蛇毒cystatin在Bac-to-Bac杆状病毒表达系统中的表达与鉴定
发布时间：2018-12-09 16:38
【摘要】：目的 探讨蛇毒cystatin 在Bac-to-Bac 杆状病毒表达系统中的表达。 方法 根据中华眼镜蛇蛇毒cystatin 蛋白的氨基酸序列合成cystatin 基因的四个片段,通过缓慢退火PCR 的方法将其拼接为完整的cystatin 基因,PCR 扩增蛇毒cystatin 基因,将其克隆到供体质粒pFastBacHTc 中,通过转化DH10Bac 菌筛选、鉴定并抽提获取高纯度的重组穿梭载体Bacmid-cystatin,经脂质体介导将重组穿梭载体转染Sf9 细胞,获取并扩增重组病毒,空斑分析确定病毒滴度,Western-blot分析适宜的重组融合蛋白表达时间和MOI 值,SDS-PAGE 分析细胞总蛋白,Western-blot 鉴定重组融合蛋白。 结果 1 构建了携带蛇毒cystatin基因片段的重组供体质粒pFastBacHTc -cystatin,经双酶切鉴定和序列分析证实蛇毒cystatin 基因已正确插入供体质粒的多克隆位点。 2 构建了携带蛇毒cystatin 基因片段的重组穿梭载体Bacmid-cystatin,经PCR 鉴定分析证实蛇毒cystatin 基因已正确转座插入穿梭载体的转座接触位点。 3 确定了重组融合蛋白在Sf9 细胞中表达的最佳时间是感染后96 h,适宜的MOI 值为10。该重组融合蛋白经SDS-PAGE、Western-blot 分析鉴定为蛇毒6×His-cystatin 融合蛋白,分子量为15000 道尔顿,与融合蛋白的理论分子量相
[Abstract]:Objective to investigate the expression of snake venom cystatin in Bac-to-Bac baculovirus expression system. Methods four fragments of cystatin gene were synthesized according to the amino acid sequence of cystatin protein from cobra venom of Cobra chinensis. The cystatin gene was spliced into complete cystatin gene by slow annealing PCR, and the cystatin gene of snake venom was amplified by PCR. It was cloned into donor plasmid pFastBacHTc. The recombinant shuttle vector Bacmid-cystatin, with high purity was identified and extracted by screening the transformed DH10Bac bacteria. The recombinant shuttle vector was transfected into Sf9 cells by liposome, and the recombinant virus was obtained and amplified. The virus titer was determined by plaque analysis, the expression time and MOI value of recombinant fusion protein were analyzed by Western-blot, the total cell protein was analyzed by SDS-PAGE, and the recombinant fusion protein was identified by Western-blot. Results 1 the recombinant donor plasmid pFastBacHTc cystatin, carrying snake venom cystatin gene fragment was identified by double enzyme digestion and sequence analysis. It was confirmed that the cystatin gene of snake venom was correctly inserted into the polyclonal site of donor plasmid. 2Recombinant shuttle vector Bacmid-cystatin, carrying snake venom cystatin gene fragment was constructed. PCR analysis confirmed that the cystatin gene of snake venom had been correctly transposed and inserted into the transposable contact site of the shuttle vector. 3The optimal time of expression of recombinant fusion protein in Sf9 cells was 96 h after infection, and the optimum MOI value was 10. 5%. The recombinant fusion protein was identified by SDS-PAGE,Western-blot as 6 脳 His-cystatin fusion protein of snake venom with molecular weight of 15000 Dalton.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346
本文编号：2369703
[Abstract]:Objective to investigate the expression of snake venom cystatin in Bac-to-Bac baculovirus expression system. Methods four fragments of cystatin gene were synthesized according to the amino acid sequence of cystatin protein from cobra venom of Cobra chinensis. The cystatin gene was spliced into complete cystatin gene by slow annealing PCR, and the cystatin gene of snake venom was amplified by PCR. It was cloned into donor plasmid pFastBacHTc. The recombinant shuttle vector Bacmid-cystatin, with high purity was identified and extracted by screening the transformed DH10Bac bacteria. The recombinant shuttle vector was transfected into Sf9 cells by liposome, and the recombinant virus was obtained and amplified. The virus titer was determined by plaque analysis, the expression time and MOI value of recombinant fusion protein were analyzed by Western-blot, the total cell protein was analyzed by SDS-PAGE, and the recombinant fusion protein was identified by Western-blot. Results 1 the recombinant donor plasmid pFastBacHTc cystatin, carrying snake venom cystatin gene fragment was identified by double enzyme digestion and sequence analysis. It was confirmed that the cystatin gene of snake venom was correctly inserted into the polyclonal site of donor plasmid. 2Recombinant shuttle vector Bacmid-cystatin, carrying snake venom cystatin gene fragment was constructed. PCR analysis confirmed that the cystatin gene of snake venom had been correctly transposed and inserted into the transposable contact site of the shuttle vector. 3The optimal time of expression of recombinant fusion protein in Sf9 cells was 96 h after infection, and the optimum MOI value was 10. 5%. The recombinant fusion protein was identified by SDS-PAGE,Western-blot as 6 脳 His-cystatin fusion protein of snake venom with molecular weight of 15000 Dalton.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346
【参考文献】
相关期刊论文 前3条
1 宋军,万榕,翁绳美,林旭,林建银;蛇毒cystatin基因合成及其在大肠杆菌的表达[J];福建医科大学学报;2004年03期
2 景志忠,王佩雅,才学鹏;杆状病毒表达系统研究进展及在寄生虫基因工程 疫苗中的应用前景[J];中国兽医科技;2001年03期
3 郭春,James H.McKerrow,Fred E.Cohen;N,N′-双取代脲类克鲁斯氏锥体虫半胱氨酸蛋白酶小分子抑制剂的合成及其活性研究[J];中国药科大学学报;2003年06期
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