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1 生物工程学报 Chin J Biotech 2009, April 25; 25(4): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 动物及兽医生物技术陈伟业 1, 曲林茂 2, 胡森 1, 胡倩倩 2, 张倩 1, 支海兵 3, 黄克和 2, 步志高 1, , , : 本研究旨在构建表达小反刍兽疫 H 蛋白的重组山羊痘病毒, 并评价其免疫效力利用筛选基因 gpt 和 egfp 并结合空斑技术, 纯化筛选了重组小反刍兽疫 H 基因的重组山羊痘病毒 (rgpv-pprv-h), 经过 PCR 鉴定重组病毒已纯化免疫荧光和蛋白印迹都表明重组病毒能够感染绵羊羔羊睾丸细胞表达小反刍兽疫 H 蛋白以 PFU 的 rgpv-pprv-h 皮内注射免疫山羊 6 只, 并于首次免疫后 28 d 以相同剂量进行二次免疫免疫后采血分离血清进行病毒中和试验, 结果表明, 一次免疫后 21 d, 山羊痘病毒中和抗体效价依次为 , 和小反刍兽疫病毒中和抗体效价全部转阳依次为 ; 二次免疫后 14 d, 山羊痘病毒中和抗体效价抗体全部大于或等于 80, 小反刍兽疫病毒中和抗体效价依次为 , 应该具有对山羊痘病毒和小反刍兽疫病毒强毒攻击的完全免疫保护作用本研究为小反刍兽疫重组山羊痘疫苗的产业化提供了参考 : 重组疫苗, 小反刍兽疫, 山羊痘病毒载体, 中和抗体 1 Recombinant goat pox virus expressing PPRV H protein Weiye Chen 1, Linmao Qu 2, Sen Hu 1, Qianqian Hu 2, Qian Zhang 1, Haibing Zhi 3, Kehe Huang 2, and Zhigao Bu 1 1 State Key Laboratory of Veterinary Biotechnology, Key Laboratory of Veterinary Public Health Ministry of Agriculture, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin , China 2 College of Veterinary Medicine, Nanjing Agricultural University, Nanjing , China 3 China Institute of Veterinary Drug Control, Beijing , China Abstract: The purpose of the study is to construct recombinant goat pox virus (GPV) expressing Peste des petits ruminants virus (PPRV) H protein, and to evaluate the immunization effect. Recombinant GPV containing PPRV H gene (rgpv-pprv-h) was selected and purified by gpt and egfp utilizing plaque purification, and the final selected recombinant GPV was proved to be purified by PCR. Immunofluorescence and Western blotting showed that the recombinant virus could express H protein of PPRV while infecting lamb testis cells. Six goats were immunized with PFU rgpv-pprv-h through intradermal injection, and were immunized for the second time at 28 days with the same dose recombinant virus after first immunization. Serum was collected after immunization, and was analyzed for the neutralization antibodies. 21 days after first immunization, the neutralization antibodies of GPV were 40, 80, 80, 80, 40, 80 in turn, and neutralization antibodies of PPRV were 80, 80, 80, 80, 40, 40, 10 in turn; 14 days after second immunization, the neutralization antibodies of GPV were all 80, and the neutralization antibodies of PPRV were Received: November 14, 2008; Accepted: January 15, 2009 Supported by: National High Technology Research and Development Program of China ( 863 Program) (No Z449). Corresponding author: Zhigao Bu. Tel: ; Fax: ; zgb@hvri.ac.cn 国家高技术研究发展计划 (863 计划 ) (No Z449) 资助

2 : H 497 >80, 80, >80, 80, 80 and 40 in turn. This study established a foundation for the industrialization of the PPRV recombinant GPV vaccine. Keywords: recombinant vaccine, Peste des petits of ruminants (PPR), goat pox virus vector, neutralization antibodies (Peste des petits of ruminants, PPR) (Goat pox, GP) (Peste des petits of ruminants virus, PPRV) (Goat pox virus, GPV) 2, (The World Organisation for Animal Health, OIE) A (World Organisation for Animal Health. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 2004) PPR, 2007,, GP,, PPR GP 2 PPRV, (Rinderpest virus, RPV),, (F)(H) PPRV GPV GPV GPV GP GP PPR-GP, GP PPR,, 150 kb,, (TK), ; GPV, ; GP, ;,, PPRV,, PPR, [1] [2 4] [5,6],, [7], GPV, PPRV H, 1 材料与方法 1.1 材料 GPV (CVCC AV41) PPRV (Nigeria/75/1) (LT) 1, 10% MEM ; Vero (ATCC NO. CCL-81) 10% MEM GPV PPRV 2% MEM, LT Vero marker Taq DNA DNA MBI, DL2000 DNA marker TaKaRa, pbluescript II KS(+) Stratagene, pires2-egfp Clontech, Zeiss, DMEM MEM Gibco, TRITC IgG IgG (MPA)(Xanthine) (Hypoxanthine) Sigma 1.2 方法 Taq DNA pires2- egfp 666 ~1973 bp ires-egfp ir-f: 5 -aaactgcaggcccctctccctcccccc-3, ir-r: 5 -aaactgcagttacttgtacagctcgtc-3, Pst I ( ) PCR pbluescript II(KS+) EcoR V, pblue-ires-egfp Pst I pblue-ires-egfp ires-egfp, ptk-gpt-gfp [7] Pst I,

