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1 微生物学通报 Microbiology China 研究报告 Jun. 0, 014, 41(6): by Institute of Microbiology, CAS DOI: /j.microbiol.china 猪圆环病毒型流行株的分离及其感染性克隆的构建陈瑞爱 1,* 邵定勇同戈黄红亮李延鹏 3 黄文科 ( ) ( ) ( ) 摘要 : 目的通过分离一株猪圆环病毒型 (PCV) 流行毒株, 并构建其感染性克隆, 为研究 PCV 基因功能提供操作平台方法通过 PCR 方法, 从疑似患断奶仔猪多系统衰竭综合症 (PMWS) 的仔猪淋巴结中鉴定为猪圆环病毒 (Porcine circovirus,pcv) 型阳性把阳性病料接种 PK-15 细胞传代培养, 在培养物中扩增出 PCV 的全基因序列对扩增出的全序列进行序列测定, 并与 GenBank 中公布的 5 株广东 PCV 分离株 (GD-pz GD-gj GD-jm GD-ss 和 GD-sz) 进行同源性分析通过 EcoRⅠ 和 SalⅠ 将 PCV 全基因组序列克隆进 puc18 载体中, 获得含 PCV GD-zq 株全基因组单拷贝的重组质粒 ppcv-gd-zq, 再通过 SalⅠ 和 Hind Ⅲ 把另一个全长拷贝克隆进 ppcv-gd-zq 质粒中, 使 PCV GD-zq 株基因组 DNA 以头尾相接的双重复方式克隆进 puc18 载体中, 获得重组质粒 ppcv-gd-zq 将 ppcv-gd-zq DNA 纯化和定量后转染 PK-15 细胞, 拯救 PCV GD-zq 病毒结果从 PMWS 感染的猪淋巴结中分离到了一株 PCV, 命名为 GD-zq 株 ; 序列分析结果显示,GD-zq 株全基因组为 bp, 与 GenBank 中公布的 5 株广东 PCV 分离株 ORF1 核苷酸一致性为 97.1% 99.7%, 编码氨基酸一致性为 98.7% 100%;ORF 核苷酸一致性为 93.% 99.6%, 编码氨基酸一致性为 9.3% 99.1%; 全基因一致性为 96.0% 99.6% ppcv-gd-zq 质粒转染 PK-15 细胞后, 其通过间接免疫荧光实验 (IFA) 能从转染细胞及其传代细胞中, 检测到拯救出的病毒结论分离了一株 PCV 广东株 GD-zq, 成功构建了 PCV GD-zq 株的感染性克隆关键词 : 猪型圆环病毒, 全基因, 感染性克隆, 序列分析 Isolation and identification of porcine circovirus type of GD-zq strain and construction of its infectious clone CHEN Rui-Ai 1,* SHAO Ding-Yong TONG Ge HUANG Hong-Liang LI Yan-Peng 3 HUANG Wen-Ke (1. College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong , China) (. Guangdong Dahuanong Animal Health Products Co. Ltd., Yunfu, Guangdong 57400, China) (3. Biotechnology Research Institute, CAAS, Beijing , China) Abstract: [Objective] To identify and construct an infectious clone for an isolation of porcine 基金项目 : (No. 011A ) * 通讯作者 : chenruiaidhn@16.com 收稿日期 : 接受日期 : 优先数字出版日期 (

