表达PCV-2 Cap和PPV-1 VP2蛋白的重组PRV的构建及鉴定
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摘要
为了构建表达猪圆环病毒2型(PCV-2)Cap蛋白和猪细小病毒1型(PPV-1)VP2蛋白的重组伪狂犬病病毒(PRV)及鉴定其免疫原性,试验采用经典同源重组技术构建重组病毒,利用密码子优化的VP2基因(VP2_(opti))和含PRV gG蛋白信号肽序列的VP2_(opti)基因分别构建重组质粒pMD18T-LR(TK)-VP2_(opti)和pMD18T-LR(TK)-VP2_(opti)(SP)。用已构建的重组病毒rPRV-TK~-/gE~-/gG~-/3Cap~+为骨架,以EGFP为标签经同源重组获得重组病毒。采用IPMA法鉴定Cap蛋白和VP2蛋白抗原在重组病毒感染的PK-15细胞中的表达,采用IFA法鉴定VP2蛋白在重组病毒感染的PK-15细胞中的表达与定位。用这2株重组病毒免疫小鼠,通过检测血清中PRV、PCV-2和PPV-1抗体水平对构建的重组病毒在小鼠体内的免疫原性进行评价。结果表明:经同源重组获得重组病毒rPRV-TK~-/VP2~+/gE~-/gG~-/2Cap~+和rPRV-TK~-/VP2~+(SP)/gE~-/gG~-/2Cap~+;在重组病毒感染的PK-15细胞中均能检测到阳性Cap蛋白和VP2蛋白抗原;重组病毒rPRV-TK~-/VP2~+/gE~-/gG~-/2Cap~+表达的VP2蛋白定位于细胞浆和细胞核,而重组病毒rPRV-TK~-/VP2~+(SP)/gE~-/gG~-/2Cap~+表达的VP2蛋白定位于细胞浆;用这2株重组病毒免疫小鼠能够检测到PRV中和抗体;用灭活的重组病毒免疫小鼠后可产生低水平的Cap和VP2抗体,而重组病毒活毒免疫的小鼠未检测到相应抗体。说明这2株重组病毒在体外能够表达Cap蛋白和VP2蛋白,PRV gG蛋白信号肽序列的加入促进了VP2蛋白从细胞核的释放。
To construct the recombinant Pseudorabies virus(PRV) expressing Cap protein of Porcine circovirus type 2(PCV-2) and VP2 protein of Porcine parvovirus 1(PPV-1) and evaluate the immunogenicity of the recombinant virus, classical homologous recombination technique was used for construction of the recombinant virus. The codon optimized VP2 gene(VP2_(opti)) and the VP2_(opti) gene containing signal peptide sequences were cloned into the recombinant plasmids pMD18 T-LR(TK)-VP2_(opti) and pMD18 T-LR(TK)-VP2_(opti)(SP), respectively. Using the constructed recombinant virus rPRV-TK~-/gE~-/gG~-/3 Cap~+ as skeleton and EGFP as tag, the recombinant virus were obtained after homologous recombination. Cap protein and VP2 protein expressed by the recombinant virus in PK-15 cells were detected by IPMA. The localization of VP2 protein expressed by the recombinant virus in PK-15 cells was identified by IFA. The immunogenicity of these two constructed recombinant virus in mice were evaluated by detecting the antibodies level of PRV, PCV-2 and PPV-1 in serum. The results showed that recombinant viruses(rPRV-TK~-/VP2~+/gE~-/gG~-/2 Cap~+ and rPRV-TK~-/VP2~+(SP)/gE~-/gG~-/2 Cap~+) were obtained by the classical homologous recombination. Both Cap protein and VP2 protein could be detected in PK-15 cells infected by the recombinant viruses. VP2 protein expressed by the recombinant virus rPRV-TK~-/VP2~+/gE~-/gG~-/2 Cap~+ located in the cytoplasm and nucleus, while VP2 protein expressed by the recombinant virus rPRV-TK~-/VP2~+(SP)/gE~-/gG~-/2 Cap~+ located in the cytoplasm. PRV neutralizing antibodies could be detected in mice serum immunized with these two recombinant viruses, and low levels of Cap and VP2 antibodies can be produced in mice immunized with inactivated recombinant viruses, but the corresponding antibodies could not be detected in mice immunized with active recombinant viruses. It indicates that these two recombinant viruses express PCV-2 Cap protein and PPV-1 VP2 protein in vitro, and the addition of PRV gG protein signal peptide sequences promote the release of VP2 protein from the nucleus to cytoplasm.