3 498 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No.4, ptk-gpt-ires- egfp-gfp Sal I ptk-gpt-ires-egfp-gfp GFP, ptk-gpt-ires-egfp( 1) 图 1 表达绿色荧光蛋白重组 GPV 构建的同源重组质粒 ptk-gpt-ires-egfp 示意图 Fig. 1 Map of plasmid ptk-gpt-ires-egfp for construction of recombinant GPV expressing egfp. PPRV (Nigeria/75/1) Vero Trizol(Invitrogen) PPRV RNA, GenBank (Accession No. X74443) 5 -GGTTTAAACGCCGCCACCATG TCCGCACAAAG 5 -TGCGTTTAAACTCAGAC TGGA TTACATG( Pme I ), RT-PCR 1.85 kb PPRV (H), H (ORF) ATG kozak (GCCGCCACC) H PCR pbluescript II(KS+) EcoR V, pb-pprv-h,, Pme I pb-pprv-h H ORF, Sal I ptk-gpt-ires-egfp,, ptk-gig-pprv-h( 2) LT 35 mm (Nunc), 80%, 0.05 (M.O.I.) GPV 100 μl, 1 h, (Invitrogen) ptk-gig-ppr-h, 12 h 10% DMSO(PBS, ph 7.2) 2.5 min, 24 h, 90% (Cytopathic effect, CPE), LT, 3, 3, 30 s,, 10, 35 mm LT, (MPA, 25 μg/ml), (Xanthine, 250 μg/ml), (Hypoxanthine, 15 μg/ml), 37 o C 10 d,, 3, 3, 30 s, 10, 35 mm LT, 100 μl, 1 h 1%,,, LT, 90%,, SDS- K DNA, TK (TK : 5 -ctt ctc gag gaa tca cca gag ccg ata ac-3, TK : 5 - acc gaa ttc aac tcg tgt ctg ata ccc-3, 40 s, 58 o C) [7], PCR PPRV H ( : 5 -AATGGCACAGGAATAGGC-3, : 5 -GAGCGACCCGTGTCATAG-3, 40 s, 58 o C) H PCR GPV PPRV H rgpv-pprv-h 图 2 用于构建表达 PPRV H 蛋白重组 GPV 的同源重组质粒 ptk-gig-pprv-h 示意图 Fig. 2 Map of plasmid ptk-gig-pprv-h for construction of recombinant GPV expressing PPRV H protein.

: H 499 1.2.3 M.O.I. 0.05 24 LT, LT 80%,, PBST( 0.05% Tween20 PBS) 3 3% 20 min,, PBST 3 1 GPV 1 PBST( 0.

4 : H M.O.I LT, LT 80%,, PBST( 0.05% Tween20 PBS) 3 3% 20 min,, PBST 3 1 GPV 1 PBST( 0.5%) 50 PPRV (PPRV Nigeria/75/1 BALB/C )100 μl, 30 min, PBST 3 PBST 200 (TRITC) IgG 100 μl, 30 min, PBST 3, M.O.I LT, LT 80%,, 2000 r/min SDS-PAGE SDS-PAGE, 4 o C 3% BSA(PBST ), PBST 3, 1% BSA(PBST ) 50 PPRV 1 h, PBST 3, PBST 5000 IgG(Sigma), 1 h PBST 3, DAB, ~2, 2, 1 (1~6 ) H, 2 (7~9 ) GPV, MEM 0.5 ml, PFU,, 250 μl 28 d d, 56 o C 30 min, 20 o C GPV PPRV GPV PPRV GPV PPRV,, 96,, O.I.E. (World Organisation for Animal Health. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 2004) 2 结果 2.1 重组病毒的克隆筛选与鉴定 gpt,, egfp LT, GPV DNA, TK [7] PCR TK, PPRV-H, GPV, TK, GPV 754 bp, 2200 bp (P7.5 P11 egfp gpt IRES ), 40 s, 2200 bp, 740 bp ( 3); PPRV-H, 461 bp, ( 3) H GPV, GPV, PPRV H rgpv-pprv-h GPV rgpv-pprv-h LT, rgpv-pprv-h, GPV ( 4) 图 3 重组 GPV 的纯化鉴定 Fig. 3 PCR assay of the purification of recombinant GPV. (A) M: DL2000 marker; P: PCR results of GPV with primers for TK gene; H: PCR results of rgpv-pprv-h with primers for TK gene. (B) 1: PCR results of GPV with primers for PPRV H gene; 2: PCR results of rgpv-pprv-h with primers for PPRV H gene.

500 ISSN1000-3061 CN11-1998/Q Chin J Biotech April 25, 2009 Vol.25 No.4 图 4 荧光显微镜下观察 rgpv-pprv-h 感染 LT 细胞表达 egfp (100 ) Fig.