2 1169 circovirus type (PCV). [Methods] PCR was used to identify an isolate, PCV GD-zq strain, from the tissues of clinical pigs with postweaning multisystemic wasting syndrome (PMWS). The complete sequence of the isolate was megaligned with other 5 PCV Guangdong isolates (GD-pz, GD-gj, GD-jm, GD-ss and GD-sz) from GenBank by DNAstar. Two copies of the whole genomic sequence was amplified and cloned into puc18 with the restriction enzymes EcoRⅠ, SalⅠ, SalⅠand Hind Ⅲ, and the positive clone ppcv-gd-zq was identified by enzyme analysis. By purification and quantitation, the ppcv-gd-zq DNA was transfected to PK-15 cell lines to rescue the infectious PCV GD-zq. [Results] PCV GD-zq strain was isolated and identified from the lymphonodus of clinical pigs with PMWS. Sequence analysis shows that the isolate s complete genome was composed of nucleotides, which shares 96.0% 99.6% homology between other 5 Guangdong reference strains, and shares 97.1% 99.7% homology in ORF1, 93.% 99.6% in ORF, respectively. Amino acid homology alignment shows 98.7% 100% in ORF1 and 93.% 99.6% in OFR. Seventy two hours post transfection of ppcv-gd-zq, GD-zq strain could be detected by immunofluorescence assay (IFA). [Conclusion] A PCV was isolated, and its infectious DNA clone was constructed successfully. Keywords: Porcine circovirus type (PCV), Complete genome, Infectious clone, Sequence analysis (Porcine circovirus PCV) PCV DNA 17 0 nm [1] PCV1 PCV PCV bp bp (Postweaning multisystemic wasting syndrome PMWS) [-4] PMWS PCV [5] PCV PCV PCV [6] PCV [7] PCV 00 nt 6 [8] ORF1 (Rep) ORF (Cap) [9] ORF3 [10] PCV ( DNA) [11] Fenaux PCV DNA PCV DNA [1] PCV PCV PCV PMWS PCV (GD-zq) GD-zq PCV

3 1170 微生物学通报 Microbiol. China 014, Vol.41, No.6 1 材料与方法 1.1 病料和细胞 50 (PMWS) PCV PK-15 (ATCC) 1. 主要试剂 LA Taq T4 DNA PCR NEB TIANGEN (Lipfectamine) TRIzol LS Reagent Invitrogen PCV PCV ORF (FITC) IgG Sigma 1.3 引物及模板制备引物 : [13-14] PCV1 PCV Oligo 6.0 PCV P1 5 -GCTGAAC TTTTGAAAGTGAGCGGG-3 P 5 -TC ACACAGTCTCAGTAGATCATCCCA-3 43 bp GenBank (PCV) PCV F (PCV-EcoR Ⅰ) 5 -GAAGAATTCGCTGGCTGAACTT TTGAAAG-3 5 EcoR Ⅰ R (PCV-Sal Ⅰ) 5 -GCAGTCGACAAATTTCTGACAAA CGTTA-3 5 SalⅠ F (PCV-SalⅠ) 5 -GTTGTCGACGCTGGCTGAACTTTTGAAAG-3 5 Sal Ⅰ R (PCV-Hind Ⅲ) 5 -GCAAAGCTTAAATTTCTGACAAACGTTA-3 5 Hind Ⅲ PCR bp 1.3. 病毒 DNA 提取 : 50 mg 1 ml PBS 1.5 ml g 10 min 537 μl 60 μl 10% SDS ( 1%) 3 μl K ( 100 mg/l) 50 C 60 min : r/min 10 min : 1/10 DNA g 10 min 0 μl DNA 0 C 1.4 PCR 及其产物的酶切鉴定 1.3 DNA PCR P1 P PCR PCR 5 μl 10 PCR buffer ( Mg + ).5 μl.5 mmol/l dntps μl 5 mmol/l MgCl 1.5 μl 1 μl (50 pmol/l) DNA 1 μl rtaq 1 μl (5 U/μL) 5 μl PCR 94 C 5 min 94 C 45 s 56 C 45 s 7 C 45 s 30 7 C 10 min PCR Nco I 1.5 病毒的分离培养及免疫荧光鉴定病毒的分离培养 : PCV 3 60% PK-15 7 h 1 PK-15 7 h 5 (IFA) 1.5. 间接免疫荧光实验 (IFA) 检测 : 4 7 h PBS (ph 7.) 3 80% 10 min 1 ml PCV (1 50 ) 37 C 45 min PBS 3 1 ml FITC- IgG (1 500 ) 37 C 45 min 1 ml PBS/ IFA PCV-GD-zq

