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1 1276 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 蔡鑫娜等 / 猪繁殖与呼吸综合征病毒 nsp4 抗体制备与鉴定 August 25, 2017, 33(8): Chin J Biotech, All rights reserved 新方法 nsp4 蔡鑫娜, 谭敏, 曹胜亮, 黄艳, 孙法超, 商营利, 刘思当, 肖一红 / ,,,. nsp4., 2017, 33(8): Cai XN, Tan M, Cao SL, et al. Preparation and identification of polyclonal antibodies specific for nsp4 protein of porcine reproductive and respiratory syndrome virus. Chin J Biotech, 2017, 33(8): 摘要 : 为了获得猪繁殖与呼吸综合征病毒 (PRRSV) nsp4 的抗体, 根据 HP-PRRSV TA-12 株 (GenBank Accession No. HQ416720) 的 nsp4 基因序列, 设计并合成一对引物用 RT-PCR 扩增后克隆到原核表达载体 pet-28a(+) 中, 构建重组质粒 pet28a-nsp4, 转化至 Trasseta(DE3), 经 IPTG 诱导重组蛋白获得了高效可溶性表达, 大小约为 26 kda 经镍离子亲和柱 (Ni + -NTA) 纯化获得了高纯度重组蛋白, 将纯化的 nsp4 蛋白免疫新西兰大白兔制备了多克隆抗体 ELISA 检测抗体效价可达 10 6,Western blotting 和 IFA 检测结果表明所制备的多克隆抗体具有良好的免疫反应特异性, 能够识别 PRRSV 感染宿主细胞中的 nsp4 蛋白本研究成功制备了针对 nsp4 的多克隆抗体, 为进一步研究 nsp4 的功能及 PRRSV 致病机制奠定了基础 : 猪繁殖与呼吸综合症病毒,nsp4, 多克隆抗体, 鉴定 Preparation and identification of polyclonal antibodies specific for nsp4 protein of porcine reproductive and respiratory syndrome virus Xinna Cai, Min Tan, Shengliang Cao, Yan Huang, Fachao Sun, Yingli Shang, Sidang Liu, and Yihong Xiao College of Veterinary Medicine and Animal Sciences, Shandong Agricultural University, Tai an , Shandong, China Abstract: To obtain specific antibodies against nsp4 protein of porcine reproductive and respiratory syndrome virus (PRRSV), nsp4 gene was amplified by RT-PCR and cloned into pet-28a(+) vector, designated pet28a-nsp4. pet28a-nsp4 Received: March 25, 2017; Accepted: June 14, 2017 Supported by: Natural Science Foundation of Shandong Province (No. ZR2014CM024), Funds of Shandong Double Tops. Corresponding author: Yihong Xiao. Tel: ; Fax: ; xiaoyihong01@163.com (No. ZR2014CM024)

2 蔡鑫娜等 / 猪繁殖与呼吸综合征病毒 nsp4 抗体制备与鉴定 1277 was transformed into Escherichia coli Trasseta (DE3) cells and expressed after induction of IPTG. SDS-PAGE analysis showed that the recombinant protein was expressed in soluble form with the molecular weight of 26 kda. The soluble fusion protein in the supernatant was purified using Ni + -NTA affinity chromatography. New Zealand rabbits were immunized by the purified nsp4 and anti-sera against nsp4 were obtained. The titer of polyclonal antibodies was about 10 6 and showed good specificity and sensitivity in the immunofluorescence assay and Western blotting analysis. The polyclonal antibodies also recognized native nsp4 form PRRSV infected Marc-145 cells, providing a useful tool in PRRSV replication mechanism study. Keywords: porcine reproductive and respiratory syndrome virus, nsp4, polyclonal antibody, identification (Porcine reproductive and respiratory syndrome PRRS) PRRS (PRRSV) 1987 [1] [2] 1996 PRRS PRRSV [3] (Highly pathogenic PRRS HP-PRRS) [4-6] (50% 100%) (20% 100%) [7-8] 2008 HP-PRRS [9] PRRSV PRRSV RNA 15 kb 9 (Open reading frame ORF) [10] ORF1 (ORF1a ORF1b) ORF2 (ORF2a ORF2b) ORF3 ORF4 ORF5 (ORF5a) ORF6 ORF7 [11-13] PRRSV 7 14 (nsp) nsp4 ORF1a pp1a pp1ab (nsp3-12) PRRSV [14-16] nsp4 PRRSV nsp4 PRRSV nsp4 1 材料与方法 1.1 毒株菌株与载体 HP-PRRSV TA12 (GenBank Accession No. HQ416720) PRRSV CH-1R Marc-145 pet-28a(+) DH5α Trasseta (DE3) TranGen Biotech 1.2 主要试剂 T4 DNA Thermo NheⅠ XhoⅠ Trans2K Plus DNA TaKaRa DNA His (Kan + ) (Amp + ) HRP-DAB Tiangen cjb@im.ac.cn

