猪繁殖与呼吸综合征病毒非结构蛋白NSP2原核表达与单克隆抗体的研制
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摘要
猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndromevirus,PRRSV)属动脉炎病毒科成员,可引起怀孕母猪繁殖障碍和仔猪呼吸道症状。PRRSV基因组为单股正链RNA,大小约15kb,含有8个开放阅读框(ORFs)。ORF1编码的聚合蛋白裂解产生13个非结构蛋白(NSP1-13),NSP2有多个B细胞表位,具有较强的免疫原性,病毒感染后能够刺激机体产生特异性抗体。本实验通过基因重组技术将截短修饰过的NSP2基因克隆至原核表达载体中进行表达,表达出的蛋白经过纯化及鉴定后免疫Balb/C小鼠,通过细胞融合技术获得了两株单克隆抗体,主要内容包括:
1.PRRSV NSP2基因在大肠杆菌中的表达:以含有PRRSV NSP2基因的pET-NSP2重组质粒为模板,参照已发表的PRRSV基因组序列,设计一对PCR引物,并在其上下游分别添加EcoRI和NotI酶切位点。获得的PCR产物用EcoRI和NotI酶切后与经同样处理的pGEX-6p-1原核表达载体质粒连接,转化DH_(5α)细胞后挑取阳性克隆。经酶切鉴定和测序验证后,转化至E.coli BL_(21)中,用0.6mM异丙基硫代-β-D-半乳糖苷(IPTG)于37℃诱导表达4b后,取细菌茵体用SDS-PAGE分析。结果表明,NSP2在大肠杆菌中得到表达,重组蛋白大小约为50kDa。将表达出来的NSP2蛋白用亲和层析法进行纯化,用猪抗PRRSV血清抗体进行Westernblot鉴定,结果表明,融合表达的GST-tNSP2蛋白能被PRRSV阳性血清特异性识别。
2.PRRSV NSP2蛋白单克隆抗体的制备:取纯化的GST-tNSP2蛋白按每只50μg的剂量与等量弗氏完全佐剂进行乳化,经皮下多点注射免疫6-8周龄Balb/c小鼠。每间隔2w再用弗氏不完全佐剂乳化的重组蛋白加强免疫2次,并于融合前3d用纯化的GST-tNSP2蛋白免疫1次。取脾细胞与SP2/0骨髓瘤细胞进行融合,融合率达到80%,首次筛选阳性率达60%,经过3次亚克隆,获得2株能稳定分泌抗NSP2蛋白抗体的杂交瘤细胞株,将其命名为285、3H3。亚型鉴定结果均为IgG1型,其轻链均为κ链。间接免疫荧光试验证明,285和3H3均能与PRRSV S1毒林产生特异性反应,而不能与SY0608毒株反应。采用间接ELISA法测得285细胞培养上清抗体效价为1:512,腹水效价为1:102400;3H3细胞培养上清为1:640,腹水效价为1:204800。将杂交瘤细胞株连续培养60d,传代20次,分别取第5、10、20代的培养上清,检测其ELISA抗体效价,结果基本一致。
综上所述,本试验通过原核表达技术和细胞融合技术获得了两株能稳定分泌单克隆抗体的杂交瘤细胞株285、3H3,为PRRSV分离株的鉴定及NSP2功能研究奠定了重要基础。
Porcine reproductive and respiratory syndrome virus(PRRSV) is a member of the Arteriviridae,can cause reproductive failure in sows and respiration disturbance in piglets.The genome is a positive sense,15 kb single strand of RNA consisting of 8 open reading frames(ORFs).The ORF1 is predicted to be anto-proteolytically cleaved at 12 sites,producing 13 non-structural proteins ultimately(NSP_(1-13)).NSP2 contains a larger number of linear B-cell epitopes,indicating that NSP2 protein of PRRSV is an immunogenic protein capable of eliciting specific antibody production during viral infections.In the study,NSP2 protein was partly expressed in E.coli and two NSP2-specific monoclonal antibodys were developed.The process was as following:
1.Expression of NSP2 gene of PRRSV in prokaryocyte system:In order to express the NSP2 gene of PRRSV,a pair of primers that have EcoRI and NotI sites were designed according to the sequence of PRRSV S1 strain.The NSP2 gene was amplified from recombinant plasmid pET-NSP2 by PCR using the primers,and cloned into a prokaryotic expression plasmid pGEX-6p-1.The ligated product was transformed into E.coli DH5αand then picked the positive clone,a recombinant plasmid named pGEX-6p-1-tNSP2 was constructed and identified with restriction enzyme digestion and sequenced.The plasmid pGEX-6p-1-tNSP2 was transformed into E.coli BL_(21) and induced by 0.6mM isopropyl-β-D-thiogalactoside(IPTG) at 37℃for 4h.SDS-PAGE of the bacteria revealed that a fusion protein GST-tNSP2 with molecular weight of 50 kDa was expressed in E.coli.The purified GST-tNSP2 protein showed a Strong reaction with the PRRSV-positive sera in Western blot assay.