To construct the recombinant Pseudorabies virus(PRV) expressing Cap protein of Porcine circovirus type 2(PCV-2) and VP2 protein of Porcine parvovirus 1(PPV-1) and evaluate the immunogenicity of the recombinant virus, classical homologous recombination technique was used for construction of the recombinant virus. The codon optimized VP2 gene(VP2_(opti)) and the VP2_(opti) gene containing signal peptide sequences were cloned into the recombinant plasmids pMD18 T-LR(TK)-VP2_(opti) and pMD18 T-LR(TK)-VP2_(opti)(SP), respectively. Using the constructed recombinant virus rPRV-TK~-/gE~-/gG~-/3 Cap~+ as skeleton and EGFP as tag, the recombinant virus were obtained after homologous recombination. Cap protein and VP2 protein expressed by the recombinant virus in PK-15 cells were detected by IPMA. The localization of VP2 protein expressed by the recombinant virus in PK-15 cells was identified by IFA. The immunogenicity of these two constructed recombinant virus in mice were evaluated by detecting the antibodies level of PRV, PCV-2 and PPV-1 in serum. The results showed that recombinant viruses(rPRV-TK~-/VP2~+/gE~-/gG~-/2 Cap~+ and rPRV-TK~-/VP2~+(SP)/gE~-/gG~-/2 Cap~+) were obtained by the classical homologous recombination. Both Cap protein and VP2 protein could be detected in PK-15 cells infected by the recombinant viruses. VP2 protein expressed by the recombinant virus rPRV-TK~-/VP2~+/gE~-/gG~-/2 Cap~+ located in the cytoplasm and nucleus, while VP2 protein expressed by the recombinant virus rPRV-TK~-/VP2~+(SP)/gE~-/gG~-/2 Cap~+ located in the cytoplasm. PRV neutralizing antibodies could be detected in mice serum immunized with these two recombinant viruses, and low levels of Cap and VP2 antibodies can be produced in mice immunized with inactivated recombinant viruses, but the corresponding antibodies could not be detected in mice immunized with active recombinant viruses. It indicates that these two recombinant viruses express PCV-2 Cap protein and PPV-1 VP2 protein in vitro, and the addition of PRV gG protein signal peptide sequences promote the release of VP2 protein from the nucleus to cytoplasm.
引文
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[8] POMERANZ L E, REYNOLDS A E,HENGARTNER C J. Molecular biology of Pseudorabies virus: impact on neurovirology and veterinary medicine [J]. Microbiol Mol Biol Rev, 2005, 69(3): 462-500.
[9] WANG Y P, DU W J, HUANG L P, et al. The Pseudorabies virus DNA polymerase accessory subunit UL42 directs nuclear transport of the holoenzyme [J]. Front Microbiol, 2015, 7: 124.
[10] LEI J L, XIA S L, WANG Y, et al. Safety and immunogenicity of a gE/gI/TK gene-deleted Pseudorabies virus variant expressing the E2 protein of classical swine fever virus in pigs [J]. Immunol Lett, 2016, 174: 63-71.
[11] BOISVERT M, BOUCHARD-LéVESQUE V, FERNANDES S, et al. Classic nuclear localization signals and a novel nuclear localization motif are required for nuclear transport of Porcine parvovirus capsid proteins [J]. J Virol, 2014, 88(20): 11748-11759.
[12] SEDLIK C, DRIDI A, DERIAUD E, et al. Intranasal delivery of recombinant parvovirus-like particles elicits cytotoxic T-cell and neutralizing antibody responses [J]. J Virol, 1999, 73(4): 2739.
[13] ZHENG L L, GUO X Q, ZHU Q L, et al. Construction and immunogenicity of a Recombinant pseudorabies virus co-expressing Porcine circovirus type 2 capsid protein and interleukin 18 [J]. Vet Res, 2015, 201: 8-15.
[2] 粟元文, 王怀禹, 吕远蓉. 猪细小病毒NS1蛋白的表达与鉴定 [J]. 黑龙江畜牧兽医, 2017(11上): 174-177.
[3] SUN J H, HUANG L P, WEI Y W, et al. Prevalence of emerging porcine parvoviruses and their co-infections with porcine circovirus type 2 in China[J]. Arch Virol, 2015, 160(5):1339-1344.
[4] CHI J N, WU C Y, CHIEN M S, et al. The preparation of Porcine circovirus type 2 (PCV2) virus-like particles using a recombinant Pseudorabies virus and its application to vaccine development [J]. J Biotechnol, 2014, 181(3): 12-19.
[5] HUANG L P, LU Y H, WEI Y W, et al. Identification of one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of Porcine circovirus type 2 [J]. BMC Microbiol, 2011, 11(1): 1-10.
[6] KONG M, PENG Y, CUI Y, et al. Development and evaluation of the rVP-ELISA for detection of antibodies against Porcine parvovirus [J]. J Virol Methods, 2014, 206: 115-118.
[7] HUANG L P, LU Y H, WEI Y W, et al. Construction and biological characterisation of recombinant Porcine circovirus type 2 expressing the V5 epitope tag[J]. Virus Res, 2011, 161(2): 115-123.
[8] POMERANZ L E, REYNOLDS A E,HENGARTNER C J. Molecular biology of Pseudorabies virus: impact on neurovirology and veterinary medicine [J]. Microbiol Mol Biol Rev, 2005, 69(3): 462-500.
[9] WANG Y P, DU W J, HUANG L P, et al. The Pseudorabies virus DNA polymerase accessory subunit UL42 directs nuclear transport of the holoenzyme [J]. Front Microbiol, 2015, 7: 124.
[10] LEI J L, XIA S L, WANG Y, et al. Safety and immunogenicity of a gE/gI/TK gene-deleted Pseudorabies virus variant expressing the E2 protein of classical swine fever virus in pigs [J]. Immunol Lett, 2016, 174: 63-71.
[11] BOISVERT M, BOUCHARD-LéVESQUE V, FERNANDES S, et al. Classic nuclear localization signals and a novel nuclear localization motif are required for nuclear transport of Porcine parvovirus capsid proteins [J]. J Virol, 2014, 88(20): 11748-11759.
[12] SEDLIK C, DRIDI A, DERIAUD E, et al. Intranasal delivery of recombinant parvovirus-like particles elicits cytotoxic T-cell and neutralizing antibody responses [J]. J Virol, 1999, 73(4): 2739.
[13] ZHENG L L, GUO X Q, ZHU Q L, et al. Construction and immunogenicity of a Recombinant pseudorabies virus co-expressing Porcine circovirus type 2 capsid protein and interleukin 18 [J]. Vet Res, 2015, 201: 8-15.