毕赤酵母表达犬长效干扰素融合蛋白的研究
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摘要
干扰素有着抗病毒、抗细胞增殖、免疫调节等多种极具医疗价值的功效。而体内半衰期短的缺点却严重限制着它的临床应用。人医临床上常用人血清白蛋白与干扰素的融合蛋白方式来延长干扰素半衰期,而动物临床上却没有这种融合蛋白长效干扰素开发应用的报道。同时现有的动物干扰素制品大都采用原核表达方式表达生产,其产品在活性和成本方面都不理想。而酵母表达系统是一种真核表达系统,其所表达的蛋白产物修饰合理,活性高。毕赤酵母的分泌表达会把目的蛋白分泌到培养液上清中,而其它杂质蛋白很少,更加有利于目的蛋白的分离纯化,有利于降低目的蛋白的生产成本。所以本实验用毕赤酵母表达系统分泌表达了犬血清白蛋白与犬alpha干扰素的融合蛋白,以此来探索应用血清白蛋白融合蛋白技术来制备犬长效干扰素的可行性。
实验共分四个主体部分,首先是表达质粒的构建,用反转录PCR法从犬组织器官中扩增出犬血清白蛋白cDNA,再对犬血清白蛋白cDNA进行修饰,首先去掉终止密码子的同时在5'端加入EcoR Ⅰ酶切位点,在3'端加入柔性Linker碱基链和BamH Ⅰ酶切位点,再对改造后的cDNA进行点突变,以此来消除不利于质粒构建的两个Bg1Ⅱ酶切位点；在犬alpha干扰素方面,为了提高酵母的表达效率,对犬alpha干扰素进行了密码子优化,优化合成过程中去除了犬alpha干扰素的起始密码子,并在其5'端引入Bg1Ⅱ酶切位点,在3'端引入EcoR Ⅰ酶切位点,利用pUC18载体为中间载体把两段经过修饰的基因以血清白蛋白—干扰素的方式融合相连,再把融合基因转入毕赤酵母表达载体pPIC3.5K中,构建犬长效干扰素融合蛋白毕赤酵母分泌表达质粒pPIC3.5K-SI,在构建过程中以犬血清白蛋白的天然信号肽为毕赤酵母分泌表达的信号肽,解决了酵母信号肽可能切割不完全的情况。
在第二部分实验中,采用原生质球转化法和电转化法两种方法将表达质粒转入酵母,对两种转化方法进行了对比,为实验室相关实验的后续工作提供了参考。转化后对多拷贝阳性克隆进行了筛选,采用了组氨酸营养缺陷培养基、Mut基因型鉴定培养基、G418抗性梯度筛选和基因组PCR等多种方法筛选出多拷贝的阳性克隆重组酵母菌株。
第三部分实验中,用以甘油为碳源的培养基扩增重组酵母,用以甲醇为碳源的培养基诱导表达目的蛋白,并分离纯化目的蛋白。表达过程中确定了最佳的诱导表达时间为96小时。表达后用超滤浓缩法、亲和层析法和脱盐层析法纯化目的蛋白,根据纯化后的SDS-PAGE电泳结果,得到了纯化的犬长效干扰素融合蛋白,浓度为252μg/ml。
第四部分,对得到的犬长效干扰素进行了体外、体内的生物活性分析。用Wish-VSV法检测了该融合蛋白体外的生物学活性,其体外生物学活性达到了2.56×105IU/ml。用荧光定量PCR法检测了融合蛋白体内激发的相关细胞因子2'5'—寡腺苷酸合成酶的转录水平,以此来判断融合蛋白动物体内的半衰期和生物学活性情况,其药效动力学显示在第8h时出现峰值,融合蛋白药效持续近10天。
本实验用动物血清白蛋白与动物干扰素相融合的方式开展动物长效干扰素的开发研究,所制备的犬长效融合蛋白干扰素在动物体外、体内均有良好的生物学活性,同时有较长的体内半衰期,为探索开发低成本、高功效的动物长效干扰素的研究奠定了基础。
Interferon(IFN) has many functions in antivirus, anti-tumor and immunoregulation. But the short half-life of IFN restricts its clinical application. In human clinic, the fusion protein of Human serum albumin(HSA) and IFN was used to prolong the half-life of IFN, but there is no report of this kind of fusion protein of IFN in animals. Now most of animal's IFN are expressed in prokaryotic organism, and their activity and cost are not reasonable. The yeast expression system is eukaryotic and the protein expressed is modified and has high activity. The secretive expression of Pichia pastoris can secrete protein in supernatant of culture with less other proteins. This expression pattern is beneficial to purification and reducing cost of objective protein. In this experiment I expressed the fusion protein of canine serum albumin(CSA) and alpha interferon(IFN-a), and explored the possibilities of long-activity IFN with this method.
