猪圆环病毒2型ORF2可溶性表达条件的优化
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摘要
为探索猪圆环病毒2型(PCV2)ORF2基因在大肠杆菌中高效可溶性表达的条件,扩增ORF2基因构建重组表达质粒p ET30a-ORF2,转化入大肠杆菌BL21(DE3)感受态获得重组表达菌;通过改变菌体培养温度和时间、诱导温度和时间、溶解氧量、IPTG及CaCl_2浓度,实现目的蛋白高效可溶性表达。结果表明:重组表达质粒p ET30a-ORF2经酶切及测序证明构建正确,重组表达质粒在大肠杆菌BL21(DE3)中实现可溶性表达,其最佳表达条件为:在500 mL培养瓶中加入200 mL LB卡那霉素阳性培养基,菌体于37℃培养4 h后,分别加入IPTG及Ca Cl2至终浓度为0.6 mmol/L及0.06 mol/L,30℃诱导表达4 h;重组Cap蛋白的最高表达量占总蛋白的55.9%。说明优化后可实现猪圆环病毒Cap蛋白的可溶性表达,这为进一步研究该蛋白的结构及其生物学特性奠定了基础。
To optimize the condition for secretory expression of ORF2 gene of porcine circovirus type 2(PCV2) in Escherichia coli,ORF2 gene was amplified from recombinant plasmid pMD-ORF2,and then the ORF2 gene was inserted into the vector pET30 a.The constructed recombinant plasmid pET30 a-ORF2 was transformed to E.coli BL21(DE3) for expression.The expression product was analyzed by SDS-PAGE.In order to improve the secretory expression level of target protein,temperature and time for culture and induction,dissolved oxygen content,concentration of IPTG and CaCl_2 were optimized.Both restriction analysis and sequencing proved that the recombinant plasmid pET30 a-ORF2 was constructed correctly,and ORF2 protein was secretory expressed in the E.coli BL21(DE3) after induction.The expression condition for ORF2 was optimized as follows:200 mL of kanamycin-positive LB medium was added into 500 mL flask and cultured at 37℃ for 4 h,then IPTG and CaCl_2 were added to the final concentration of 0.6 mmol/L and 0.06 mol/L,and induced at 30 ℃ for 4 h.Under this condition,expression of ORF2 protein was the highest,up to 55.9 percent of total protein content.Finally,the condition for expression of Cap protein was optimized,which provided a basis for the further mechanistic study of the structure and biological characteristics of Cap protein.
To optimize the condition for secretory expression of ORF2 gene of porcine circovirus type 2(PCV2) in Escherichia coli,ORF2 gene was amplified from recombinant plasmid pMD-ORF2,and then the ORF2 gene was inserted into the vector pET30 a.The constructed recombinant plasmid pET30 a-ORF2 was transformed to E.coli BL21(DE3) for expression.The expression product was analyzed by SDS-PAGE.In order to improve the secretory expression level of target protein,temperature and time for culture and induction,dissolved oxygen content,concentration of IPTG and CaCl_2 were optimized.Both restriction analysis and sequencing proved that the recombinant plasmid pET30 a-ORF2 was constructed correctly,and ORF2 protein was secretory expressed in the E.coli BL21(DE3) after induction.The expression condition for ORF2 was optimized as follows:200 mL of kanamycin-positive LB medium was added into 500 mL flask and cultured at 37℃ for 4 h,then IPTG and CaCl_2 were added to the final concentration of 0.6 mmol/L and 0.06 mol/L,and induced at 30 ℃ for 4 h.Under this condition,expression of ORF2 protein was the highest,up to 55.9 percent of total protein content.Finally,the condition for expression of Cap protein was optimized,which provided a basis for the further mechanistic study of the structure and biological characteristics of Cap protein.
引文
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[15]Mahe D,Blanchard P,Truong C,et al.Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes[J].Journal of General Virology,2000,81(7):1815-1824.
[16]Wang X,Jiang P,Li Y,et al.Protection of pigs against post-weaningmultisystemic wasting syndrome by a recombinant adenovirus expressing the capsid protein of porcine circovirus type 2[J].Vet Microbiol,2007,121(3-4):215-224.
