CDV强毒BJ16B2株H基因在昆虫杆状病毒中的表达与鉴定
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摘要
试验旨在获得具有天然构象且抗原性良好的犬瘟热病毒(canine distemper virus,CDV)强毒株的H蛋白。以分离的CDV强毒株BJ16B2为模板,RT-PCR特异扩增H基因,克隆至表达载体pFastBacTMHTA上,酶切及测序鉴定正确后比对分析H基因同源性,并将重组质粒pFastBacTMHTA-H转化大肠杆菌DH10BacTM感受态细胞,同源重组构建穿梭质粒rBacmid-H,将重组穿梭质粒转染Sf9昆虫细胞,拯救重组杆状病毒。利用IFA和Western blotting对获得的重组蛋白进行鉴定及抗原性分析。结果显示,重组质粒pFastBacTMHTA-H酶切测序鉴定正确,该H基因属于Asia-1强毒株,与参考毒株的核苷酸和氨基酸同源性分别为89.8%~99.3%和89.6%~99.5%。在杆状病毒中所表达的H蛋白能与CDV阳性血清及His-tag抗体发生特异性反应,IFA鉴定能够产生特异性绿色荧光,且在68ku左右出现蛋白条带,表明H蛋白成功表达且具有良好的反应原性。本研究获得了具有天然构象及良好抗原性的CDV重组H蛋白,为后续蛋白结构和抗原特性差异分析、单抗制备、CDV亚单位疫苗的开发奠定了基础。
To obtain the native conformation and antigenicity of hemagglutinin(H)protein of canine distemper virus(CDV)virulent strain,the H gene which referred to BJ16 B2 strain was accurately amplified by RT-PCR and cloned into pFastBacTMHTA vector.The recombinant plasmid was confirmed by restriction enzyme digestion and DNA sequencing,H gene was analyzed and the recombinant plasmid was transformed into E.coli DH10 BacTM competent cell,and constructed shuttle vector,which was transfected into Sf9 cell to save baculovirus expressing H protein.To determine the antigenicity of recombinant protein,Western blotting and IFA analysis were used to detect the interesting protein.The results demonstrated that H gene belongs to Asia-1 virulent strain,and the homology of nucleotide and amino acid among the reference strains was 89.8%to99.3% and 89.6%to 99.5%,respectively.The fusion protein generated by recombinant baculovirus was reacted with both His-tag monoclonal antibodies and CDV positive serum by Western blotting test,and presented specific green fluorescence in IFA test(68 ku),which showed that this protein had sufficient antigenicity.The resluts served as a strong foundation for the the subsequent analysis of protein structure and antigenicity differences,the preparation of monoclonal antibodies and the development of CDV subunit vaccine.
To obtain the native conformation and antigenicity of hemagglutinin(H)protein of canine distemper virus(CDV)virulent strain,the H gene which referred to BJ16 B2 strain was accurately amplified by RT-PCR and cloned into pFastBacTMHTA vector.The recombinant plasmid was confirmed by restriction enzyme digestion and DNA sequencing,H gene was analyzed and the recombinant plasmid was transformed into E.coli DH10 BacTM competent cell,and constructed shuttle vector,which was transfected into Sf9 cell to save baculovirus expressing H protein.To determine the antigenicity of recombinant protein,Western blotting and IFA analysis were used to detect the interesting protein.The results demonstrated that H gene belongs to Asia-1 virulent strain,and the homology of nucleotide and amino acid among the reference strains was 89.8%to99.3% and 89.6%to 99.5%,respectively.The fusion protein generated by recombinant baculovirus was reacted with both His-tag monoclonal antibodies and CDV positive serum by Western blotting test,and presented specific green fluorescence in IFA test(68 ku),which showed that this protein had sufficient antigenicity.The resluts served as a strong foundation for the the subsequent analysis of protein structure and antigenicity differences,the preparation of monoclonal antibodies and the development of CDV subunit vaccine.
