【摘要】：目的 探討蛇毒cystatin 在Bac-to-Bac 桿狀病毒表達系統(tǒng)中的表達。 方法 根據(jù)中華眼鏡蛇蛇毒cystatin 蛋白的氨基酸序列合成cystatin 基因的四個片段,通過緩慢退火PCR 的方法將其拼接為完整的cystatin 基因,PCR 擴增蛇毒cystatin 基因,將其克隆到供體質(zhì)粒pFastBacHTc 中,通過轉(zhuǎn)化DH10Bac 菌篩選、鑒定并抽提獲取高純度的重組穿梭載體Bacmid-cystatin,經(jīng)脂質(zhì)體介導將重組穿梭載體轉(zhuǎn)染Sf9 細胞,獲取并擴增重組病毒,空斑分析確定病毒滴度,Western-blot分析適宜的重組融合蛋白表達時間和MOI 值,SDS-PAGE 分析細胞總蛋白,Western-blot 鑒定重組融合蛋白。 結(jié)果 1 構(gòu)建了攜帶蛇毒cystatin基因片段的重組供體質(zhì)粒pFastBacHTc -cystatin,經(jīng)雙酶切鑒定和序列分析證實蛇毒cystatin 基因已正確插入供體質(zhì)粒的多克隆位點。 2 構(gòu)建了攜帶蛇毒cystatin 基因片段的重組穿梭載體Bacmid-cystatin,經(jīng)PCR 鑒定分析證實蛇毒cystatin 基因已正確轉(zhuǎn)座插入穿梭載體的轉(zhuǎn)座接觸位點。 3 確定了重組融合蛋白在Sf9 細胞中表達的最佳時間是感染后96 h,適宜的MOI 值為10。該重組融合蛋白經(jīng)SDS-PAGE、Western-blot 分析鑒定為蛇毒6×His-cystatin 融合蛋白,分子量為15000 道爾頓,與融合蛋白的理論分子量相
[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.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R346

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