猪圆环病毒2型ORF2与猪繁殖及呼吸综合征病毒ORF5嵌合抗原表位在乳酸乳球菌中的表达
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摘要
目的乳酸乳球菌中表达猪圆环病毒2型(porcine circovirus type 2,PCV2)ORF2与猪繁殖及呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)ORF5的嵌合抗原表位。方法按乳酸乳球菌密码子使用偏好设计并合成PCV2 ORF2及PRRSV ORF5的串联抗原表位基因,插入至乳酸菌表达质粒p NZ8148,构建重组表达质粒,转化乳酸乳球菌NZ9000中,经乳酸菌素(nisin)诱导表达嵌合蛋白,采用Ni-NTA亲和层析纯化,进行12%SDS-PAGE分析。结果重组表达质粒经双酶切鉴定,构建正确。嵌合蛋白相对分子质量约25 000,在乳酸乳球菌中主要以可溶性形式表达,纯化后纯度达96%以上,浓度约1 mg/m L,产量可达60 mg/L细菌培养物。结论 PCV2ORF2与PRRSV ORF5的嵌合抗原表位在乳酸乳球菌中得到高效可溶性表达。
Objective To express the epitope chimaera of porcine circovirus type 2(PCV2) ORF2 and porcine reproductive and respiratory syndrome virus(PRRSV)ORF5 in Lactococcus lactis. Methods Tandem antigenic epitope gene of PCV2 ORF2 and PRRSV ORF5 was designed and synthesized according to the codon preference of Lactococcus lactis,and inserted into vector pNZ8148. The constructed recombinant plasmid was transformed into L. lactis NZ9000 for expression under induction of nisin. The expressed product was purified by Ni-NTA affinity chromatography and analyzed by 12% SDS-PAGE. Results Restriction analysis showed that recombinant plasmid was constructed correctly. The chimaera,with a relative molecular mass of about 25 000,was mainly expressed in a soluble form,and reached a purity of more than 96% after purification,of which the concentration was about 1 mg/m L,and the yield was 60 mg/L bacterial culture. Conclusion The epitope chimaera of PCV2 ORF2 and PRRSV ORF5 was highly expressed in a soluble form in L. lactis.
Objective To express the epitope chimaera of porcine circovirus type 2(PCV2) ORF2 and porcine reproductive and respiratory syndrome virus(PRRSV)ORF5 in Lactococcus lactis. Methods Tandem antigenic epitope gene of PCV2 ORF2 and PRRSV ORF5 was designed and synthesized according to the codon preference of Lactococcus lactis,and inserted into vector pNZ8148. The constructed recombinant plasmid was transformed into L. lactis NZ9000 for expression under induction of nisin. The expressed product was purified by Ni-NTA affinity chromatography and analyzed by 12% SDS-PAGE. Results Restriction analysis showed that recombinant plasmid was constructed correctly. The chimaera,with a relative molecular mass of about 25 000,was mainly expressed in a soluble form,and reached a purity of more than 96% after purification,of which the concentration was about 1 mg/m L,and the yield was 60 mg/L bacterial culture. Conclusion The epitope chimaera of PCV2 ORF2 and PRRSV ORF5 was highly expressed in a soluble form in L. lactis.
引文
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[14]ZHANG T J.Construction and protective immunogenicity of a recombinant PRRSV expressing ORF2 of porcine circovirus type2[D].Nanjing:Nanjing Agricultural University,2015.(in Chinese)张挺杰.表达PCV2 ORF2基因的重组猪繁殖与呼吸综合征病毒的构建及其免疫保护效力研究[D].江苏南京:南京农业大学,2015.
[2]杨洁.猪流行性腹泻病毒COE基因重组乳酸菌的构建及免疫原性分析[C].中国畜牧兽医学会动物微生态学分会中国畜牧兽医学会动物微生态学分会第五届第十二次全国学术研讨会论文集.北京:中国畜牧兽医学会动物微生态学分会,2016:1.