2.The development of monoclonal antibodies against NSP2 protein:Six to eight -week-old Balb/c mice were hypodermically immunized three times at 2-week interval with 50ug of the GST-tNSP2 protein emulsified in Freund'scomplete(first injection) or incomplete adjuvant(second and third injections).The mice received a final injection of 50 ug of the protein in PBS before fusion.The splenocytes of the immunized mice were fused with murine myeloma cells SP2/0,and the rate of fusion was 80%,the positive rate of the first screen was 60%.After subcloning by 3 times, two hybridoma clones which produced McAbs steadily were screened by ELISA, named 3H3 and 2B5.Both of the McAbs belong to IgG1 isotype,and their light chains belongκtype.They all reacted strongly with the PRRSV S1 infected Marc-145 cells in IFA,but not with the PRRSV SY0608 strain.The ELISA titers of 2B5 and 3H3 in the ascites were 1:102400 and 1:204800 respectively,meanwhile the ELISA titers in cell cultural supernatant were 1:512 and 1:640 respectively,and the titers of 5、10、20 generations were also the same.
In summary,in this experiment we get two hybridoma clones which produced McAbs steadily,and the expressed GST-tNSP2 protein and McAbs could be used for identification of PRRSV isolates and functional analysis of NSP2.
1.PRRSV NSP2基因在大肠杆菌中的表达:以含有PRRSV NSP2基因的pET-NSP2重组质粒为模板,参照已发表的PRRSV基因组序列,设计一对PCR引物,并在其上下游分别添加EcoRI和NotI酶切位点。获得的PCR产物用EcoRI和NotI酶切后与经同样处理的pGEX-6p-1原核表达载体质粒连接,转化DH_(5α)细胞后挑取阳性克隆。经酶切鉴定和测序验证后,转化至E.coli BL_(21)中,用0.6mM异丙基硫代-β-D-半乳糖苷(IPTG)于37℃诱导表达4b后,取细菌茵体用SDS-PAGE分析。结果表明,NSP2在大肠杆菌中得到表达,重组蛋白大小约为50kDa。将表达出来的NSP2蛋白用亲和层析法进行纯化,用猪抗PRRSV血清抗体进行Westernblot鉴定,结果表明,融合表达的GST-tNSP2蛋白能被PRRSV阳性血清特异性识别。
2.PRRSV NSP2蛋白单克隆抗体的制备:取纯化的GST-tNSP2蛋白按每只50μg的剂量与等量弗氏完全佐剂进行乳化,经皮下多点注射免疫6-8周龄Balb/c小鼠。每间隔2w再用弗氏不完全佐剂乳化的重组蛋白加强免疫2次,并于融合前3d用纯化的GST-tNSP2蛋白免疫1次。取脾细胞与SP2/0骨髓瘤细胞进行融合,融合率达到80%,首次筛选阳性率达60%,经过3次亚克隆,获得2株能稳定分泌抗NSP2蛋白抗体的杂交瘤细胞株,将其命名为285、3H3。亚型鉴定结果均为IgG1型,其轻链均为κ链。间接免疫荧光试验证明,285和3H3均能与PRRSV S1毒林产生特异性反应,而不能与SY0608毒株反应。采用间接ELISA法测得285细胞培养上清抗体效价为1:512,腹水效价为1:102400;3H3细胞培养上清为1:640,腹水效价为1:204800。将杂交瘤细胞株连续培养60d,传代20次,分别取第5、10、20代的培养上清,检测其ELISA抗体效价,结果基本一致。
综上所述,本试验通过原核表达技术和细胞融合技术获得了两株能稳定分泌单克隆抗体的杂交瘤细胞株285、3H3,为PRRSV分离株的鉴定及NSP2功能研究奠定了重要基础。
Porcine reproductive and respiratory syndrome virus(PRRSV) is a member of the Arteriviridae,can cause reproductive failure in sows and respiration disturbance in piglets.The genome is a positive sense,15 kb single strand of RNA consisting of 8 open reading frames(ORFs).The ORF1 is predicted to be anto-proteolytically cleaved at 12 sites,producing 13 non-structural proteins ultimately(NSP_(1-13)).NSP2 contains a larger number of linear B-cell epitopes,indicating that NSP2 protein of PRRSV is an immunogenic protein capable of eliciting specific antibody production during viral infections.In the study,NSP2 protein was partly expressed in E.coli and two NSP2-specific monoclonal antibodys were developed.The process was as following:
1.Expression of NSP2 gene of PRRSV in prokaryocyte system:In order to express the NSP2 gene of PRRSV,a pair of primers that have EcoRI and NotI sites were designed according to the sequence of PRRSV S1 strain.The NSP2 gene was amplified from recombinant plasmid pET-NSP2 by PCR using the primers,and cloned into a prokaryotic expression plasmid pGEX-6p-1.The ligated product was transformed into E.coli DH5αand then picked the positive clone,a recombinant plasmid named pGEX-6p-1-tNSP2 was constructed and identified with restriction enzyme digestion and sequenced.The plasmid pGEX-6p-1-tNSP2 was transformed into E.coli BL_(21) and induced by 0.6mM isopropyl-β-D-thiogalactoside(IPTG) at 37℃for 4h.SDS-PAGE of the bacteria revealed that a fusion protein GST-tNSP2 with molecular weight of 50 kDa was expressed in E.coli.The purified GST-tNSP2 protein showed a Strong reaction with the PRRSV-positive sera in Western blot assay.