引文
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[23]王安平,朱善元,王永娟,等.鸭源新城疫病毒F基因在昆虫细胞中的表达和鉴定[J].中国畜牧兽医,2015,42(11):2862-2866.WANG A P,ZHU S Y,WANG Y J,et al.Expressiong and identification of F gene of duck newcastle disease virus in insect cells[J].China Animal Husbandry&Veterinary Medicine,2015,42(11):2862-2866.(in Chinese)
[24]郭慧娟,樊翠,张英,等.猪圆环病毒2型CAP蛋白在flashBAC杆状病毒表达系统中的表达与鉴定[J].中国畜牧兽医,2016,43(7):1876-1883.GUO H J,FAN C,ZHANG Y,et al.Expression and identification of porcine circivirus type 2capsid protein using flashBAC baculovirus expression system[J].China Animal Husbandry&Veterinary Medicine,2016,43(7):1876-1883.(in Chinese)
[25]刘春菊,任炜杰,王志亮.杆状病毒表达系统在兽用亚单位疫苗领域应用的研究进展[J].中国畜牧兽医,2013,40(9):101-105.LIU C J,REN W J,WANG Z L.Research progress on application of baculovirus expressin system in veterinary subunit vaccine field[J].China Animal Husbandry&Veterinary Medicine,2013,40(9):101-105.(in Chinese)
[26]GLENN G M,SMITH G,FRIES L,et al.Safety and immunogenicity of a Sf9insect cell-derived respiratory syncytial virus fusion protein nanoparticle vaccine[J].Vaccine,2013,31(3):524-532.
[2]SAWATSKY B,CATTANEO R,VON MESSLING V.Canine distemper virus spread and transmission to na6ve ferrets:Selective pressure on SLAM-dependent entry[J].Journal of Virology,2018,Epub ahead of print.
[3]SAKAI K,YOSHIKAWA T,SEKI F,et al.Canine distemper virus associated with a lethal outbreak in monkeys can readily adapt to use human receptors[J].Journal of Virology,2013,87(12):7170-7175.
[4]HIRMA K,TOGASHI K,WAKASA C,et al.Cytotoxic T-lymphocyte activity specific for hemagglutinin(H)protein of canine distemper virus in dogs[J].The Journal of Veterinary Medical Science,2003,65(1):109-112.
[5]ANIS E,HOLFORD A L,GALYON G D,et al.Antigenic analysis of genetic variants of canine distemper virus[J].Veterinary Microbiology,2018,219(6):154-160.
[6]BI Z,WANG Y,WANG X,et al.Phylogenetic analysis of canine distemper virus in domestic dogs in Nanjing,China[J].Archives Virology,2015,160(2):523-527.
[7]STOLT-BERGNER P,BENDA C,BERGBREDE T,et al.Baculovirus-driven protein expression in insect cells:A benchmarking study[J].Journal of Structural Biology,2018,Epub ahead of print.
[8]MANSOURI M,BERGER P.Baculovirus for gene delivery to mammalian cells:Past,present and future[J].Plasmid,2018,98(5):1-7.
[9]FUTATSYMORI-SUGAI M,TSUMOTO K.Signal peptide design for improving recombinant protein secretion in the baculovirus expression vector system[J].Biochemical and Biophysical Research Communications,2010,391(1):931-935.
[10]PAN Z,LIU J,MA J,et al.The recombinant EHV-1vector producing CDV hemagglutinin as potential vaccine against canine distemper[J].Microbial Pathogenesis,2017,111(10):388-394.
[11]BI Z,XIA X,WANG Y,et al.Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein[J].Microbiology Immunology,2015,59(4):202-208.