4 病毒全基因组的扩增及感染性克隆构建 PCV 全基因组序列的扩增 : 5 DNA F (PCV-EcoR Ⅰ) R (PCV-SalⅠ) F (PCV-SalⅠ) R (PCV-Hind Ⅲ) PCV PCR 94 C 5 min 94 C 1 min 48 C 1 min 7 C 3 min 30 7 C 10 min 4 C ORF PCR 94 C 5 min 94 C 1 min 56 C 1 min 7 C 1 min 30 7 C 10 min DNA DNA 1.6. PCV 感染性克隆的构建 : EcoR Ⅰ Sal Ⅰ F (PCV-EcoRⅠ) R (PCV-SalⅠ) PCR puc18 ppcv-gd-zq Sal Ⅰ Hind Ⅲ F (PCV-SalⅠ) R (PCV-Hind Ⅲ) PCR ppcv-gd-zq ppcv-gd-zq 1.7 序列分析和比对 DNAStar ppcv-gd-zq GenBank 5 (GD-pz GD-gj GD-jm GD-ss GD-sz) 1.8 质粒转染及拯救病毒的检测重组质粒转染 : Invitrogen ppcv-gd-zq PCV PK-15 puc18 7 h PK 拯救病毒的检测 : [15-16] PK-15 PCV ORF FITC- IgG (IFA) 1.9 拯救病毒的生长特性 ppcv-gd-zq 5 K ml/ h DMEM 7 h 7 h 1.8. IFA Reed-Muench TCID 50 5 DNA 1.6. 结果与分析.1 PCR 扩增及分型鉴定结果 P1 P 43 bp PCR Nco I DL500 marker 168 bp 75 bp ( 1) PCV PCV1. 组织传代病毒的间接免疫光试验结果 5 PK h PCV FTTC- IgG 图 1 病料组织的 PCR 鉴定 Figure 1 PCR identification for the PCV-suspected tissue samples 1 NcoⅠ PCR 3 PCR 4 DL500 marker. Note: 1: PCR product digested by NcoⅠ;, 3: PCR products; 4: DL500 marker.

微生物学通报 Microbiol. China 117 置显微镜下观察结果视野中有 50%以上的细胞.4 呈现特异性荧光对照细胞无荧光表明分离的病 014, Vol.41, No.6 序列分析与比对结果 ppcv-gd-zq 质粒送上海生工生物工程技术毒为猪圆环病毒型(图 ) 服务有限公司进行测序验证结果正确用 DNAStar.

7% 编码氨基酸一致性为 Ⅰ双酶切 ppcv-gd-zq 质粒后获得大小分别为 98.7% 100% 与 GD-ss 株的氨基酸序列完全一致.7 kb 和 1.7 kb 左右的预期片段 SalⅠ和 Hind Ⅲ ORF 核苷酸一致性为 93.% 99.6% 编码氨基酸双酶切 ppcv-gd-zq 质粒后获得大小分别为一致性为 9.3% 99.

5 GD-zq感染性克隆转染及病毒拯救将 ppcv-gd-zq 质粒转染 PK-15 细胞 7 h 后通过 IFA 检测结果显示多数转染细胞的胞浆和部分转染细胞的胞浆和胞核中可观察特异性亮绿色荧光对照载体质粒转染样品无荧光出现(图 5) 图病料传代样品的间接免疫荧光试验结果(100 ) Figure The detection result for PCV with IFA

图 4 6 株 PCV 广东分离株全基因组序列的核苷酸同源性比较 Figure 4 Identity comparison of the nucleotides of 6 PCV isolates 图3 PCV 重组质粒酶切鉴定图 Figure 3 The restriction enzyme analysis for the recombinants of ppcv- GD-zq and

