猪细小病毒NS1基因与VP2基因的克隆、表达及VP2基因免疫的研究
摘要
猪细小病毒(porcine parvovirus，PPV)感染是一种严重危害养猪业的病毒性疾病，广泛分布于世界各地，至今尚未得到有效的控制。因此研制有效疫苗来防制本病依然是一项十分紧迫的任务。
NS1蛋白是PPV的非结构蛋白之一，由NS1基因编码。NS1蛋白对PPV早期和晚期的基因转录都发挥着重要的调节作用。由于病毒灭活后不表达NS1蛋白，利用该蛋白作为诊断抗原可以区分灭活疫苗免疫猪和野毒感染猪。本实验室在1999年从污染了猪细小病毒的PK15细胞系中分离得到了一株PPV，定名为SY-99株。以SY-99株的基因组DNA为模板，利用PCR扩增出了NS1基因，将其克隆到原核表达载体pET28a中，获得重组质粒pET28a／SYNS1。序列测定结果显示，NS1基因全长1989核苷酸，编码662个氨基酸。在推导的氨基酸序列中含有在PPV复制过程中发挥重要作用的保守基序GKRN，并有三个潜在的糖基化位点，分别位于356-359、446-449和513-516位氨基酸。序列比较表明，SY-99株与NADL2-1株、NADL2-2株和Kresse株NS1基因的核苷酸同源性分别为98％、99％和99％，氨基酸同源性分别为97％、99％和99％。将重组质粒pET28a／SYNS1转化到大肠杆菌BL21(DE3)株中，所得的重组菌株经IPTG诱导后，表达出高水平的NS1蛋白，表达率可达17.8％。经ELISA和Western Blotting检测，表达的重组蛋白具有免疫学活性。经过优化选择确定了NS1蛋白高效表达的最适参数。表达产物带有组氨酸标签，适于用His·Bind柱亲和纯化。重组NS1蛋白为制备PPV诊断抗原用于本病的流行病学调查提供了一种检测工具。
当前用于PPV免疫防制的疫苗主要是油乳剂灭活苗和冻干弱毒苗，这两种常规疫苗都存在一定的缺陷，如生产疫苗所用的猪源传代细胞系或原代细胞很容易污染其它病原，PPV的体外培养滴度低，难于生产出高效价的疫苗，而且弱毒疫苗还存在毒力返强的可能，所以研制新型疫苗十分有必要。PPV VP2蛋白可以形成病毒样粒子(vires-like particles，VLPs)，其免疫原性与PPV病毒粒子近似，可以使接种动物获得保护性免疫。因此本研究拟用体外表达的重组VP2蛋白作为预防PPV的候选亚单位疫苗。以SY-99株基因组DNA为模板，用PCR扩增得到了VP2基因，将其克隆到酵母表达载体pPICZαB中，经测序后与PPV参考毒株NADL-2株进行序列比较，结果表明二者同源性达99.2％。经定点突变后，VP2基因中的SacI位点被缺失，构建了重组酵母转移载体pPICZαB／mVP2，经SacI线性化后，导入酵母受体菌GS115中，经甲醇诱导培养后，用SDS-PAGE检测重组菌株裂解产物，结果未检出目的蛋白条带。然后将VP2基因克隆到了另一酵母表达载体pPIC9K中，构建了酵母转移载体pPIC9K／mVP2，经转化和诱导培养后，也未检测目的条带。我们又将VP2基因靠近5，端部分进行了序列改造，构建了酵母转移载体pPICZαA／MVP2，转化酵母受体菌后诱导培养，检测结果显示，VP2仍然没
博土学位论文 摘 要 东北农业大学
有得到成功表达。在利用酵母表达 PPV VPZ的试验研究中，我们均得到了含有重组子的
阳性酵母菌株，我们对酵母生长条件进行了变化摸索，但在SDS－PAGE胶上始终没有肉
眼可见的目的条带。所以我们初步推测SY－99株WZ基因中存在不适于在酵母体系表达
的某种元件，抑或表达量过低而难于检测。
作为一种新型疫苗，基因疫苗具有安全可靠、生产简单、储运方便等优点，可望克
服常规疫苗的某些不足。我们将猪细小病毒VPZ基因及猪Y－干扰素（IFN－Y）基因同时
克隆到真核表达载体pIRESlneo中，构建了共表达VPZ和IFN－Y的基因免疫质粒
pIRESI／VPZ／Y，用此表达质粒单独或加入脂质体肌注接种 BABUC小鼠，三次兔疫后，
采取接种鼠血清，用ELISA检测特异性抗体，结果表明，脂质体可以加强表达质粒接种
小鼠的兔疫应答水平，为今后研制猪细小病毒基因疫苗提供了依据。
Porcine parvovirus (PPV) infection is one of the most important viral diseases, and characterized by reproductive failures in swine. PPV is epidemic in most regions of the world, including China, causing great economic loss in swine industry. Prevention and control of PPV infection is an urgent task.