3 1278 ISSN CN /Q Chin J Biotech August 25, 2017 Vol.33 No.8 Clarity Max Western ECL Substrate Bio-Rad HRP IgG (H+L) Beyotime Cy3 IgG FITC IgG Jackson Sigma (PVDF) Millipore Ni + -NTA 1.3 引物的设计与合成 GenBank HP-PRRSV TA12 (Accession No. HQ417620) nsp4 NheⅠ XhoⅠ 612 bp 5 -CCCGCTAGCG GTGCTTTCAGAACTCA-3 5 -CCCTCGAGTTCCAGTTCGGGTTTGGC-3 ( ) 1.4 病毒 RNA 提取及 RT-PCR HP-PRRSV TA-12 Marc-145 TRIzol RNA 20 μl 5 2 μl 0.01 mol/l dntps 4 μl RNA 1 μl Oligo(dT)18 primer 1 μl AMV 1 μl RNase-free dh 2 O 1 μl RNA 10 μl min 70 5 min PCR 50 μl 1 μl TaKaRa Premix Taq DNA 25 μl cdna 2 μl ddh 2 O 50 μl 94 5 min s s s min 4 PCR 1.5 重组表达质粒的构建 pet-28a(+) nsp4 NheⅠ XhoⅠ DH5α Kan + 5 ml Kan + LB 37 5 h PCR PCR NheⅠ XhoⅠ ( ) pet28a-nsp4 1.6 重组蛋白表达纯化及 SDS-PAGE 分析 pet28a-nsp4 Trasseta(DE3) Kan + LB 37 LB 1% Kan + LB OD mmol/l IPTG 5 h r/min 5 min SDS-PAGE Ni + -NTA 1.7 Western blotting 鉴定结果 nsp4 SDS-PAGE PVDF His HRP IgG Bio-Rad ECL DAB 1.8 nsp4 蛋白多抗血清的制备 nsp mg/

蔡鑫娜等 / 猪繁殖与呼吸综合征病毒 nsp4 抗体制备与鉴定 1279 3 1 1 2 1 mg/ 3 4 80 1.

4 OD 450 1.10 多抗血清特异性检测 1.10.1 Western blotting 分析 HP-PRRSV PRRSV Marc-145 24 h SDS-PAGE PVDF 1 200 1 2 000 HRP IgG DAB 1.

1 目的基因的获取 HP-PRRSV TA-12 RNA cdna PCR 612 bp ( 1) 2.

1 Amplification of PRRSV nsp4 gene by PCR.

4 蔡鑫娜等 / 猪繁殖与呼吸综合征病毒 nsp4 抗体制备与鉴定 mg/ ELISA 效价的测定 ELISA nsp4 200 ng/ μl/ 37 1 h HRP IgG 100 μl 37 1 h 3 mol/l H 2 SO 4 OD 多抗血清特异性检测 Western blotting 分析 HP-PRRSV PRRSV Marc h SDS-PAGE PVDF HRP IgG DAB IFA Marc /cm h 4% 1% trixon h Cy3 FITC 37 1 h 2 结果与分析 2.1 目的基因的获取 HP-PRRSV TA-12 RNA cdna PCR 612 bp ( 1) 2.2 表达载体的构建与鉴定 NheⅠ XhoⅠ 1% bp 612 bp ( 2) 100% ( ) pet28a-nsp4 图 1 HP-PRRSV TA-12 株 nsp4 的扩增 Fig. 1 Amplification of PRRSV nsp4 gene by PCR. M: DNA marker 5000; 1: PCR product of the nsp4 gene; 2: negative control. 图 2 重组质粒 pet28a-nsp4 酶切鉴定结果 Fig. 2 Identification of the recombinant plasmid pet28ansp4. M: DNA marker 5000; 1: undigested pet28ansp4; 2: pet28a-nsp4 digested by NheⅠand XhoⅠ cjb@im.ac.cn

1280 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech August 25, 2017 Vol.33 No.8 2.3 nsp4 重组蛋白的表达 12% SDS-PAGE pet28a-nsp4 Trasseta(DE3) 26 kda ( 3) 2.