5 微生物学通报 Microbiol. China 117 置显微镜下观察结果视野中有 50%以上的细胞.4 呈现特异性荧光对照细胞无荧光表明分离的病 014, Vol.41, No.6 序列分析与比对结果 ppcv-gd-zq 质粒送上海生工生物工程技术毒为猪圆环病毒型(图 ) 服务有限公司进行测序验证结果正确用 DNAStar.3 单拷贝和双拷贝PCV-GD-zq株DNA克隆的鉴定用限制性核酸内切酶 EcoRⅠ和 SalⅠ酶切鉴软件将本研究分离和克隆 PCV-GD-zq 株全基因组定单拷贝质粒 ppcv-gd-zq 用 SalⅠ和 Hind Ⅲ 比较结果 GD-zq 株 ORF1 与 5 株广东分离株核苷酶切鉴定 ppcv-gd-zq 质粒结果 EcoRⅠ和 Sal 酸一致性在 97.1% 99.7% 编码氨基酸一致性为 Ⅰ双酶切 ppcv-gd-zq 质粒后获得大小分别为 98.7% 100% 与 GD-ss 株的氨基酸序列完全一致.7 kb 和 1.7 kb 左右的预期片段 SalⅠ和 Hind Ⅲ ORF 核苷酸一致性为 93.% 99.6% 编码氨基酸双酶切 ppcv-gd-zq 质粒后获得大小分别为一致性为 9.3% 99.1% ORF 的核苷酸一致性在 4.4 kb 和 1.7 kb 左右的预期片段(图 3) 93.% 99.6% 全基因的一致性在 96.0% 99.6%之序列与 GenBank 公布的其它 5 株广东分离株 (GD-pz GD-gj GD-jm GD-ss 和 GD-sz)序列进行间全基因组同源性分析结果见图 4.5 GD-zq感染性克隆转染及病毒拯救将 ppcv-gd-zq 质粒转染 PK-15 细胞 7 h 后通过 IFA 检测结果显示多数转染细胞的胞浆和部分转染细胞的胞浆和胞核中可观察特异性亮绿色荧光对照载体质粒转染样品无荧光出现(图 5) 图病料传代样品的间接免疫荧光试验结果(100 ) Figure The detection result for PCV with IFA (100 ) 注 A 接毒样品 B 未接毒的对照样品. Note: A: PK-15 cells infected by the isolate; B: Un-infected PK-15 cells. 图 4 6 株 PCV 广东分离株全基因组序列的核苷酸同源性比较 Figure 4 Identity comparison of the nucleotides of 6 PCV isolates 图3 PCV 重组质粒酶切鉴定图 Figure 3 The restriction enzyme analysis for the recombinants of ppcv- GD-zq and ppcv-gd-zq 注 1 BM5000 DNA marker ppcv-gd-zq EcoRⅠ酶切 3 ppcv-gd-zq EcoRⅠ酶切 4 ppcv-gd-zq EcoRⅠ和图5 SalⅠ双酶切 5 ppcv-gd-zq SalⅠ和Hind Ⅲ双酶切. Note: 1: BM5000 DNA marker; : ppcv-gd-zq digested by EcoRⅠ; 3: ppcv-gd-zq digested by EcoRⅠ; 4: ppcv-gd-zq digested by EcoRⅠand SalⅠ; 5: ppcv-gd-zq digested by SalⅠ and Hind Ⅲ. Figure 5 Identification of the infectious DNA clone of PCV by IFA (400 ) PCV 拯救病毒的 IFA 检测(400 ) 注 A ppcv-gd-zq 转染拯救的病毒 B 空载体对照. Note: A: Transfection of ppcv-gd-zq; B: Transfection of puc18 (control).

6 拯救病毒 TCID 50 的测定及序列分析 5 IFA TCID 50 /0.1 ml PCV ppcv-gd-zq 99.89% 164 (C A) (A G) 164 ORF1 (Ser) ORF Val Ala 3 讨论 (PMWS) PCV PCV PRV PPV PRRSV PMWS [17-18] PMWS PCV PPV PRV PRRSV PCV PCV NcoⅠ PCV1 [13] PCR 3 PCV (168 bp 75 bp) PCV1 1 ( 43 bp) 3 ( bp) PCV1 PCV PCR ( 1) PCV PCV1 PCV 94% [19-] DNA PCR ppcv-gd-zq GenBank % 99.6% GD-zq 5 ORF1 ORF ORF1 97.1% 99.7% ORF 93.% 99.6% ORF ORF1 ORF1 5 ( GD-ss ) OFR GD-jm 9.3% PCV ORF GD-zq ppcv-gd-zq 99.89% ORF1 ORF ORF Val Ala Fenaux PCV DNA PCV PCV DNA PCV PCV DNA PCV DNA PCV DNA [1] PCV DNA PCV [11] PCV PCR PCV DNA PCV PCV PCV DNA PCV