The main non-structural protein of PPV, NS1 encoded by NS1 gene, is associated with the early and late transcription of PPV. NS1 can be used as a differential antigen for differentiating the pigs immunized with "dead" PPV vaccine from pigs infected with wild-type PPV. A PPV designated SY-99 strain was isolated from PK15 cells in 1999. The NS1 gene was amplified from the genomic DNA of SY-99, and then cloned into a prokaryotic expression vector pET28a, resulting in a recombinant plasmid pET28a/SYNS1. The NS1 gene was shown to contain 1989 base pairs (bp) and encode 662 amino acids (aa). A conserved motif GKRN, an important element in PPV transcription, was identified in the deduced NS1 protein sequence of SY-99. There are three potential glycosalation sites in the NS1 protein, at 356-359, 446-449, 513-516aa, respectively. The nucleotide identities of NS1 between SY-99 and PPV strains NADL2-1, NADL2-2 and Kresse are 98%, 99% and 99%, respectively, and the amino acid identities 97%, 99% and 99%, respectively. T
he pET28a/SYNSl was transformed into E. coli. BL21 (DE3). The NS1 protein was expressed with high-level in the transformed BL21 (DE3) after induction with IPTG. The expressed His-fusion protein was shown to be immunologically active by Western blotting. The IPTG concentration and induction time were optimized. The His-fusion recombinant protein was purified with His'Band column chromatography. The expressed NS1 protein will be beneficial to study of PPV infection.
The vaccines available against PPV infection are mostly derived from inactivated virus or modified live virus. These conventional vaccines show many disadvantages, including incidental reversion and less safety and efficacy. So it's essential to develop more effective and safer new-type vaccines against PPV infection.
The structural protein VP2 of PPV can form virus-like particles (VLPs). The VLPs are similar in antigenicity to PPV virions, so it can be used as a subunite vaccine candidate for protecting pigs from PPV infection. The VP2 gene of SY-99 was amplified and cloned into a yeast expression vector pPICZaB to obtain a recombinant plasmid pPICZaB/VP2. The pPICZaB/mVP2, modified from pPICZaB/VP2 by site-directed mutation to delete the Sad site in VP2 gene, was linearized by Sad and transformed into the host yeast GS115. The target protein was not detectable by SDS-PAGE analysis after induction by methanol. We mutated some codons of the VP2 gene based on the yeast codon preference. The modified VP2 gene
was then cloned into pPICZctA, resulting in pPICZ Gene vaccine represents another new-generation vaccine and has some advantages over the traditional vaccines. The VP2 gene and the porcine IFN-y gene were cloned into a eukaryotic expression plasmid pIRESl玡o, obtaining a recombinant pIRESl/VP2/y co-expressing VP2 and IFN-y. BABL/c mice were immunized with the pIRESl/VP2/Y alone or plus lipofectin. After three times of immunization, the mice sera were collected and assayed by ELISA. The mice inoculated with the plasmid plus lipofectin showed stronger immune response than those inoculated with the plasmid alone. The results implied that VP2 gene might be a promising gene vaccine candidate against PPV infection.
