重组杆状病毒高效表达猪圆环病毒2型Cap蛋白及其免疫特性分析
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摘要
目的利用重组杆状病毒高效表达猪圆环病毒2型(porcine circovirus 2,PCV2)Cap蛋白,并分析该蛋白的免疫特性。方法将PCV2 Cap基因克隆至转移载体pFastBacⅠ,转化感受态E.coli DH10Bac细胞,获得重组杆粒r Bacmid-Cap,转染Sf9细胞,SDS-PAGE和Western blot法检测重组杆状病毒,并于High Five(H5)细胞中进行高效表达。将小鼠随机分为PBS对照组、杆状病毒组、重组杆状病毒组及疫苗对照组,均经小鼠肌内注射,100μL/只,分别于第0和14天进行免疫,于首免后第21、28、35及42天经小鼠眼眶静脉丛采血,分离血清,ELISA法进行检测。结果 PCR和测序分析结果表明,重组转移质粒和重组杆粒构建正确。重组PCV2 Cap蛋白的相对分子质量约28 000,可与抗His标签单克隆抗体及抗PCV2 Cap多克隆抗体发生特异性结合。重组杆状病毒按MOI=5及1感染H5细胞约72 h,PCV2 Cap蛋白表达量最高,表达产量约150μg/mL。首免后第21、28、35及42天,重组杆状病毒组小鼠血清抗体水平明显高于PBS对照组及杆状病毒组(P <0. 001);首免后第21、28、35天,重组杆状病毒组小鼠血清抗体水平明显高于疫苗对照组(P <0. 05),首免后第42天,两组差异无统计学意义(P> 0. 05)。结论成功利用杆状病毒表达系统高效表达了PCV2 Cap,重组蛋白可诱导小鼠产生较高水平抗体,本研究为PCV疫苗的研制奠定了基础。
Objective To highly express the capsid(Cap) protein of porcine circovirus type 2(PCV2) by baculovirus expression system and analyze the immune property of expressed product. Methods PCV2 Cap gene was cloned into transfer vector pFastBacⅠ. The constructed recombinant plasmid was transformed to competent E.coli DH10 Bac,and the obtained recombinant bacmid was transfected to Sf9 cells. The recombinant baculovirus was identified by SDS-PAGE and Western blot,and expressed in High Five(H5) cells. Kunming mice were randomly divided into four groups and immunized with PBS(control),wild baculovirus,recombinant baculovirus and commercid PCV2 vaccine respectively by intramuscular injection for 2 times at an interval of 2 weeks,100 μL for each. Serum samples were collected on days 21,28,35 and 42 after the first injection and determined for specific IgG level against recombinant protein by indirect ELISA. Results PCR analysis and sequencing showed that the recombinant transfer vector and recombinant bacmid were constructed correctly. Recombinant PCV2 Cap protein,with a relative molecular mass of about 28 000,showed specific binding to anti-His tag monoclonal antibody and anti-PCV2/Cap polyclonal antibody. The expression level of PCV2 Cap protein reached the maximum of about 150 μg/mL in H5 cells 72 h after infection with the recombinant baculovirus at MOIs of 5 and 1. The serum antibody levels of mice on days 21,28,35 and 42 after the first immunization with recombinant baculovirus were significantly higher than those with PBS and with wild baculovirus(P < 0. 001). However,serum antibody levels of mice on days 21,28 and 35 after the first immunization with recombinant baculovirus was significantly higher than those with commercial PCV2 vaccine(P < 0. 05),while showed no significant difference on day 42(P > 0. 05). Conclusion PCV2 Cap was highly expressed in baculovirus,which induced high antibody level in mice.It laid a foundation of preparation of PCV vaccine.
