAGE和Western Blotting鉴定,BALB/c小鼠免疫2次,初次后4w检测免疫血清中HPV52中和抗体滴度。[结果]获得纯度90%的HPV52L1蛋白,将获得的HPV52L1蛋白进行解组装和重组装形成VLPs,动态光散射观察到粒径约为70nm,透射电镜观察到50～60nm的均一VLPs,中和抗体滴度达25600。[结论]大肠杆菌系统表达人乳头瘤病毒52型(HPV52) L1蛋白,并经过一步柱层析纯化获得纯度达90%的L1蛋白,经解组装重组装获得粒径大小为50～60nm的VLPs,具有良好免疫原性。
[Objective]To express the major structural protein L1 of human papillomavirus type 52( HPV52) in E. coli and re-assemble L1 protein into virus-like particles( VLPs).[Method]HPV52 L1 gene was optimized and synthesized to construct p ET-30 a-52 L1 expression vector which was transformed into E. coli BL21 StarTM( DE3). The HPV52 L1 protein with purity of more than 90% was expressed that induced by IPTG,purified by cation exchange chromatography and identified by SDS-PAGE and Western Blotting. The HPV52 L1 protein was subjected to self-assembly VLPs with disassembly and reassembly.BALB/c mice were immunized for two times,HPV52 neutralizing antibody titer in immune serum was detected 4 weeks after the first immunization.[Result]Observation by dynamic light scattering showed that the uniform VLPs was formed in a round shape with a diameter about 70 nm and transmission electronic microscopy showed that about 50-60 nm. HPV52 neutralizing antibody titer in immune serum reached 25,600. [Conclusion]The human papillomavirus type 52( HPV52) L1 protein was expressed in E. coli system and purified by one-step column chromatography to obtain L1 protein with a purity of 90%. After disassembly and assembly,VLPs with particle size of 50-60 nm were obtained,has good immunogenicity.
[Objective]To express the major structural protein L1 of human papillomavirus type 52( HPV52) in E. coli and re-assemble L1 protein into virus-like particles( VLPs).[Method]HPV52 L1 gene was optimized and synthesized to construct p ET-30 a-52 L1 expression vector which was transformed into E. coli BL21 StarTM( DE3). The HPV52 L1 protein with purity of more than 90% was expressed that induced by IPTG,purified by cation exchange chromatography and identified by SDS-PAGE and Western Blotting. The HPV52 L1 protein was subjected to self-assembly VLPs with disassembly and reassembly.BALB/c mice were immunized for two times,HPV52 neutralizing antibody titer in immune serum was detected 4 weeks after the first immunization.[Result]Observation by dynamic light scattering showed that the uniform VLPs was formed in a round shape with a diameter about 70 nm and transmission electronic microscopy showed that about 50-60 nm. HPV52 neutralizing antibody titer in immune serum reached 25,600. [Conclusion]The human papillomavirus type 52( HPV52) L1 protein was expressed in E. coli system and purified by one-step column chromatography to obtain L1 protein with a purity of 90%. After disassembly and assembly,VLPs with particle size of 50-60 nm were obtained,has good immunogenicity.
引文
[1]高波,张靖,张学峰,等.人乳头瘤病毒31和33型L1蛋白类病毒颗粒的制备及其免疫原性[J].中国生物制品学杂志,2014,27(12):1508-1511.
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[12]刘微,郭明旸,纪莉婷.人乳头瘤病毒16型L1的表达、病毒样颗粒组装及其免疫原性研究[J].中国人兽共患病学报,2018,34(8):684-688.
[13]Modis,Y.Atomic model of the papillomavirus capsid[J].EMBO(European Molecular Biology Organization)Journal,2002,21(18):4754-4762.
[14]Giovanni Cardone,Adam L Moyer,Naiqian Cheng,et al.Maturation of the human papillomavirus 16 capsid[J].Mbio,2014,5(4):01104-01114.
[15]杨旭,黄惟巍,姚宇峰,等.人乳头瘤病毒16型L1基因在毕赤酵母中表达的影响因素分析[J].中国生物工程杂志,2017,37(10):1-7.
[16]Patrick C Hanington,Daniel R Barreda,Miodrag Belosevic.A novel hematopoietic granulin induces proliferation of goldfish(Carassius auratus L.)macrophages[J].Journal of Biological Chemistry,2006,281(15):9963-9970.