4 SDS-PAGE and Western blotting analysis of the recombinant protein.

1 mg/ml, 2.3 mg/ml, 0.8 mg/ml, 0.5 mg/ml, 0.35 mg/ml, and 0.21 mg/ml; 10: Western blotting analysis of the recombinant nsp4 protein.

M: protein marker; 1: the whole bacteria of pet-28a(+)transformed Trasseta (DE3) before induction; 2: the whole bacterium of pet-28a(+) transformed

after induction; 5: supernatant of pet28a-nsp4 after induction; 6: precipitation of pet28a-nsp4 after induction; 7: Western blotting analysis of the

5 1280 ISSN CN /Q Chin J Biotech August 25, 2017 Vol.33 No nsp4 重组蛋白的表达 12% SDS-PAGE pet28a-nsp4 Trasseta(DE3) 26 kda ( 3) 2.4 nsp4 的纯化 (Ni + -NTA) ( 4) 2.5 重组蛋白 nsp4 抗血清的 ELISA 效价测定 ELISA nsp4 HRP ( 5) 图 4 nsp4 纯化产物 SDS-PAGE 及 Western blotting 鉴定 Fig. 4 SDS-PAGE and Western blotting analysis of the recombinant protein. M: protein marker; 1: supernatant before purification; 2: supernatant after purification; 3: wash buffer; 4 9: the purified recombinant nsp4 protein 3.1 mg/ml, 2.3 mg/ml, 0.8 mg/ml, 0.5 mg/ml, 0.35 mg/ml, and 0.21 mg/ml; 10: Western blotting analysis of the recombinant nsp4 protein. 图 3 SDS-PAGE 分析 nsp4 在 Trasseta(DE3) 表达 Fig. 3 SDS-PAGE analysis of recombinant nsp4 expressed in Trasseta (DE3). M: protein marker; 1: the whole bacteria of pet-28a(+)transformed Trasseta (DE3) before induction; 2: the whole bacterium of pet-28a(+) transformed Trasseta (DE3) after induction; 3: the whole bacterium of pet28a-nsp4 transformed Trasseta (DE3) before induction; 4: the whole bacterium of pet28a-nsp4 after induction; 5: supernatant of pet28a-nsp4 after induction; 6: precipitation of pet28a-nsp4 after induction; 7: Western blotting analysis of the recombinant nsp4 protein after induction; 8: Western blotting analysis of the whole bacteria of pet-28a(+) after induction. 图 5 用间接 ELISA 检测兔抗 nsp4 抗体滴度 Fig. 5 Titration of the rabbit anti-nsp4 sera by ELISA. 2.6 多抗血清特异性检测 Western blotting nsp4 HP-PRRSV PRRSV Marc-145 HP-PRRSV PRRSV nsp4 25 kda PRRSV ( 6)

蔡鑫娜等 / 猪繁殖与呼吸综合征病毒 nsp4 抗体制备与鉴定 1281 2.6.

microscopy(630 ). 3 讨论图 6 用 Western blotting 鉴定兔抗 nsp4 血清特异性 Fig.

M: protein marker; 1: TA-12-infected Marc-145 cell lysates; 2:

6 蔡鑫娜等 / 猪繁殖与呼吸综合征病毒 nsp4 抗体制备与鉴定 IFA HP-PRRSV PRRSV Marc-145 IFA ( 7) nsp4 PRRSV ( 8) 图 8 用共聚焦显微镜观察 nsp4 的亚细胞定位 (630 ) Fig. 8 Localization of the nsp4 protein in the PRRSVinfected cellsby confocal microscopy(630 ). 3 讨论图 6 用 Western blotting 鉴定兔抗 nsp4 血清特异性 Fig. 6 Identification of the rabbit anti-nsp4 sera by Western blotting. M: protein marker; 1: TA-12-infected Marc-145 cell lysates; 2: CH-1R-infected Marc-145 cell; 3: uninfected Marc-145 cell lysates. 图 7 用 IFA 鉴定兔抗 nsp4 抗血清 (200 ) Fig. 7 Identification of the rabbit anti-nsp4 sera by IFA (200 ). PRRSV nsp4 nsp1 nsp2 nsp4 nsp7 B [17-22] nsp2 nsp7 PRRSV nsp4 B PRRSV nsp4 nsp4 [23-27] nsp4 nsp4 3 nsp IFA Western blotting nsp4 HP- PRRSV Marc-145 nsp4 nsp cjb@im.ac.cn

7 1282 ISSN CN /Q Chin J Biotech August 25, 2017 Vol.33 No.8 [28] nsp4 PRRSV nsp4 GFP GFP nsp4 GFP nsp4 B NF-κB IFN-β [29-30] nsp4 PRRSV DNAStar nsp4 97.5% Western blotting IFA PRRSV nsp4 nsp4 REFERENCES [1] Paton DJ, Brown IH, Edwards S, et al. Blue ear disease of pigs. Vet Rec, 1991, 128(26): 617. [2] Wensvoort G, Terpstra C, Pol JMA, et al. Mystery swine disease in the Netherlands: the isolation of Lelystad virus. Vet Q, 1991, 13(3): [3] Guo BQ, Chen ZS, Liu WX, et al. Study on isolation of PRRSV from aborted fetuses suspected of PRRS. Chin J Prev Vet Med, 1996, (2): 1 5 (in Chinese).,,,. PRRS PRRSV., 1996, (2): 1 5. [4] Tong GZ, Zhou YJ, Hao XF, et al. Highly pathogenic porcine reproductive and respiratory syndrome, China. Emerg Infect Dis, 2007, 13(9): [5] Tian KG, Yu XL, Zhao TZ, et al. Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark. PLoS ONE, 2007, 2(6): e526. [6] Li YF, Wang XL, Bo KT, et al. Emergence of a highly pathogenic porcine reproductive and respiratory syndrome virus in the Mid-Eastern region of China. Vet J, 2007, 174(3): [7] An TQ, Tian ZJ, Xiao Y, et al. Origin of highly pathogenic porcine reproductive and respiratory syndrome virus, China. Emerg Infect Dis, 2010, 16(2): [8] Jiang YF, Zhou YJ, Tian ZJ, et al. Identification of aspecific epitope on nonstructural protein 2 of an attenuated strain of highly pathogenic porcine reproductive and respiratory syndrome virus. Chin J Anim Infect Dis, 2009, 17(3): 1 7 (in Chinese).,,,. HuN4., 2009, 17(3): 1 7. [9] Hao XF, Zhou YJ, Tian ZJ, et al. Development of a RT-PCR method for differentiation of the highly pathogenic PRRSVs and the classical PRRSVs. Chin J Prev Vet Med, 2007, 29(9): (in Chinese).,,,. RT-PCR., 2007, 29(9): [10] Dea S, Gagnon CA, Mardassi H, et al. Antigenic variability among North American and European strains of porcine reproductive and respiratory syndrome virus as defined by monoclonal antibodies to the matrix protein. J Clin Microbiol, 1996, 34(6): [11] Dea S, Gagnon CH, Pirzadeh B, et al. Current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (PRRS) virus: comparison of the North American and European isolates. Arch Virol, 2000, 145(4): [12] Wu WH, Fang Y, Farwell R, et al. A 10-kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b. Virology, 2001, 287(1): [13] Allende R, Lewis TL, Lu Z, et al. North American and European porcine reproductive and respiratory