Objective To highly express the capsid(Cap) protein of porcine circovirus type 2(PCV2) by baculovirus expression system and analyze the immune property of expressed product. Methods PCV2 Cap gene was cloned into transfer vector pFastBacⅠ. The constructed recombinant plasmid was transformed to competent E.coli DH10 Bac,and the obtained recombinant bacmid was transfected to Sf9 cells. The recombinant baculovirus was identified by SDS-PAGE and Western blot,and expressed in High Five(H5) cells. Kunming mice were randomly divided into four groups and immunized with PBS(control),wild baculovirus,recombinant baculovirus and commercid PCV2 vaccine respectively by intramuscular injection for 2 times at an interval of 2 weeks,100 μL for each. Serum samples were collected on days 21,28,35 and 42 after the first injection and determined for specific IgG level against recombinant protein by indirect ELISA. Results PCR analysis and sequencing showed that the recombinant transfer vector and recombinant bacmid were constructed correctly. Recombinant PCV2 Cap protein,with a relative molecular mass of about 28 000,showed specific binding to anti-His tag monoclonal antibody and anti-PCV2/Cap polyclonal antibody. The expression level of PCV2 Cap protein reached the maximum of about 150 μg/mL in H5 cells 72 h after infection with the recombinant baculovirus at MOIs of 5 and 1. The serum antibody levels of mice on days 21,28,35 and 42 after the first immunization with recombinant baculovirus were significantly higher than those with PBS and with wild baculovirus(P < 0. 001). However,serum antibody levels of mice on days 21,28 and 35 after the first immunization with recombinant baculovirus was significantly higher than those with commercial PCV2 vaccine(P < 0. 05),while showed no significant difference on day 42(P > 0. 05). Conclusion PCV2 Cap was highly expressed in baculovirus,which induced high antibody level in mice.It laid a foundation of preparation of PCV vaccine.
引文
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[2]SEGALES J.Best practice and future challenges for vaccination against porcine circovirus type 2[J].Expert Rev Vaccines,2015,14(3):473-487.
[3]REN L,CHEN X,OUYANG H.Interactions of porcine circovirus 2 with its hosts[J].Virus Genes,2016,52(4):437-444.
[4]MENG X J.Porcine circovirus type 2(PCV2):pathogenesis and interaction with the immune system[J].Annu Rev Anim Biosci,2013,1(1):43-64.
[5]CHAE C.An emerging porcine circovirus type 2b mutant(m PCV2b)originally known as PCV2d[J].Vet J,2015,203(1):6-9.
[6]ELLIS J,CLARK E,HAINES D,et al.Porcine circovirus-2and concurrent infections in the field[J].Vet Microbiol,2004,98(2):159-163.
[7]GU J,XING G,LEI J,et al.Porcine circovirus type 2 and PCV2-systemic disease-a review[J].Sheng Wu Gong Cheng Xue Bao,2015,31(6):880-891.(in Chinese)
[8]PENG Z,MA T,PANG D,et al.Expression,purification and antibody preparation of PCV2 Rep and ORF3 proteins[J].Int JBiol Macromol,2016,86(5):277-281.
[9]WU P C,CHEN T Y,CHI J N,et al.Efficient expression and purification of porcine circovirus type 2 virus-like particles in Escherichia coli[J].J Biotechnol,2016,220(1):78-85.
[10]JUHAN N M,LEROITH T,OPRIESSNIG T,et al.The open reading frame 3(ORF3)of porcine circovirus type 2(PCV2)is dispensable for virus infection but evidence of reduced pathogenicity is limited in pigs infected by an ORF3-null PCV2mutant[J].Virus Res,2010,147(1):60-66.
[11]GAO Z,DONG Q,JIANG Y E A.ORF4-protein deficient PCV2 mutants enhance virus-induced apoptosis and show differential expression of m RNAs in vitro[J].Virus Res,2014,183(1):56-62.
[12]韦平,秦爱建.重要动物病毒分子生物学[M].北京:科学出版社,2008:477-500.
[13]LI L,YUAN W,GUO H,et al.Prevalence and genetic variation of porcine circovirus type 2 in Hebei,China from 2004 to2014[J].Gene,2016,586(2):222-227.
[14]XIAO C T,HALBUR P G,OPRIESSNIG T.Global molecular genetic analysis of porcine circovirus type 2(PCV2)sequences confirms the presence of four main PCV2 genotypes and reveals a rapid increase of PCV2d[J].J Gen Virol,2015,96(Pt 7):1830-1841.