[17]James CCook,Joseph GJoyce,Huge A George,et al.Purification of virus-like particles of recombinant human papilloma virus type 11major capsid protein L1 from Saccharomyces cerevisiae[J].Protein Expression&Purification,1999,17(3):477-484.
[18]史晶洁,宋硕,王大宁,等.人乳头瘤病毒样颗粒组装研究进展[J].病毒学报,2018,34(2):252-258.
[19]付丁伊.人乳头瘤病毒(HPV)衣壳蛋白L1的体外自组装调控及内在机理研究[D].长春:吉林大学,2018.
[2]陈雪,王志荣,刘洪洋,等.一种可诱发高滴度的针对人乳头瘤病毒HPV16、HPV52、HPV58中和抗体反应的HPV16嵌合病毒样颗粒疫苗的研究[J].病毒学报,2018,34(4):505-514.
[3]Franco Maria Buonaguro,Maria Lina Tornesello,Luigi Buonaguro.Virus-like particle vaccines and adjuvants:the HPV paradigm.[J].Expert Review of Vaccines,2016,8(10):1379-1398.
[4]Henryk Mach,David B Volkin,Robert D Troutman,et al.Disassembly and reassembly of yeast-derived recombinant human papillomavirus virus-like particles(HPV VLPs)[J].J Pharm Sci,2006,95(10):2195-2206.
[5]AntónioRoldao,Maria Candida M Mellado,Leda R Castilho,et al.Virus-like particles in vaccine development[J].Expert Review of Vaccines,2010,9(10):1149-1176.
[6]Pan D,Zha X,Yu X,et al.Enhanced expression of soluble human papillomavirus L1 through coexpression of molecular chaperonin in Escherichia coli[J].Protein Expression and Purification,2016,120:92-98.
[7]Zhang Z,Zhang J,Xia N,et al.Expanded strain coverage for a highly successful public health tool:prophylactic 9-valent human papillomavirus vaccine[J].Hum Vaccin Immunother,2017,13(1):2280-2291.
[8]Christopher B.Buck,Diana V.Pastrana,Douglas R.Lowy,et al.Efficient intracellular assembly of papillomaviral vectors[J].Journal of Virology,2004,78(2):751-757.
[9]Diana V Pastrana,Christopher B Buck,Yuk-Ying S Pang,et al.Reactivity of human sera in a sensitive,high-throughput pseudovirus-based papillomavirus neutralization assay for HPV16 and HPV18[J].Virology,2004,321(2):205-216.
[10]马伟红,贾超颖,王鹤.表达人乳头瘤病毒58型E6E7融合基因的慢病毒系统的建立及其真核表达[J].广西医科大学学报,2017,34(1):6-11.
[11]王亚婷,李文生,闫小飞,等.人乳头瘤病毒58型L1/E7“假病毒”疫苗的制备及免疫效果评价[J].现代检验医学杂志,2018,33(03):35-37+43.
[12]刘微,郭明旸,纪莉婷.人乳头瘤病毒16型L1的表达、病毒样颗粒组装及其免疫原性研究[J].中国人兽共患病学报,2018,34(8):684-688.
[13]Modis,Y.Atomic model of the papillomavirus capsid[J].EMBO(European Molecular Biology Organization)Journal,2002,21(18):4754-4762.
[14]Giovanni Cardone,Adam L Moyer,Naiqian Cheng,et al.Maturation of the human papillomavirus 16 capsid[J].Mbio,2014,5(4):01104-01114.
[15]杨旭,黄惟巍,姚宇峰,等.人乳头瘤病毒16型L1基因在毕赤酵母中表达的影响因素分析[J].中国生物工程杂志,2017,37(10):1-7.
[16]Patrick C Hanington,Daniel R Barreda,Miodrag Belosevic.A novel hematopoietic granulin induces proliferation of goldfish(Carassius auratus L.)macrophages[J].Journal of Biological Chemistry,2006,281(15):9963-9970.
[17]James CCook,Joseph GJoyce,Huge A George,et al.Purification of virus-like particles of recombinant human papilloma virus type 11major capsid protein L1 from Saccharomyces cerevisiae[J].Protein Expression&Purification,1999,17(3):477-484.
[18]史晶洁,宋硕,王大宁,等.人乳头瘤病毒样颗粒组装研究进展[J].病毒学报,2018,34(2):252-258.
[19]付丁伊.人乳头瘤病毒(HPV)衣壳蛋白L1的体外自组装调控及内在机理研究[D].长春:吉林大学,2018.
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