7 1174 微生物学通报 Microbiol. China 014, Vol.41, No.6 PCV GD-zq puc18 参考文献 [1] Tischer I, Gelderhlom H, Vettermann W, et al. A very small porcine virus with circular single-stranded DNA[J]. Nature, 198, 95(5844): [] Hamel AL, Lin LL, Nayar GPS. Nucleotide sequence of the porcine circovirus associated with post weaning multisystemic wasting syndrome in pigs[j]. Journal of Virology, 1998, 7(6): [3] Allan GM, Meehan B, Todd D, et al. Novel porcine circoviruses from pigs with wasting disease syndromes[j]. Veterinary Record, 1998, 14(17): [4] Bolin SR, Stoffregen WC, Nayar GP. Postweaning multisystemic wasting syndrome induced after experimental inclution of cesarean-derived, colostrums-deprived piglets with type porcine circovirus[j]. Journal of Veterinary Diagnostic Investigation, 001, 13(3): [5] Opriessnig T, Meng XJ, Halbur PG. Porcine circoviruses type -associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies[j]. Journal of Veterinary Diagnostic Investigation, 007, 19: [6] Walker IW, Konoby CA, Jewhurst VA, et al. Development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type [J]. Journal of Veterinary Diagnostic Investigation, 000, 1(4): [7] Kennedy S, Moffett D, Mcneilly F, et al. Reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type alone or incombination with porcine parvovirus[j]. Journal of Comparative Pathology, 000, 1(1): 9-4. [8] Segalés J, Allan GM, Domingo M. Porcine circovirus diseases[j]. Animal Health Research Reviews, 005, 6(): [9] Mankert A, Caliskan R, Hattermann K, et al. Molecular biology of Porcine circovirus: analyses of gene expression and viral replication[j]. Veterinary Microbiology, 004, 98(): [10] Liu J, Zhu Y, Chen I, et al. The ORF3 protein of porcine circovirus type interacts porcine ubiquitin E3 ligase Pirh and facilitates p53 expressi on in viral infection[j]. Journal of Virology, 007, 81(17): [11] Cheung AK. Rolling-circle replication of an animal circovirus genome in a theta-replicating bacterial plasmid in Escherichia coli[j]. Journal of Virology, 006, 80(17): [1] Fenaux M, Opriessnig T, Halbur PG, et al. Cloned genomic DNA of type porcine circovirus is infectious when injected directly into the liver and lymph nodes of pigs: characterization of clinical disease, virus distribution, and pathologic lesions[j]. Journal of Virology, 00, 76(): [13] Fenaux M, Halbur PG, Gill M, et al. Genetic characterization of type porcine circovirus (PCV-) from pigs with post-weaning multisystemic wasting syndrome in different geographic regions of North America and development of a differential PCR-restriction PCV-1 and PCV-[J]. Journal of Clinical Microbiology, 000, 38(7): [14]. [D]. :, 01. [15],,,. 1 [J]., 011, 41(): [16],,,. II DNA [J]., 01, 8(3): [17] Ellis JA, Bratanich A, Clark EG, et al. Coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome[j]. Journal of Veterinary Diagnostic Investigation, 000, 1(1): 1-7. [18] Sirinarumitr T, Sorden SD, Morozov I, et al. Double in situ by hybridization for simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV)[J]. Journal of Veterinary Diagnostic Investigation, 001, 13(1): [19] Kim HH, Park SI, Hyun BH, et al. Genetic diversity of porcine circovirus type in Korean pigs with postweaning multisystemic wasting syndrome during [J]. Journal of Veterinary Medical Science, 009, 71(3): [0] Cortey M, Pileri E, Sibila M, et al. Genotypic shift of porcine circovirus type from PCV-a to PCV-b in Spain from 1985 to 008[J]. Veterinary Journal, 011, 187(3): [1] Harding JC, Ellis JA, McIntosh KA, et al. Dual heterologous porcine circovirus genogroup a/b infection induces severe disease in germ-free pigs[j]. Veterinary Microbiology, 010, 145(3/4): [],,,. II SD/008 [J]., 013, 33(4):

材料方法

材料方法生物技术通报张发洲杨慧兰为了构建单纯疱疹病毒型感染细胞多肽真核表达质粒应用技术从株的基因组中扩增基因并连接至真核表达载体对阳性克隆进行菌落酶切和测序鉴定后成功构建了重组质粒用转染试剂盒将重组质粒转染至细胞中并用及检测其表达情况结果显示基因在细胞中得到正确表达真核表达质粒的构建成功为进一步研究对宿主细胞的影响奠定了基础单纯疱疹病毒型感染细胞多肽