[3]MOHSENI A H,RAZAVILAR V,KEYVANI H,et al.Codon usage optimization and construction of plasmid encoding Iranian human papillomavirus type 16 E7 oncogene for Lactococcus lactis subsp.Cremoris MG1363[J].Asian Pac J Cancer Prev,2017,18(3):783-788.
[4]FAN P H,WEI Y W,GUO L J,et al.Studies on synergetic pathogenicity of co-infection with highly pathogenic PRRSV and PCV2[J].Sci Agri Sin,2012,45(18):3859-3872.(in Chinese)范培虎,危艳武,郭龙军,等.高致病性PRRSV与PCV2共感染协同致病性研究[J].中国农业科学,2012,45(18):3859-3872.
[5]OBER R A,THISSEN J B,JAING C J,et al.Increased microbiome diversity at the time of infection is associated with improved growth rates of pigs after co-infection with porcine reproductive and respiratory syndrome virus(PRRSV)and porcine circovirus type 2(PCV2)[J].Vet Microbiol,2017,208(9):203-211.
[6]YANG R,FU L Z,YANG L,et al.The tandem expressing of the antigen epitope genes of PCV2-ORF2 and PRRSV-ORF5and primary identification of its immunogenicity[J].Chin J Vet Sci,2014,34(11):1721-1724,1747.(in Chinese)杨睿,付利芝,杨柳,等.PCV2 ORF2与PRRSV ORF5抗原表位区串联原核表达及免疫原性[J].中国兽医学报,2014,34(11):1721-1724,1747.
[7]NIEDERWERDER M C,JAING C J,THISSEN J B,et al.Microbiome associations in pigs with the best and worst clinical outcomes following co-infection with porcine reproductive and respiratory syndrome virus(PRRSV)and porcine circovirus type2(PCV2)[J].Vet Microbiol,2016,188(1):1-11.
[8]MARTELLI P,ARDIGO P,FERRARI L,et al.Concurrent vaccinations against PCV2 and PRRSV:study on the specific immunity and clinical protection in naturally infected pigs[J].Vet Microbiol,2013,162(2-4):558-571.
[9]贾晓娟.猪Gp96N增强PRRSV合成肽疫苗、亚单位疫苗及PCV2灭活疫苗免疫效应的分析[D].北京:中国农业大学,2014.
[10]PIEYRO P E,KENNEY S P,GIMNEZ-LIROLA L G,et al.Expression of antigenic epitopes of porcine reproductive and respiratory syndrome virus(PRRSV)in a modified live-attenuated porcine circovirus type 2(PCV2)vaccine virus(PCV1-2a)as a potential bivalent vaccine against both PCV2 and PRRSV[J].Virus Res,2015,210(1):154-164.
[11]NIEDERWERDER M C,BAWA B,SERAO N V,et al.Vaccination with a porcine reproductive and respiratory syndrome(PRRS)modified live virus vaccine followed by challenge with PRRS virus and porcine circovirus type 2(PCV2)protects against PRRS but enhances PCV2 replication and pathogenesis compared to results for nonvaccinated cochallenged controls[J].Clin Vaccine Immunol,2015,22(12):1244-1254.
[12]HAN J,MA H,CAO L,et al.Immunogenicity of recombinant vaccinia virus vaccines co-expressing GP3/GP5 of European PRRSV and Cap protein of PCV2 in pigs[J].Appl Microbiol Biotechnol,2018,102(3):1145-1154.
[13]SUN W C.Molecular epidemiological investigation and the immunogenicity of recombinant adenovirus vaccines of PRRSVand PCV2[D].Nanning:Guangxi University,2017.(in Chinese)孙文超.PRRSV与PCV2的分子流行病学调查及其重组腺病毒疫苗实验免疫研究[D].广西南宁:广西大学,2017.
[14]ZHANG T J.Construction and protective immunogenicity of a recombinant PRRSV expressing ORF2 of porcine circovirus type2[D].Nanjing:Nanjing Agricultural University,2015.(in Chinese)张挺杰.表达PCV2 ORF2基因的重组猪繁殖与呼吸综合征病毒的构建及其免疫保护效力研究[D].江苏南京:南京农业大学,2015.