NS1蛋白是PPV的非结构蛋白之一，由NS1基因编码。NS1蛋白对PPV早期和晚期的基因转录都发挥着重要的调节作用。由于病毒灭活后不表达NS1蛋白，利用该蛋白作为诊断抗原可以区分灭活疫苗免疫猪和野毒感染猪。本实验室在1999年从污染了猪细小病毒的PK15细胞系中分离得到了一株PPV，定名为SY-99株。以SY-99株的基因组DNA为模板，利用PCR扩增出了NS1基因，将其克隆到原核表达载体pET28a中，获得重组质粒pET28a／SYNS1。序列测定结果显示，NS1基因全长1989核苷酸，编码662个氨基酸。在推导的氨基酸序列中含有在PPV复制过程中发挥重要作用的保守基序GKRN，并有三个潜在的糖基化位点，分别位于356-359、446-449和513-516位氨基酸。序列比较表明，SY-99株与NADL2-1株、NADL2-2株和Kresse株NS1基因的核苷酸同源性分别为98％、99％和99％，氨基酸同源性分别为97％、99％和99％。将重组质粒pET28a／SYNS1转化到大肠杆菌BL21(DE3)株中，所得的重组菌株经IPTG诱导后，表达出高水平的NS1蛋白，表达率可达17.8％。经ELISA和Western Blotting检测，表达的重组蛋白具有免疫学活性。经过优化选择确定了NS1蛋白高效表达的最适参数。表达产物带有组氨酸标签，适于用His·Bind柱亲和纯化。重组NS1蛋白为制备PPV诊断抗原用于本病的流行病学调查提供了一种检测工具。
当前用于PPV免疫防制的疫苗主要是油乳剂灭活苗和冻干弱毒苗，这两种常规疫苗都存在一定的缺陷，如生产疫苗所用的猪源传代细胞系或原代细胞很容易污染其它病原，PPV的体外培养滴度低，难于生产出高效价的疫苗，而且弱毒疫苗还存在毒力返强的可能，所以研制新型疫苗十分有必要。PPV VP2蛋白可以形成病毒样粒子(vires-like particles，VLPs)，其免疫原性与PPV病毒粒子近似，可以使接种动物获得保护性免疫。因此本研究拟用体外表达的重组VP2蛋白作为预防PPV的候选亚单位疫苗。以SY-99株基因组DNA为模板，用PCR扩增得到了VP2基因，将其克隆到酵母表达载体pPICZαB中，经测序后与PPV参考毒株NADL-2株进行序列比较，结果表明二者同源性达99.2％。经定点突变后，VP2基因中的SacI位点被缺失，构建了重组酵母转移载体pPICZαB／mVP2，经SacI线性化后，导入酵母受体菌GS115中，经甲醇诱导培养后，用SDS-PAGE检测重组菌株裂解产物，结果未检出目的蛋白条带。然后将VP2基因克隆到了另一酵母表达载体pPIC9K中，构建了酵母转移载体pPIC9K／mVP2，经转化和诱导培养后，也未检测目的条带。我们又将VP2基因靠近5，端部分进行了序列改造，构建了酵母转移载体pPICZαA／MVP2，转化酵母受体菌后诱导培养，检测结果显示，VP2仍然没
博土学位论文 摘 要 东北农业大学
有得到成功表达。在利用酵母表达 PPV VPZ的试验研究中，我们均得到了含有重组子的
阳性酵母菌株，我们对酵母生长条件进行了变化摸索，但在SDS－PAGE胶上始终没有肉
眼可见的目的条带。所以我们初步推测SY－99株WZ基因中存在不适于在酵母体系表达
的某种元件，抑或表达量过低而难于检测。
作为一种新型疫苗，基因疫苗具有安全可靠、生产简单、储运方便等优点，可望克
服常规疫苗的某些不足。我们将猪细小病毒VPZ基因及猪Y－干扰素（IFN－Y）基因同时
克隆到真核表达载体pIRESlneo中，构建了共表达VPZ和IFN－Y的基因免疫质粒
pIRESI／VPZ／Y，用此表达质粒单独或加入脂质体肌注接种 BABUC小鼠，三次兔疫后，
采取接种鼠血清，用ELISA检测特异性抗体，结果表明，脂质体可以加强表达质粒接种
小鼠的兔疫应答水平，为今后研制猪细小病毒基因疫苗提供了依据。
Porcine parvovirus (PPV) infection is one of the most important viral diseases, and characterized by reproductive failures in swine. PPV is epidemic in most regions of the world, including China, causing great economic loss in swine industry. Prevention and control of PPV infection is an urgent task.