[17]Kamstrup S,Barfoed A M,Frimann T H,et al.Immunization against PCV2 structural protein by DNA vaccination of mice[J].Vaccine,2004,22:1358-1361.
[18]杨杰,韩勇,高花,等.大肠杆菌表达SLA-3蛋白与口蹄疫病毒多肽的复性研究[J].微生物学通报,2017,44(2):394-401.
[19]Segalés J,Kekarainen T,Cortey M.The natural history of porcine circovirus type2:From an inoffensive virus to a devastating swine disease[J].Vet Microbiol,2013,165(1-2):13-20.
[2]Ellis J,Krakowka S,Lairmore M,et al.Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets[J].Journal of Veterinary Diagnostic Investigation,1999,11(1):3-14.
[3]Clark E G.Postweaning multisystemic wasting syndrome[C]//Proceedings of American Association of Swine Practitioners.Quebec City,Canada.1997:499-501.
[4]郎洪武,张广川,吴发权,等.断奶猪多系统衰弱综合征血清抗体检测[J].中国兽医科技,2000,30(3):3-5.
[5]He J,Cao J,Zhou N,et al.Identification and functional analysis of the novel ORF4 protein encoded by porcinecircovirus type 2[J].J Virol,2013,87(3):1420-1429.
[6]Cheung A K.Porcine circovirus:transcription and DNA replication[J].Virus Res,2012,164(1-2):46-53.
[7]Nygaard F B,Harlow K W.Heterologous expression of soluble,active proteins in Escherichia coli:the human estrogen receptor hormone-binding domain as paradigm[J].Protein Expression and Purification,2001,21(3):500-509.
[8]Segalés J.Porcine circovirus type 2(PCV2)infections:clinical signs,pathology and laboratory diagnosis[J].Virus Res,2012,164(1-2):10-19.
[9]Fan H,Xiao S,Tong T,et al.Immunogennicity of porcine circovirus type 2 capsid protein targeting to different subcellular compartments[J].Molecular Immunology,2008,45(3):653-660.
[10]Beach N M,Meng X J.Efficacy and future prospects of commercially available and experimental vaccines against porcine circovirus type 2(PCV2)[J].Virus Res,2012,164(1-2):33-42.
[11]Choi C,Chae C.Colocalitzation of porcine reproductive and respiratory syndrome virus and porcine circovirus 2 in porcine dermatitis and nephropathy syndrome by double-lableing technique[J].Veterinary Pathology,2001,38(4):436-441.
[12]Blanchard P,MahéD,Cariolet R,et al.Protection of swine against post-weaning multisystemic wasting syndrome(PMWS)by porcine circovirus type 2(PCV2)proteins[J].Vaccine,2003,21(31):4565-4575.
[13]杨克礼,时代,段正赢,等.猪圆环病毒2型Cap基因的克隆与原核表达[J].江西农业学报,2017,29(1):96-101.
[14]Nawagitgul P,Harms P A,Morozov I,et al.Modified indirect porcine circovirus(PCV)type 2-based and recombinant capsid protein(ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to PCV[J].Clinical and Diagnostic Laboratory Immunology,2002,9(1):33-40.
[15]Mahe D,Blanchard P,Truong C,et al.Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes[J].Journal of General Virology,2000,81(7):1815-1824.
[16]Wang X,Jiang P,Li Y,et al.Protection of pigs against post-weaningmultisystemic wasting syndrome by a recombinant adenovirus expressing the capsid protein of porcine circovirus type 2[J].Vet Microbiol,2007,121(3-4):215-224.
[17]Kamstrup S,Barfoed A M,Frimann T H,et al.Immunization against PCV2 structural protein by DNA vaccination of mice[J].Vaccine,2004,22:1358-1361.
[18]杨杰,韩勇,高花,等.大肠杆菌表达SLA-3蛋白与口蹄疫病毒多肽的复性研究[J].微生物学通报,2017,44(2):394-401.
[19]Segalés J,Kekarainen T,Cortey M.The natural history of porcine circovirus type2:From an inoffensive virus to a devastating swine disease[J].Vet Microbiol,2013,165(1-2):13-20.
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