[12]蔡孜萌,范威宏,张洪亮,等.犬瘟热病毒重组H蛋白间接ELISA检测方法的建立[J].畜牧与兽医,2017,49(11):69-74.CAI Z M,FAN W H,ZHANG H L,et al.Development of an indirect ELISA for dection of antibody against the recombinant H protein of canine distemper cirus[J].Animal Husbandry&Veterinary Medicine,2017,49(11):69-74.(in Chinese)
[13]袁颖烁,宋菲菲,张乐萃,等.犬瘟热病毒H蛋白的原核表达及免疫原性的初步鉴定[J].中国畜牧兽医,2013,40(4):40-45.YUAN Y S,SONG F F,ZHANG L C,et al.Prokaryotic expression and identificaiton of the preliminary immunogenicity of the H protein of caninne distemper virus[J].China Animal Husbandry&Veterinary Medicine,2013,40(4):40-45.(in Chinese)
[14]薛磊.犬瘟热病毒GZ-Z株H基因原核表达载体的构建[J].贵州畜牧兽医,2015,39(6):16-24.XUE L.Construction prokaryotic expression vector of canine distemper virus GZ-Z strain of H gene[J].Guizhou Journal of Animal Husbandry&Veterinary Medicine,2015,39(6):16-24.(in Chinese)
[15]周莉,刘志杰,曾智勇,等.犬瘟热病毒H基因原核表达质粒的构建[J].贵州农业科学,2011,39(9):174-176.ZHOU L,LIU Z J,ZENG Z Y,et al.Construction of prokaryotic expression vector of H gene of canine distemper virus[J].Guizhou Agricultural Sciences,2011,39(9):174-176.(in Chinese)
[16]袁颖烁,宋菲菲,刘国强,等.犬瘟热病毒H蛋白基因重组腺病毒的构建及免疫效果评估[J].中国生物工程杂志,2014,34(1):64-70.YUAN Y S,SONG F F,LIU G Q,et al.Construction and immune efficacy of recombinant adenovirus expression H gene of canine distemper virus[J].China Biotechnology,2014,34(1):64-70.(in Chinese)
[17]田美杰,葛金英,王喜军,等.表达犬瘟热病毒H基因重组新城疫病毒的构建及其免疫原性研究[J].中国预防兽医学报,2012,34(4):257-261.TIAN M J,GE J Y,WANG X J,et al.Construction of newcastle disease virus-vectored canine distemper virus(CDV)vaccine and induction of CDV neutralizing antibody[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(4):257-261.(in Chinese)
[18]虞一聪.犬瘟热病毒M、F、H基因重组杆状病毒的构建、鉴定与表达研究[D].长春:吉林农业大学,2015.YU Y C.Construction,identification and expression of recombinant baculovirus of M,F,and H genes of canine distemper virus[D].Changchun:Jilin Agricultural University,2015.(in Chinese)
[19]冯佩平.犬瘟热病毒主要结构蛋白基因在昆虫细胞中的表达及间接ELISA检测方法的建立[D].长春:吉林农业大学,2012.FENG P P.Expression of CDV major structural protein in Sf9cells and establishment of indirect ELISA assay[D].Changchun:Jilin Agricultural University,2012.(in Chinese)
[20]赵静.表达犬瘟热病毒H基因重组狂犬病病毒生物学特性的研究[D].广州:华南农业大学,2016.ZHAO J.Biological characterization of recombinant rabies virus expressing H protein of canine parvovirus[D].Guangzhou:South China Agricultural University,2016.(in Chinese)
[21]COX M M,HASHIMOTO Y.A fast track influenza virus vaccine produced in insect cells[J].Journal of Invertebrate Pathology,2011,107(7):S31-S41.
[22]GRANT R J,KELLEY K L,MARUNIAK J E,et al.Expression from baculovirus and serological reactivity of the nucleocapsid protein of dolphin morbillivirus[J].Veterinary Microbiology,2010,143(2-4):384-388.
[23]王安平,朱善元,王永娟,等.鸭源新城疫病毒F基因在昆虫细胞中的表达和鉴定[J].中国畜牧兽医,2015,42(11):2862-2866.WANG A P,ZHU S Y,WANG Y J,et al.Expressiong and identification of F gene of duck newcastle disease virus in insect cells[J].China Animal Husbandry&Veterinary Medicine,2015,42(11):2862-2866.(in Chinese)
[24]郭慧娟,樊翠,张英,等.猪圆环病毒2型CAP蛋白在flashBAC杆状病毒表达系统中的表达与鉴定[J].中国畜牧兽医,2016,43(7):1876-1883.GUO H J,FAN C,ZHANG Y,et al.Expression and identification of porcine circivirus type 2capsid protein using flashBAC baculovirus expression system[J].China Animal Husbandry&Veterinary Medicine,2016,43(7):1876-1883.(in Chinese)
[25]刘春菊,任炜杰,王志亮.杆状病毒表达系统在兽用亚单位疫苗领域应用的研究进展[J].中国畜牧兽医,2013,40(9):101-105.LIU C J,REN W J,WANG Z L.Research progress on application of baculovirus expressin system in veterinary subunit vaccine field[J].China Animal Husbandry&Veterinary Medicine,2013,40(9):101-105.(in Chinese)
[26]GLENN G M,SMITH G,FRIES L,et al.Safety and immunogenicity of a Sf9insect cell-derived respiratory syncytial virus fusion protein nanoparticle vaccine[J].Vaccine,2013,31(3):524-532.