The main non-structural protein of PPV, NS1 encoded by NS1 gene, is associated with the early and late transcription of PPV. NS1 can be used as a differential antigen for differentiating the pigs immunized with "dead" PPV vaccine from pigs infected with wild-type PPV. A PPV designated SY-99 strain was isolated from PK15 cells in 1999. The NS1 gene was amplified from the genomic DNA of SY-99, and then cloned into a prokaryotic expression vector pET28a, resulting in a recombinant plasmid pET28a/SYNS1. The NS1 gene was shown to contain 1989 base pairs (bp) and encode 662 amino acids (aa). A conserved motif GKRN, an important element in PPV transcription, was identified in the deduced NS1 protein sequence of SY-99. There are three potential glycosalation sites in the NS1 protein, at 356-359, 446-449, 513-516aa, respectively. The nucleotide identities of NS1 between SY-99 and PPV strains NADL2-1, NADL2-2 and Kresse are 98%, 99% and 99%, respectively, and the amino acid identities 97%, 99% and 99%, respectively. T
he pET28a/SYNSl was transformed into E. coli. BL21 (DE3). The NS1 protein was expressed with high-level in the transformed BL21 (DE3) after induction with IPTG. The expressed His-fusion protein was shown to be immunologically active by Western blotting. The IPTG concentration and induction time were optimized. The His-fusion recombinant protein was purified with His'Band column chromatography. The expressed NS1 protein will be beneficial to study of PPV infection.
The vaccines available against PPV infection are mostly derived from inactivated virus or modified live virus. These conventional vaccines show many disadvantages, including incidental reversion and less safety and efficacy. So it's essential to develop more effective and safer new-type vaccines against PPV infection.
The structural protein VP2 of PPV can form virus-like particles (VLPs). The VLPs are similar in antigenicity to PPV virions, so it can be used as a subunite vaccine candidate for protecting pigs from PPV infection. The VP2 gene of SY-99 was amplified and cloned into a yeast expression vector pPICZaB to obtain a recombinant plasmid pPICZaB/VP2. The pPICZaB/mVP2, modified from pPICZaB/VP2 by site-directed mutation to delete the Sad site in VP2 gene, was linearized by Sad and transformed into the host yeast GS115. The target protein was not detectable by SDS-PAGE analysis after induction by methanol. We mutated some codons of the VP2 gene based on the yeast codon preference. The modified VP2 gene
was then cloned into pPICZctA, resulting in pPICZ
引文
[1] Arella, M., Garzon, S., Bergeron, J., et al. Physico-hemical properties, production, and purification of parvovirus. In: Tijssen, P., CRS Handbook of parvowirus, CRC Press, Boca Roton, FA. 1990,11-29．
[2] Bachmann, P.A., and Danner,K. Porcine parvovirus infectilon in vitro: A study model for the replication of parvoviruses. II. Kinetics of virus and antigen production. Zentralbl Veterinarmed(B). 1976,23:355-363．
[3] Bergeron J, Heberrrt B, Tijssen P. Genome orgnization of the kresse strain of porcine parvovirus: Identification of the allotropic determinant and comparison with those of NADL-2 and field isolates. Journal of Virology. 1996, 70(4) :2508-2515．
[4] Bolt DM, Hani H, Muller E. Non-suppurative myocarditis in piglets associated with porcine parvovirus infection. J Comp Pathol. 1997, 117(2) :107-118．
[5] Cartwright, S.F.; and Huck, R.A. Viruses isolation in association with herd infertility, abortions and stillbirths in pigs. Vet Rec.1967, 81:196-197．
[6] Choi C S,Molitor T W,Joo H S. Inhibition of Porcine Parvovirus Replication by Empty Virus Particles.Arch.Virol,1987,96:75-87．
[7] Cotmore S F, Sturzenbecker L JJ, Tattersall P. The Autonomous Parvovirus MVME Encodes Two Nonstructural Proteins in Addition to Its Capsid Polypeptides. Virol. 1983, 129:333-343．
[8]Cotmore S F,tattersall P.The Automously Replicating Parvoviruses of Vertebrates.Adv.Virus Res.1987,33:91-174.