The study consists of four parts. First, construction of expression plasmid. I amplified the CSA's cDNA from canine tissue using RT-PCR and modified the cDNA. The EcoR I cutting site is inserted into the5' end of cDNA after removing the terminator codon and the linker and BamH Ⅰ cutting site are inserted into3' end. The recombinated cDNA is mutated to remove two Bgl Ⅱ cutting sites that unfit for the construction of plasmid. For increasing the expression of yeast, canine IFN-a is optimized and synthesized. In optimized process the initiation codon of IFN-α was removed, the Bgl Ⅱ cutting site is inserted into5'end. and the EcoR I cutting site is inserted into3' end. The recombinated DNA is connected as CSA-IFN using pUC18vector. Transforming the fusion DNA into pPIC3.5K which is the vector of Pichia pastoris constructs pPIC3.5K-SI that is the fusion protein's secreted expressive plasmid of canine long-activity IFN within Pichia pastoris. CSA's inherent signal peptide was used as secreted expression signal peptide of Pichia pastoris to avoid non-completed remove of yeast'signal peptide.
In second part of the experiment, the expressive plasmid is transformed into yeast using the methods of protoplasts-transformation and electro-transformation. Comparative analyzing these two methods that provides the references to the next study of laboratory. After transforming, the positive clones of recombinationed yeast were screened by His culture, Mut culture, G418culture and gene PCR.
In third part of experiment, the recombinationed yeast was amplified in culture whose carbon source is glycerin. The objective protein was expressed inductively by culture whose carbon source is methanol, and was isolated and purified. In expression process, the best inductive time was determined as96h. The objective protein was purified by the methods of ultrafiltration, affinity chromatography and desalination. Using SDS-PAGE, the fusion protein of canine long-activity IFN "s concentration was determined as252μg/ml.
In fourth part of the experiment, the activity of canine long-activity IFN was detected in vitro and in vivo. The activity in vitro of the fusion protein is detected by using Wish-VSV, and the result is2.56×105IU/ml. The transcriptional level of2'5'-OAS which is the fusion associated cytokines which detected using RTFQ PCR. The Pharmacodynamic effects of the fusion protein in vivo reach the peak at8h, and the duration is close to10days.
The experiment focus on animal long-activity IFN using the fusion protein of animal serum albumin and IFN. The objective protein has good activity in vitro and in vivo, and has long half-life in vivo. This experiment lays a foundation for the research in developing animal long-activity IFN that has low cost and good function.
实验共分四个主体部分,首先是表达质粒的构建,用反转录PCR法从犬组织器官中扩增出犬血清白蛋白cDNA,再对犬血清白蛋白cDNA进行修饰,首先去掉终止密码子的同时在5'端加入EcoR Ⅰ酶切位点,在3'端加入柔性Linker碱基链和BamH Ⅰ酶切位点,再对改造后的cDNA进行点突变,以此来消除不利于质粒构建的两个Bg1Ⅱ酶切位点；在犬alpha干扰素方面,为了提高酵母的表达效率,对犬alpha干扰素进行了密码子优化,优化合成过程中去除了犬alpha干扰素的起始密码子,并在其5'端引入Bg1Ⅱ酶切位点,在3'端引入EcoR Ⅰ酶切位点,利用pUC18载体为中间载体把两段经过修饰的基因以血清白蛋白—干扰素的方式融合相连,再把融合基因转入毕赤酵母表达载体pPIC3.5K中,构建犬长效干扰素融合蛋白毕赤酵母分泌表达质粒pPIC3.5K-SI,在构建过程中以犬血清白蛋白的天然信号肽为毕赤酵母分泌表达的信号肽,解决了酵母信号肽可能切割不完全的情况。
在第二部分实验中,采用原生质球转化法和电转化法两种方法将表达质粒转入酵母,对两种转化方法进行了对比,为实验室相关实验的后续工作提供了参考。转化后对多拷贝阳性克隆进行了筛选,采用了组氨酸营养缺陷培养基、Mut基因型鉴定培养基、G418抗性梯度筛选和基因组PCR等多种方法筛选出多拷贝的阳性克隆重组酵母菌株。
第三部分实验中,用以甘油为碳源的培养基扩增重组酵母,用以甲醇为碳源的培养基诱导表达目的蛋白,并分离纯化目的蛋白。表达过程中确定了最佳的诱导表达时间为96小时。表达后用超滤浓缩法、亲和层析法和脱盐层析法纯化目的蛋白,根据纯化后的SDS-PAGE电泳结果,得到了纯化的犬长效干扰素融合蛋白,浓度为252μg/ml。
第四部分,对得到的犬长效干扰素进行了体外、体内的生物活性分析。用Wish-VSV法检测了该融合蛋白体外的生物学活性,其体外生物学活性达到了2.56×105IU/ml。用荧光定量PCR法检测了融合蛋白体内激发的相关细胞因子2'5'—寡腺苷酸合成酶的转录水平,以此来判断融合蛋白动物体内的半衰期和生物学活性情况,其药效动力学显示在第8h时出现峰值,融合蛋白药效持续近10天。
本实验用动物血清白蛋白与动物干扰素相融合的方式开展动物长效干扰素的开发研究,所制备的犬长效融合蛋白干扰素在动物体外、体内均有良好的生物学活性,同时有较长的体内半衰期,为探索开发低成本、高功效的动物长效干扰素的研究奠定了基础。
