H5N1禽流感病毒HA基因在昆虫细胞中的表达及生物活性鉴定

中国生物工程杂志

2007, Vol. 27

Issue (3): 42-46

研究报告

H5N1禽流感病毒HA基因在昆虫细胞中的表达及生物活性鉴定

张晓霁刘明张云刘春国

中国农业科学院哈尔滨兽医研究所哈尔滨兽医研究所兽医生物技术国家重点实验室中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室

The HA gene of H5N1 Avian Influenza Virus Expressed in Insect Cells and Idenfication of its Immunological Activity

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摘要：

经RT-PCR扩增了H5N1亚型禽流感病毒血凝素基因(HA)片断，限制性内切酶酶切后将其克隆到pFastBacHTA杆状病毒转座载体，经酶切鉴定及测序，筛选出阳性重组转座载体pFastBac-H5。将pFastBacH5转化含有杆状病毒穿梭载体（bacmid）的DH10Bac感受态细胞，通过蓝白斑筛选和PCR鉴定获得重组杆状病毒穿梭载体rBacmid-H5。rBacmid-H5在脂质体介导下转染sf9昆虫细胞，SDS－PAGE蛋白电泳、Western blot、血凝试验和血凝抑制试验分析表明：分子量约63Kd重组血凝素蛋白（rH5）在sf9昆虫细胞中实现了高效表达。rH5具有血凝活性，而且其血凝活性能够被H5N1禽流感病毒高免血清所抑制；rH5免疫鸡诱导产生针对H5N1禽流感病毒亚型特异的血凝抑制抗体，说明表达的重组蛋白具有与天然蛋白相似的生物活性。

Abstract:

The completed DNA of 1.7 kb HA gene was prepared from the cDNA by RT-PCR. The fragment was subcloned into the pFastBacHTa donor plasmids. the recombinant pFastBacHTa was cut by restriction enzyme and sequenced. then The purified plasmid DNA was transform into DH10Bac E. coli for transposition into the bacmid and the colonies was identified by blue/white selection. The recombinant bacmid was verified by PCR analysis. The recombinant baculovirus was transfected into sf9 cells by Cellfectin reagent, the expressed HA protein was analyzed by SDS-PAGE、Western-blot、HA test and HI test and it have good immunological activity.

收稿日期: 2006-07-26 出版日期: 2007-03-25

通讯作者: 刘明

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引用本文:

张晓霁,刘明,张云,刘春国. H5N1禽流感病毒HA基因在昆虫细胞中的表达及生物活性鉴定[J]. 中国生物工程杂志, 2007, 27(3): 42-46.

. The HA gene of H5N1 Avian Influenza Virus Expressed in Insect Cells and Idenfication of its Immunological Activity. China Biotechnology, 2007, 27(3): 42-46.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2007/V27/I3/42

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