[9]Kradowka S,Ellis JA,Meehan B.Viral wasting syndrome of swine:experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfectoon with porcine circovirus 2 and porcine parvovirus.Vet Pathol.2000,37(3):254-263.
[10]Martinez C.,Dalsgaard,K.,De Turiso,J.A.L.,et al.Production of porcine parvovirus empty capsids with high immunogenic activity.Vaccine.1992,10(10):684-690.
[11]Mayr,A.et al.,Arch.ges.Virusforsch.,1968,25,38～51.
[12]Molitor,T.W.,Joo,H.S.and Collett,M.S.Porcine parvorirus DNA:Characterization of the Genomic and Replicative Form DNA of Two Virus Isolates.Virology,1984,137:241～254.
[13]Molitor,T.W.,Joo,H.S.and Collett,M.S.Porcine parvorirus:Virus purification and structural and antigenic properties of viron polypeptides.J.Virology.1983,45:842～854.
[14]Molitor,T.W.,Joo,H.S.& Collett,M.S.Identification and characterization of a porcine parvivirus nonstructural polypeptide.Journal of Virology 1985,55:554～559.
[15]Oraveerakul K,Choi C S.Molotor T W.Restrictilon of Parvovirus Replication in Nonpermissive Cells.J.Virol.1992,66:715-722.
[16]Paradiso P R.Identification of Multiple Forms of the Noncapsid Parvovirus.Protein NCVP1 in H-1 Parvovirus Infected Cells.J.Virol.1984,52:82-87.
[17]Ranz A I,Manclus J J,Diaz-Aroca E,et al,Porcine Parvorirus:DNA Sequence and Genome Organization.J.Gen.Viol,1989,70:2541～2553.
[18]Thacker,B.,and Leman,A.D.Evaluation of graviduteri at slaughter for porcine parvovirus infection.Proc Int Congr Pig Vet Soc.1978,5:M-49.
[19]Vasudecvacharya J.et al.Virol.1989.173,368～377.
[20]Vasudevacharya,J.,Basak,S.,Basak,S.,and Compans,R.W.The complete nucleotide sequence of an infectious clone of porcine parvovirus,strain NADL-2.Virology 1990,178,611～618.
[21]Vasudevachjarya J,Basak S,Srinivas RV.et al,Nucleotide Sequence Analysis of the Capsid Genes and the Right-Hand Terminal Palindrome of Porcine Parvorirus,Straion-2.Virol,1989,173:368～377.