Interferon(IFN) has many functions in antivirus, anti-tumor and immunoregulation. But the short half-life of IFN restricts its clinical application. In human clinic, the fusion protein of Human serum albumin(HSA) and IFN was used to prolong the half-life of IFN, but there is no report of this kind of fusion protein of IFN in animals. Now most of animal's IFN are expressed in prokaryotic organism, and their activity and cost are not reasonable. The yeast expression system is eukaryotic and the protein expressed is modified and has high activity. The secretive expression of Pichia pastoris can secrete protein in supernatant of culture with less other proteins. This expression pattern is beneficial to purification and reducing cost of objective protein. In this experiment I expressed the fusion protein of canine serum albumin(CSA) and alpha interferon(IFN-a), and explored the possibilities of long-activity IFN with this method.
The study consists of four parts. First, construction of expression plasmid. I amplified the CSA's cDNA from canine tissue using RT-PCR and modified the cDNA. The EcoR I cutting site is inserted into the5' end of cDNA after removing the terminator codon and the linker and BamH Ⅰ cutting site are inserted into3' end. The recombinated cDNA is mutated to remove two Bgl Ⅱ cutting sites that unfit for the construction of plasmid. For increasing the expression of yeast, canine IFN-a is optimized and synthesized. In optimized process the initiation codon of IFN-α was removed, the Bgl Ⅱ cutting site is inserted into5'end. and the EcoR I cutting site is inserted into3' end. The recombinated DNA is connected as CSA-IFN using pUC18vector. Transforming the fusion DNA into pPIC3.5K which is the vector of Pichia pastoris constructs pPIC3.5K-SI that is the fusion protein's secreted expressive plasmid of canine long-activity IFN within Pichia pastoris. CSA's inherent signal peptide was used as secreted expression signal peptide of Pichia pastoris to avoid non-completed remove of yeast'signal peptide.
In second part of the experiment, the expressive plasmid is transformed into yeast using the methods of protoplasts-transformation and electro-transformation. Comparative analyzing these two methods that provides the references to the next study of laboratory. After transforming, the positive clones of recombinationed yeast were screened by His culture, Mut culture, G418culture and gene PCR.
In third part of experiment, the recombinationed yeast was amplified in culture whose carbon source is glycerin. The objective protein was expressed inductively by culture whose carbon source is methanol, and was isolated and purified. In expression process, the best inductive time was determined as96h. The objective protein was purified by the methods of ultrafiltration, affinity chromatography and desalination. Using SDS-PAGE, the fusion protein of canine long-activity IFN "s concentration was determined as252μg/ml.
In fourth part of the experiment, the activity of canine long-activity IFN was detected in vitro and in vivo. The activity in vitro of the fusion protein is detected by using Wish-VSV, and the result is2.56×105IU/ml. The transcriptional level of2'5'-OAS which is the fusion associated cytokines which detected using RTFQ PCR. The Pharmacodynamic effects of the fusion protein in vivo reach the peak at8h, and the duration is close to10days.
The experiment focus on animal long-activity IFN using the fusion protein of animal serum albumin and IFN. The objective protein has good activity in vitro and in vivo, and has long half-life in vivo. This experiment lays a foundation for the research in developing animal long-activity IFN that has low cost and good function.
引文
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