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H5N1亚型禽流感病毒血凝素基因的原核表达及间接ELISA方法的初步建立
引用本文:郑其升,张晓勇,刘华雷,李鹏,陈溥言.H5N1亚型禽流感病毒血凝素基因的原核表达及间接ELISA方法的初步建立[J].微生物学报,2005,45(1):58-61.
作者姓名:郑其升  张晓勇  刘华雷  李鹏  陈溥言
作者单位:1. 南京农业大学,农业部动物疫病诊断与免疫重点开放实验室,南京,210095
2. 南京农业大学,农业部动物疫病诊断与免疫重点开放实验室,南京,210095;河北省畜牧兽医总站,保定,071000
摘    要:根据GenBank公布的H5N1亚型禽流感病毒(AIV)血凝素(HA)基因序列设计引物,用PCR方法扩增H5N1亚型禽流感病毒HA1基因, 将该片段定向插入到原核表达载体pET_32a(+)中,构建原核表达载体pET_HA1。阳性质粒转化宿主菌BL21(DE3), 经IPTG诱导, HA1基因获得表达, 重组蛋白以包涵体的形式存在。通过改变IPTG的浓度和诱导时间 , 确定了表达HA1基因的最佳诱导条件: IPTG终浓度为0.8mmol/L,诱导时间为3h。Western blot分析表明表达产物具有良好的免疫学活性。以纯化的表达产物作为诊断抗原建立了检测H5亚型AIV抗体的iHA_ELISA方法。结果表明,抗原的最佳包被浓度为4μg/mL,血清的最佳稀释度为1∶200, 阳性标准初步定为:OD待检血清>05,且 OD待检血清/OD阴性血清>2。

关 键 词:H5N1亚型禽流感病毒,血凝素基因,原核表达,iHA_ELISA
文章编号:0001-6209(2005)01-0058-04
修稿时间:2004年5月18日

The prokaryotic expression and the establishment of the putative indirect ELISA assay for the HA gene for Avian influenza virus (AIV) H5N1 subtype
ZHENG Qi-sheng ZHANG Xiao-yong LIU Hua-lei LI Peng , CHEN Pu-yan.The prokaryotic expression and the establishment of the putative indirect ELISA assay for the HA gene for Avian influenza virus (AIV) H5N1 subtype[J].Acta Microbiologica Sinica,2005,45(1):58-61.
Authors:ZHENG Qi-sheng ZHANG Xiao-yong LIU Hua-lei LI Peng  CHEN Pu-yan
Institution:Key Laboratory of Animal Disease Diagnosis and Immunology, Nanjing Agricultural University, Nanjing 210095, China.
Abstract:Using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank, the HA1 gene of H5N1 subtype AIV was amplified with PCR method. The PCR product was cloned into pET-32a(+) to get a prokaryotic recombinant plasmid pET-HA1. The target gene was successfully expressed in the host cell BL21 (DE3) when induced with IPTG. The expression was optimized with proper inducing conditions of 0.8 mmol/L IPTG and 3 hours induction. The highest expression of the target protein added up to 32.7% of the total bacterial protein. Western blot analysis proved the recombinant protein has good reactive ability against H5N1 subtype AIV positive serum. The optional working circumstances for the iHA-ELISA assay (antigenicity concentration: 4 microg/mL; serum dilution: 1:200) was tried out with chess titration. The positive criterion of this ELISA assay is OD(the tested serum) > 0.5 and OD(the tested serum)/OD(the negative serum) > 2.0.
Keywords:H5N1 subtype Avian Influenza Virus(AIV)  HA gene  Prokaryotic expression  iHA-ELISA
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