H1 ELISA. / (2008) 48(2) 221 DNA Marker TaKaRa ; (OPD) ; CellFectin Invitrogen ; His ; IgG/ (HRP) ; IgG/ (HRP) Southern Biotech ; PCR M J Research ; C

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1 Research Paper 研究报告 Acta Microbiologica Sinica 48(2) 220~225; 4 February 2008 ISSN ; CN /Q H1 亚型猪流感病毒血凝素基因在昆虫细胞中的表达及其间接 ELISA 方法的初步建立万春和, 刘明, 刘春国, 张晓霁, 杨涛, 刘大飞, 陈浩, 齐金龙, 乔传玲 (,,, ) 摘要 GenBank H1 HA, HA, pfastbacgp67b, pfastbacgp67b-h1, (bacmid) DH10Bac, (rbacmid-h1), sf9, (rbv-h1),,, ELISA, H1 ELISA, 93, 31.18%, H1 关键词 H1 ; ; ; ELISA 中图分类号 S 文献标识码 A 文章编号 (2008) (Swine influenza virus, SIV) A, 1918 SI, 1930 Shope H1N1, SIV H1N1 H1N2 H1N7 H3N2 H3N1 H4N6 H5N1 H9N2 [1~5] SI, (HA), HA,, [6] 16 HA 9 NA [7],,, H1N1 H1N2 H3N2 3, H1 HA, H1 ELISA 1 材料和方法 1.1 材料 pfastbacgp67b, (Escherichia coli)dh10bac Top Ex-Taq T4 DNA 基金项目 : 973 (2005CB523202); (2006BAD06A05) * Tel: ; liuming04@126.com 作者简介 : (1982 ),,,, chunhewan@126.com 收稿日期 : ; 修回日期 :

pfastbacgp67b-h1, (bacmid) DH10Bac, (rbacmid-h1), sf9, (rbv-h1),,, ELISA, H1 ELISA, 93, 31.18%, H1 关键词 H1 ; ; ; ELISA 中图分类号 S852.

2 H1 ELISA. / (2008) 48(2) 221 DNA Marker TaKaRa ; (OPD) ; CellFectin Invitrogen ; His ; IgG/ (HRP) ; IgG/ (HRP) Southern Biotech ; PCR M J Research ; Cole Parmer ; Bio-Rad ; Beckman A/Swine/ Guangdong/LM/05(H1N1)( LM ), (PRRS) (PR) (HC) (PP) (FMD) (TGE) (PC) (JE) GenBank H1 HA, HA, 5' BamHⅠ Hind Ⅲ,, 6*His, 5'-CGCGGA- TCCGACACACTATGTATAGGTTAC-3'; 5'-GCGAAGCTTCTAGTGATGGTGATGGTGAT- GGATCGCCAAAATTTGGTAAAT-3' (M13) Bac-to-Bac, 1.2 杆状病毒表达载体的构建和重组杆状病毒的制备 [8] LM RNA, cdna, HA PCR PCR 94 4min; 94 30s, 56 45s, 72 2min, 30 ; 72 10min PCR ( ) PCR pfastbacgp67b BamHⅠ HindⅢ, 16 1h, Top10, (pfastbacgp67b H1),,, pfastbacgp67b-h1, (bacmid) DH10Bac,, rbacmid- H1, DNA, PCR Invitrogen Bac-to-Bac Baculovirus Expression System, rbacmid-h1 DNA sf9 3, HA (rbv-h1) 1.3 表达蛋白的生物学活性检测 (HA) rbv-h1 10 (MOI=10) sf9 84h, PBS 3, PBS,,, PBS SDS-PAGE Western blot [9], 12% SDS-PAGE, H1 (1:100), IgG/ (HRP) (1:5000),, Western blot : [10], rbv-h1 sf9 72h, PBS 3, His (1:1000) IgG/ (HRP) (1:3000),, 1.4 间接 ELISA 的初步建立 [11], ELISA, H1 ELISA OD { (X)+3 (S)}, OD (X+2S),,, (X+3S), 1.5 特异性试验,

2 杆状病毒表达载体的构建和重组杆状病毒的制备 1.2.1 [8] LM RNA, cdna, HA PCR PCR 94 4min; 94 30s, 56 45s, 72 2min, 30 ; 72 10min PCR ( ) 1.2.2 PCR pfastbacgp67b BamHⅠ HindⅢ, 16 1h, Top10, (pfastbacgp67b H1),,, 1.