2.The development of monoclonal antibodies against NSP2 protein:Six to eight -week-old Balb/c mice were hypodermically immunized three times at 2-week interval with 50ug of the GST-tNSP2 protein emulsified in Freund'scomplete(first injection) or incomplete adjuvant(second and third injections).The mice received a final injection of 50 ug of the protein in PBS before fusion.The splenocytes of the immunized mice were fused with murine myeloma cells SP2/0,and the rate of fusion was 80%,the positive rate of the first screen was 60%.After subcloning by 3 times, two hybridoma clones which produced McAbs steadily were screened by ELISA, named 3H3 and 2B5.Both of the McAbs belong to IgG1 isotype,and their light chains belongκtype.They all reacted strongly with the PRRSV S1 infected Marc-145 cells in IFA,but not with the PRRSV SY0608 strain.The ELISA titers of 2B5 and 3H3 in the ascites were 1:102400 and 1:204800 respectively,meanwhile the ELISA titers in cell cultural supernatant were 1:512 and 1:640 respectively,and the titers of 5、10、20 generations were also the same.
In summary,in this experiment we get two hybridoma clones which produced McAbs steadily,and the expressed GST-tNSP2 protein and McAbs could be used for identification of PRRSV isolates and functional analysis of NSP2.
引文
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[1]孙庆申,仇华吉,童光志等.猪繁殖与呼吸综合征单克隆抗体研究进展[J].动物医学进展,2005,22(4):25-28.
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[3]De Lima M,Pattnaik A K,Flores E F,et al.Serologic marker candidates identified among B-cell linear epitopes of Nsp2 and structural proteins of a North American strain of porcine reproductive and respiratory syndrome virus[J].Virology,2006,353(2):410-421.
[4]Oleksiewicz M B,Botner A,Toft P,et al.Epitope mapping porcine reproductive and respiratory syndrome virus by phage display:the nsp2 fragment of the repliease polyprotein contains a cluster of B-Cell epitopes[J].J Virol,2001,75(7):3277-3290.
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[1]孙庆申,仇华吉,童光志等.猪繁殖与呼吸综合征单克隆抗体研究进展[J].动物医学进展,2005,22(4):25-28.
[2]Wensvoort G,Terpstra C,Pol J M,et al.Mystery swine disease in the Netherlands:the isolation of Lelystad virus[J].Vet Q,1991,13(3):121-130.
[3]De Lima M,Pattnaik A K,Flores E F,et al.Serologic marker candidates identified among B-cell linear epitopes of Nsp2 and structural proteins of a North American strain of porcine reproductive and respiratory syndrome virus[J].Virology,2006,353(2),410-421.
[4]Oleksiewicz M B,Bother A,Toft P,et al.Epitope mapping porcine reproductive and respiratory syndrome virus by phage display:the nsp2 fragment of the replicase polyprotein contains a cluster of B-Cell epitopes.Journal of virology[J],2001,75(7):3277-3290.
[5]Yan Y,Guo X,Ge X,et al.Monoclonal antibody and porcine antisera recognized B-cell epitopes of Nsp2 protein of a Chinese strain of porcinereproductive and respiratory syndrome virus[J].Virus.Res,2007,126:207-215.
[6]Kohler G,Milstein C.Continuous culture of fused cells secreting antibodies of predefined specificity [J].Nature,1975,256:495-497.
[7]刘书亮,王红宁.PRRS的病原学和免疫学研究进展[J].四川畜牧兽医,2000,27(7):79-83
[8]Li Y F,Wang X L,Tang B,et al.Emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the Mid-Eastern region of China[J].The Veterinary Journal,2007,174(3):577-584.