8 蔡鑫娜等 / 猪繁殖与呼吸综合征病毒 nsp4 抗体制备与鉴定 1283 syndrome viruses differ in non-structural protein coding regions. J Gen Virol, 1999, 80(2): [14] Snijder EJ, Meulenberg JJ. The molecular biology of arteriviruses. J Gen Virol, 1998, 79(5): [15] Snijder EJ, Wassenaar AL, van Dinten LC, et al. The arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases. J Biol Chem, 1996, 271(9): [16] van Dinten LC, Rensen S, Gorbalenya AE, et al. Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication. J Virol, 1999, 73(3): [17] Wassenaar AL, Spaan WJ, Gorbalenya AE, et al. Alternative proteolytic processing of the arterivirus replicase ORF1a poly protein: evidence that NSP2 acts as a cofactor for the nsp4 serine protease. J Virol, 1997, 71(12): [18] Xu AT, Zhou YJ, Li GX, et al. Characterization of the biochemical properties and identification of amino acids forming the catalytic center of 3C-like proteinase of porcine reproductive and respiratory syndrome virus. Biotechnol Lett, 2010, 32(12): [19] Snijder EJ, Wassenaar ALM, Spaan WJM. Proteolytic processing of the N-terminal region of the equine arteritis virus replicase. Adv Exp Med Biol, 1993, 342: [20] Snijder EJ, Wassenaar AL, Spaan WJ. Proteolytic processing of the replicase ORF1a protein of equine arteritis virus. J Virol, 1994, 68(9): [21] Van Dinten LC, Wassenaar AL, Gorbalenya AE, et al. Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains. J Virol, 1996, 70(10): [22] Yuan SZ, Zhang N, Xu L, et al. Induction of apoptosis by the nonstructural protein 4 and 10 of porcine reproductive and respiratory syndrome virus. PLoS ONE, 2016, 11(6): e [23] Mardassi H, Mounir S, Dea S, et al. Structural gene analysis of a Quebec reference strain of porcine reproductive and respiratory syndrome virus (PRRSV). Adv Exp Med Biol, 1995, 380: [24] Dokland T. The structural biology of PRRSV. Virus Res, 2010, 154(1/2): [25] Ma ZT, Wang YL, Zhao HY, et al. Porcine reproductive and respiratory syndrome virus nonstructural protein 4 induces apoptosis dependent on its 3C-like serine protease activity. PLoS ONE, 2013, 8(7): e [26] Huang C, Du YP, Yu ZB, et al. Highly Pathogenic porcine reproductive and respiratory syndrome virus Nsp4 cleaves VISA to impair antiviral responses mediated by RIG-I-like receptors. Sci Rep, 2016, 6: [27] Zhang LL, Liu J, Bai J, et al. Poly (I:C) inhibits porcine reproductive and respiratory syndrome virus replication in MARC-145 cells via activation of IFIT3. Antiviral Res, 2013, 99(3): [28] Liu HL, Fang Y. Nuclear localization of porcine reproductive and respiratory syndrome virus nonstructural protein 4. J Nanjing Agric Univ, 2011, 34(4): (in Chinese).,. nsp4., 2011, 34(4): [29] Zhang LJ, Li JN, Hu L, et al. PRRSV nsp4 inhibits type I interferon production by cleaving NF-κB essential modulator. Chin J Prev Vet Med, 2014, 36(3): (in Chinese).,,,. NSP4 NEMO I., 2014, 36(3): [30] Li CY, Wang SN, Li JN, et al. The inhibition of the interferon-β expression by porcine reproductive and respiratory syndrome virus NSP4 via cleaving IKKα. Chin J Prev Vet Med, 2016, 38(6): (in Chinese).,,,. NSP4 IKKα β., 2016, 38(6): ( 本文责编陈宏宇 ) cjb@im.ac.cn

Invitrogen 1.4 pet-gr, ICE-LGR, PCR TA, pmd18-t/gr pet-21a(+) (Nde I Xho I), E. coli DH-5,,,, pet-gr GR ORF cdna, GR, : PU: 5 -GGAATTCCAT

Invitrogen 1.4 pet-gr, ICE-LGR, PCR TA, pmd18-t/gr pet-21a(+) (Nde I Xho I), E. coli DH-5,,,, pet-gr GR ORF cdna, GR, : PU: 5 -GGAATTCCAT 42 6 Vol.42, No.6 2011 11 OCEANOLOGIA ET LIMNOLOGIA SINICA Nov., 2011 南极衣藻 (Chlamydomonas sp. ICE-L) 谷胱 * 甘肽还原酶基因的原核表达及其条件优化 1 1 燏 1 1 2 (1. 524025; 2. 266061) RT-PCR GR ORF cdna, ; IPTG,, pet-gr BL21,