[22]董长衡，现代分子病毒学，1996，140-152。
[23]郭正，等，中国兽医杂志，1990，16(5)：47～49。
[24]金岳，等，家畜传染病，1985，4：17～20。
[25]潘雪珠．猪细小病毒S-1株的分离和鉴定．上海畜牧兽医通讯．1983(1)1-3．
[26]殷震等．动物病毒学．北京：科学出版社。1985．804～810。
[2] Bachmann, P.A., and Danner,K. Porcine parvovirus infectilon in vitro: A study model for the replication of parvoviruses. II. Kinetics of virus and antigen production. Zentralbl Veterinarmed(B). 1976,23:355-363．
[3] Bergeron J, Heberrrt B, Tijssen P. Genome orgnization of the kresse strain of porcine parvovirus: Identification of the allotropic determinant and comparison with those of NADL-2 and field isolates. Journal of Virology. 1996, 70(4) :2508-2515．
[4] Bolt DM, Hani H, Muller E. Non-suppurative myocarditis in piglets associated with porcine parvovirus infection. J Comp Pathol. 1997, 117(2) :107-118．
[5] Cartwright, S.F.; and Huck, R.A. Viruses isolation in association with herd infertility, abortions and stillbirths in pigs. Vet Rec.1967, 81:196-197．
[6] Choi C S,Molitor T W,Joo H S. Inhibition of Porcine Parvovirus Replication by Empty Virus Particles.Arch.Virol,1987,96:75-87．
[7] Cotmore S F, Sturzenbecker L JJ, Tattersall P. The Autonomous Parvovirus MVME Encodes Two Nonstructural Proteins in Addition to Its Capsid Polypeptides. Virol. 1983, 129:333-343．
[8]Cotmore S F,tattersall P.The Automously Replicating Parvoviruses of Vertebrates.Adv.Virus Res.1987,33:91-174.
[9]Kradowka S,Ellis JA,Meehan B.Viral wasting syndrome of swine:experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfectoon with porcine circovirus 2 and porcine parvovirus.Vet Pathol.2000,37(3):254-263.
[10]Martinez C.,Dalsgaard,K.,De Turiso,J.A.L.,et al.Production of porcine parvovirus empty capsids with high immunogenic activity.Vaccine.1992,10(10):684-690.
[11]Mayr,A.et al.,Arch.ges.Virusforsch.,1968,25,38～51.
[12]Molitor,T.W.,Joo,H.S.and Collett,M.S.Porcine parvorirus DNA:Characterization of the Genomic and Replicative Form DNA of Two Virus Isolates.Virology,1984,137:241～254.
[13]Molitor,T.W.,Joo,H.S.and Collett,M.S.Porcine parvorirus:Virus purification and structural and antigenic properties of viron polypeptides.J.Virology.1983,45:842～854.
[14]Molitor,T.W.,Joo,H.S.& Collett,M.S.Identification and characterization of a porcine parvivirus nonstructural polypeptide.Journal of Virology 1985,55:554～559.
[15]Oraveerakul K,Choi C S.Molotor T W.Restrictilon of Parvovirus Replication in Nonpermissive Cells.J.Virol.1992,66:715-722.
[16]Paradiso P R.Identification of Multiple Forms of the Noncapsid Parvovirus.Protein NCVP1 in H-1 Parvovirus Infected Cells.J.Virol.1984,52:82-87.
[17]Ranz A I,Manclus J J,Diaz-Aroca E,et al,Porcine Parvorirus:DNA Sequence and Genome Organization.J.Gen.Viol,1989,70:2541～2553.
[18]Thacker,B.,and Leman,A.D.Evaluation of graviduteri at slaughter for porcine parvovirus infection.Proc Int Congr Pig Vet Soc.1978,5:M-49.
[19]Vasudecvacharya J.et al.Virol.1989.173,368～377.
[20]Vasudevacharya,J.,Basak,S.,Basak,S.,and Compans,R.W.The complete nucleotide sequence of an infectious clone of porcine parvovirus,strain NADL-2.Virology 1990,178,611～618.
[21]Vasudevachjarya J,Basak S,Srinivas RV.et al,Nucleotide Sequence Analysis of the Capsid Genes and the Right-Hand Terminal Palindrome of Porcine Parvorirus,Straion-2.Virol,1989,173:368～377.
[22]董长衡，现代分子病毒学，1996，140-152。
[23]郭正，等，中国兽医杂志，1990，16(5)：47～49。
[24]金岳，等，家畜传染病，1985，4：17～20。
[25]潘雪珠．猪细小病毒S-1株的分离和鉴定．上海畜牧兽医通讯．1983(1)1-3．
[26]殷震等．动物病毒学．北京：科学出版社。1985．804～810。