3 222 Chunhe Wan et al. /Acta Microbiologica Sinica (2008) 48(2) (PRRS) (PR) (HC) (PP) (FMD) (TGE) (PC) (JE)8 H3, H1, ELISA,, 1.6 临床送检样品的检测 ELISA, 93,,, OD H1 SIV (HI) 50% (RBCs) 4 30min, 2000 r/min 10 min,, (RDE)37,, 56 1h RDE 2 结果 2.1 杆状病毒表达载体的构建和重组杆状病毒的制备 pfastbacgp67b-h1 pfastbacgp 67B-H1 BamHⅠ Hind Ⅲ, 4.8kb 1.6kb, LM HA, rbacmid-h1 PCR rbacmid-h1 DNA, M13 PCR 4.1kb(2.5 kb +1.6 kb),, HA 2.2 表达蛋白的生物学活性检测 rbv-h1 Sf9 84h,, 2 2, SDS-PAGE Western blot 12% SDS-PAGE, 57kDa, ( 1-A ) Western-blot, 57kDa, sf9 ( 1-B ) 图 1 HA 蛋白的 SDS-PAGE(A) 和 Western-blot(B) Fig.1 SDS-PAGE analysis (A) and Western blot analysis (B) of the HA expression. 1. Control of sf9 cells infected by Bacmid; 2. Control of 1 SDS Buffer; 3. HA protein expressed in Sf9 cells infected recombinant baculovirus; M. protein Marker ( 2 ), rbv-h1 ; 图 2 野生型病毒感染的细胞 (A) 和 rbv-h1 (B) (400 ) Fig.2 A Bacmid transfected sf9 cells (A) and rbv-h1 transfected sf9 cells (B) (400 ).

8kb 1.6kb, LM HA, 2.1.2 rbacmid-h1 PCR rbacmid-h1 DNA, M13 PCR 4.1kb(2.5 kb +1.6 kb),, HA 2.2 表达蛋白的生物学活性检测 2.2.1 rbv-h1 Sf9 84h,, 2 2, 2.2.2 SDS-PAGE Western blot 12% SDS-PAGE, 57kDa, ( 1-A ) Western-blot, 57kDa, sf9 ( 1-B ) 图 1 HA 蛋白的 SDS-PAGE(A) 和 Western-blot(B) Fig.

4 H1 ELISA. / (2008) 48(2) 223, 2.3 间接 ELISA 条件的初步确立 ( ) 1:800, (HRP IgG) 1:3000, 1.5 h, 37 8 min X , S , OD (X+3S 0.2), OD (X+2S 0.15),,, OD 0.2, 2.4 特异性试验 ( 1), 8 OD 0.15, H3 OD 0.15,, H1 OD 0.978, 2.5 送检样品的结果分析 93, 2, 29, 31.18% HI 24, 25.81%, ELISA 29, 94.62%, ELISA HI Table 1 1 Results of cross test of indirect ELISA Other diseases positive sera in pig Swine influenza control Cell control PRRS PR HC PP FMD TGE PC JE Negative control(h3) Positive control(h1) Negative control(bacmid) Table 2 The result of sera test Sample Positive Negative Sum Seropositivity rates/% ELISA HI test ELISA HI test ELISA HI test Inner Mongolia( ) Liaoning( ) Heilongjiang( ) In sum 讨论,, HA, HA,,, [12, 13] Bac-to-Bac H1 SIV HA, 57kDa HA, ( ), HA Western blot HA H1 SIV, HA, His,,, [14,15], [16~20],,,

62%, ELISA HI Table 1 1 Results of cross test of indirect ELISA Other diseases positive sera in pig Swine influenza control Cell control PRRS PR HC PP FMD TGE PC JE Negative control(h3) Positive

