| 鸡γ-干扰素基因在昆虫细胞/杆状病毒载体系统中的表达 |
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| 引用本文： | 王海艳,刘胜旺,孔宪刚,赵振华.鸡γ-干扰素基因在昆虫细胞/杆状病毒载体系统中的表达[J].农业生物技术学报,2004,12(6):639-643. |
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| 作者姓名： | 王海艳 刘胜旺 孔宪刚 赵振华 |
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| 作者单位： | 1. 内蒙古农业大学动物科学与医学学院,呼和浩特,010018
2. 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,哈尔滨,150001 |
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| 基金项目： | 国家高技术研究与发展计划(863)项目(No.2001AA249032)资助. |
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| 摘 要： | 摘要：将鸡（Gallus gallus）的γ-干扰素（interferon-γ, IFN-γ）基因亚克隆到杆状病毒转移载体pMelBacB的BamHⅠ位点上，构建真核转移载体pMelBacB-ChIFN-γ，经限制性内切酶消化、ChIFN-γ特异引物PCR鉴定和序列测定，证明目的基因正确克隆到载体的预期位点。将纯化的pMelBacB-ChIFN-γ质粒与杆状病毒DNA（Bac-N-BlueTM DNA）共转染sf9昆虫细胞，经3轮蓝斑筛选纯化，获得了重组杆状病毒rBaculovirus-ChIFN-γ。提取病毒DNA经ChIFN-γ特异引物和重组杆状病毒特异引物PCR鉴定，证明获得了纯化的重组杆状病毒，命名为rBaculovirus-ChIFN-γ。用该重组病毒接种sf9昆虫细胞，收获接种后不同时间的细胞进行SDS-PAGE,结果表明ChIFN-γ基因在昆虫细胞中获得了表达，表达的重组蛋白分子量约为19 kD。应用在大肠杆菌(Escherichia coli )原核表达系统中表达的重组蛋白制备的兔抗ChIFN-γ多克隆抗体进行Western blot分析，表明表达的重组蛋白具有抗原性。
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| 关 键 词： | 关键词：鸡IFN-γ 昆虫细胞/杆状病毒 重组蛋白 |
| 修稿时间： | 2003年12月29 |
Expression of Recombinant Chicken IFN-γGene in Baculovirus Vector System |
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| WANG Hai-Yan,LIU Sheng-Wang,KONG Xian-gang,ZHAO Zhen-Hua.Expression of Recombinant Chicken IFN-γGene in Baculovirus Vector System[J].Journal of Agricultural Biotechnology,2004,12(6):639-643. |
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| Authors: | WANG Hai-Yan LIU Sheng-Wang KONG Xian-gang ZHAO Zhen-Hua |
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| Abstract: | Abstract：The cDNA of ChIFN-γ gene was subcloned into BamHⅠ site of pMelBacB under the control of baculovirus polyhedrin to construct recombinant transfer vector pMelBacB-ChIFN-γ. The recombinant transfer vector pMelBacB-ChIFN-γ was then transfected into sf9 insect cells with wild-type Bac-N-Blue linear DNA using cationic liposomes. The cultures were harvested and plaque-assayed with x-gal, and blue plaques were selected as recombinant viruses. After being purified for three times and identified by PCR, the purified viruses were named rBaculovirus-ChIFN-γ. The Sf9 cells were infected with rBaculovirus-ChIFN-γ and incubated at 28 ℃, their and cultures were harvested at 24, 48, 72, 96, 120 and 144 h post-inoculation, respectively. The lysates of the cells were analyzed by SDS-PAGE, a band with a apparent molecular weight of 19 kD was observed. To further confirm this band, the lysates of sf9 insect cells infected with rBaculovirus-ChIFN-γ were analyzed by Western blot using polyclonal antibodies against ChIFN-γ, the result showed that this band represented ChIFN-γ. It was demonstrated that about 19 kD recombinant ChIFN-γ protein with activity was expressed in sf9 insect cells. |
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