2 免疫荧光和蛋白印迹试验 PPRV H, (TRITC) IgG, rgpv-pprv-h, GPV ( 5) 6, PPRV-H rgpv-pprv-h LT, 67 kd,, rgpv-pprv-h PPRV H 2.

5 500 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No.4 图 4 荧光显微镜下观察 rgpv-pprv-h 感染 LT 细胞表达 egfp (100 ) Fig. 4 Observation of expression of egfp by rgpv-pprv-h infecting LT cells using fluorescence microscope (100 ). 2.2 免疫荧光和蛋白印迹试验 PPRV H, (TRITC) IgG, rgpv-pprv-h, GPV ( 5) 6, PPRV-H rgpv-pprv-h LT, 67 kd,, rgpv-pprv-h PPRV H 2.3 免疫试验 GPV rgpv-pprv-h PFU, 4 2 GPV GPV PPRV (<1:10) rgpv-pprv-h, 1 21 d, GPV , 2 14 d, 80;, 21, PPRV 6/6, , 14, PPRV 6/6, ( 1) GPV PPRV (<1:10)( 1) 图 5 免疫荧光检测 rgpv-pprv-h 感染 LT 细胞表达 PPRV H 蛋白 (100 ) Fig. 5 Analysis of the expression of PPRV F protein by rgpv- PPRV-H infecting LT cells using immunofluorescence test (100 ). 表 1 重组病毒免疫后 GPV 和 PPRV 特异中和抗体检测结果 Table 1 Neutralization antibodies against GPV and PPRV of goats immunized with recombinant virus Virus rgpv-pprv-h GPV Serial number of goats Neutralization antibodies at 21 d after first immunization Neutralization antibodies at 14 d after second immunization GPV PPRV GPV PPRV <10 80 < <10 80 < <10 80 <10

: H 501, 0.1 PFU,, PPRV H 图 6 蛋白印迹检测 rgpv-pprv-h 感染 LT 细胞表达 H 蛋白 Fig. 6 Analysis of the expression of PPRV H protein by rgpv-pprv-h infecting LT cells using Western blotting.

6 : H 501, 0.1 PFU,, PPRV H 图 6 蛋白印迹检测 rgpv-pprv-h 感染 LT 细胞表达 H 蛋白 Fig. 6 Analysis of the expression of PPRV H protein by rgpv-pprv-h infecting LT cells using Western blotting. M: prestained protein marker; P: GPV; H: rgpv-pprv-h. 3 讨论 GPV LT, TK,,,, IRES, p11 gpt egfp,, PPRV O.I.E. A,,,, O.I.E. PPRV (10) PPRV,, PPRV,, O.I.E., PPRV 100%(>1:10) H, 4 PPRV 100%(6/6) 2 [5, 6], PPRV F 1~10000 PFU, PPRV, PPRV 1000 PFU, 100%( 10), ; 10 PFU, PPRV,,,, PPRV, H HN G [8] F GPV(), H GPV, ;, H F, H F,,,, : (CVCC AV41),, ;,, PPRV [9] REFERENCES [1] Perrin A, Albina E, Breard E, et al. Recombinant capripoxviruses expressing proteins of bluetongue virus: Evaluation of immune responses and protection in small ruminants. Vaccine, 2007, 25(37-38): [2] Romero CH, Barrett T, Kitching RP, et al. Protection of cattle against rinderpest and lumpy skin disease with a recombinant capripoxvirus expressing the fusion protein gene of rinderpest virus. Vet Rec, 1994, 135(7): [3] Romero CH, Barrett T, Evans SA, et al. Single capripoxvirus recombinant vaccine for the protection of cattle against rinderpest and lumpy skin disease. Vaccine, 1993, 11(7):

7 502 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No.4 [4] Romero CH, Barrett T, Chamberlain RW, et al. Recombinant capripoxvirus expressing the hemagglutinin protein gene of rinderpest virus: Protection of cattle against rinderpest and lumpy skin disease viruses. Virology, 1994, 204(1): [5] Berhe G, Minet C, Goff C Le, et al. Development of a dual recombinant vaccine to protect small ruminants against peste-des-petits-ruminants virus and capripoxvirus infections. J Virol, 2003, 77(2): [6] Diallo A, Minet C, Berhe G, et al. Goat immune response to capripox vaccine expressing the hemagglutinin protein of peste des petits ruminants. Ann N Y Acad Sci, 2002, 969: [7] Cheng XW, Hu S, Wang XJ, et al. Construction and purification of recombinant goatpox virus expressing exotic green fluorescence protein. J Agric Biotech, 2006, 14(5): ,,,.., 2006, 14(5): [8] Wang XJ, Wang QH, Ge JY, et al. Generation of recombinant vaccinia virus expressing attachment glycoprotein of Nipah virus. Acta Microbiol Sin, 2006, 46(4): ,,,. G., 2006, 46(4): [9] Hosamani M, Singh SK, Mondal B, et al. A bivalent vaccine against goat pox and Peste des Petits ruminants induces protective immune response in goats. Vaccine, 2006, 24(35/36): yangrf@nic.bmi.ac.cn 2 4 SNIP /isham2009 : yangrf@nic.bmi.ac.cn bdhy2009@163.com : :

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