5 224 Chunhe Wan et al. /Acta Microbiologica Sinica (2008) 48(2) (AGP) HI ELISA, AGP HI,,,, HI ELISA, [20, 21] HA ELISA, H1 SIV HA, HA ELISA,, 93 ELISA, 31.18%, H1, (NA) N1 N2, ( ), 参考文献 [1] Qi X, Lu CP. Genetic characterization of novel reassortant H1N2 influenza A viruses isolated from pigs in southeastern China. Arch Virol, 2006, 151(11): [2] Brown IH, Hill ML, Harris PA, et al. Genetic characterization of an influenza A virus of unusual subtype (H1N7) isolated from pigs in England. Arch Virol, 1997, 142(6): [3] Karasin AI, Brown IH, Carman S, et al. Isolation and characterization of H4N6 avian influenza viruses from pigs with pneumonia in Canada. J Virol, 2000, 74(19): [4] Xu C, Fan W, Wei R, et al. Isolation and identification of swine influenza recombinant A/Swine/Shandong/1/2003 (H9N2) virus. Microbes Infect, 2004, 6(10): [5],,, H5N1 H9N2 (Chinese Journal of preventive Veterinary Medicine), 2004, 26(1): 1 6 [6] Shvartsmman DE, Kotler M, Tall RD, et al. Differently anchored influenza hemagglutinin mutans display distinct interaction dynamics with mutual rafts. Cell Biol, 2003, 163(4): [7] Fouchier RA, Munster V, Wallensten A, et al. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol, 2005, 79(5): [8],,, H5N1 (Journal of Agricultural Biotechnology), 2006, 14(6): [9] Sambrook J, Russel DW. Molecular Cloning A Laboratory Manual. 3 rd ed. New York: Cold Spring Harbor Laboratory Press, [10] Ausubel FM, Brent R, Kingston RE, et al. Short Protocols in Molecular Biology. 3 rd ed. New York: John Wiley & Sons, Inc, [11],,, H5N1 ELISA (Acta Microbiologica Sinica), 2005, 45(1): [12] Kost TA, Condreay JP, Jarvis DL, et al. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nature Biotechnology, 2005, 123(5): [13],,, H5N1 HA (Journal of Chinese Biotechnology), 2007, 27(3): [14] Ito T, Couceiro JN, Kelm S, et al. Molecular Basis for the Generation in Pigs of Influenza A Virus with Pandemic Potential. J Virol, 1998, 72(9): [15] Kamps B, Hoffmann C, Preiser W. Influenza Report 2006.Paris: Flying Publisher [16] Olsen, CW. The emergence of novel swine influenza viruses in North America. Virus Res,2002, 85(2), [17] Lipatov AS, Govorkova EA, Webby RJ, et al. Influenza: emergence and control. J Virol, 2004, 78(17): [18] Ma WJ, Gramer M, Rossow K, et al. Isolation and genetic characterization of new reassortant H3N1 swine influenza virus from pigs in the Midwestern United States, J Virol, 2006,80 (10): [19] Chen JM, Ma HC, Chen JW, et al. A preliminary panorama of the diversity of N1 subtypes influenza viruses. Virus Genes, 2007, 35(1): [20] Yoon KJ, Janke BH, Swalla RW, et al. Comparison of a commercial H1N1enzyme-linked immunosorbent assay and hemagglutination inhibition test in decting serum antibody against swine influenza virus. J Vet Diagn Invest, 2004, 16(3): [21] Jung K, Song DS, Kang BK, et al. Serologic surveillance of swine H1 and H3 and avian H5 and H9 influenza A virus infections in swine population in Korea. Preventive Veterinary Medicine, 2007, 79(4):

Arch Virol, 2006, 151(11): 2289 2299. [2] Brown IH, Hill ML, Harris PA, et al. Genetic characterization of an influenza A virus of unusual subtype (H1N7) isolated from pigs in England.

6 H1 ELISA. / (2008) 48(2) 225 Baculovirus expression and establishment of the indirect ELISA for the HA gene of swine influenza virus H1 subtype Chunhe Wan, Ming Liu, Chunguo Liu, Xiaoji Zhang, Tao Yang, Dafei Liu, Hao Chen, Jinlong Qi, Chuanling Qiao (Animal Influenza Laboratory of the Ministry of Agriculture, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of CAAS, Harbin , China) Abstract: The hemagglutinin (HA) gene fragment of swine influenza virus A/Swine/Guangdong/LM/05(H1N1) was amplified with HA gene specific primers and cloned into baculovirus transfer plasmid pfastbacgp67b. The recombinant plasmid pfastbacgp67b-h1 was identified by restriction enzyme digestion and gene sequencing. Following the transformation of DH10Bac Escherichia coli component cells by pfastbacgp67b-h1, recombinant bacmids rbacmid-h1 were identified by blue/white selection and PCR analysis. Then recombinant baculovirus rbv-h1 was rescued by lipofectant reagent Cellfectin induced rbacmid-h1 DNA transfection of long-phage sf9 insect cells. The recombinant HA protein was characterized by hemagglutination test, western-blot and immunohistochemistry. An indirect enzyme-linked immunosorbent assay (ELISA) was assessed to detect in pigs IgG against H1 subtype SIV present in Inner Mongolia, Liaoning and Heilongjiang provinces. Positive was found in 31.15% (29 of 93) serum samples tested from swine reared in commercial herds. However, all irrelevant control sera tested were negative. We conclude, therefore, that ELISA performed with recombinant HA as coating antigen was a better tool for swine influenza surveillance in China. Keywords: H1 subtype SIV; HA gene; baculovirus/insect cell system; indirect ELISA Supported by the Key Project of China National Programs for Fundamental Research and Development (2005CB523202) and the 11 th Five Years Programs for Science and Technology Development Support of China (2006BAD06A05) * Corresponding author. Tel: ; liuming04@126.com Received: 7 June 2007/Revised:12 November 2007 ˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇ 答作者问问 : 我想问一下作者及单位署名顺序的修改问题投稿后经过专家审查通过后, 即将发表, 如果想调整作者并且修改作者及单位署名顺序是否可以? 是否需要提供什么证明或者相关的材料? 答 :(1), ( ), (2), (3),

Liu, Hao Chen, Jinlong Qi, Chuanling Qiao (Animal Influenza Laboratory of the Ministry of Agriculture, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of

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