期刊文献+

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 被引量:9

Baculovirus expression and establishment of the indirect ELISA for the HA gene of swine influenza virus H1 subtype
下载PDF
导出
摘要 根据GenBank发表的H1亚型猪流感HA基因序列设计引物,扩增出HA基因片段,将其克隆到pFastBacGP67B杆状病毒载体上,筛选阳性重组转座载体pFastBacGP67B-H1,转化含有杆状病毒穿梭载体(bacmid)的DH10Bac感受态细胞,构建杆状病毒表达载体获得重组转座子(rBacmid-H1),在脂质体介导下转染sf9昆虫细胞,获得重组杆状病毒(rBV-H1),再感染细胞,收获目的蛋白。通过血凝试验、免疫印迹法、免疫组化分析表明该蛋白得到表达,且具有良好的生物学活性。利用表达的蛋白作为猪流感间接ELISA的抗原,初步建立H1亚型猪流感的间接ELISA检测方法,并对内蒙古、辽宁和黑龙江等地送检的93份猪血清进行了检测,阳性率为31.18%,为研制开发快速、准确、简便的H1亚型猪流感鉴别诊断试剂盒奠定基础。 The hemagglutinin (HA) gene fragment of swine influenza virus A/Swine/Guangdong/LM/05(HIN1) 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 DHIOBac 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.
出处 《微生物学报》 CAS CSCD 北大核心 2008年第2期220-225,共6页 Acta Microbiologica Sinica
基金 国家“973项目”(2005CB523202) 国家“十一五”科技支撑计划项目(2006BAD06A05)~~
关键词 H1亚型猪流感病毒 血凝素基因 昆虫细胞杆状病毒表达 间接ELISA H 1 subtype SIV HA gene baculovirus/insect cell system indirect ELISA
  • 相关文献

参考文献21

  • 1Qi x. Lu CP. Genetic characterization of novel reassortant H1N2 influenza A viruses isolated from pigs in southeastern China. Arch Virol, 2006, 151 ( 11 ): 2289-2299.
  • 2Brown 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): 1045-1050.
  • 3Karasin 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): 9322-9327.
  • 4Xu 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): 919-925.
  • 5李海燕,于康震,杨焕良,辛晓光,陈君彦,赵朴,毕英佐,陈化兰.中国猪源H5N1和H9N2亚型流感病毒的分离鉴定[J].中国预防兽医学报,2004,26(1):1-6. 被引量:106
  • 6Shvartsmman DE, Kotler M, Tall RD, et al. Differently anchored influenza hernagglutinin mutans display distinct interaction dynamics with mutual rafts. Cell Biol, 2003, 163(4):879-888.
  • 7Fouchier RA, Munster V, WaUensten A, et al. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol, 2005, 79(5): 2814-2822.
  • 8潘蔚绮,刘明,刘春国,张云,杨涛,李景鹏.一株对小鼠具有高致病性的H5N1亚型禽流感病毒的拯救[J].农业生物技术学报,2006,14(6):831-835. 被引量:2
  • 9Sambrook J, Russel DW. Molecular Cloning A Laboratory Manual. 3^rd ed. New York: Cold Spring Harbor Laboratory Press, 2001.
  • 10Ausubel FM, Brent R, Kingston RE, et al. Short Protocols in Molecular Biology. 3^rd ed. New York: John Wiley & Sons, Inc, 1995.

二级参考文献40

共引文献131

同被引文献171

引证文献9

二级引证文献22

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部