人白介素-1受体拮抗剂融合蛋白的构建、表达及生物学活性鉴定
发布时间：2018-08-14 16:03
【摘要】：IL1ra是生物体内的一种天然的蛋白质分子,与IL-1α、IL-1β同为IL-1家族的成员,其本身无任何激动剂的作用,但能特异性地与IL-1受体结合,从而拮抗IL-1的各种生物学效应。一系列的证据证明IL-1在RA发病的慢性炎症过程中具有重要的作用,IL1ra由于可以特异性地抑制IL-1的生物学功能,因此在RA的治疗中备受重视。2001年,由Amgen公司生产的重组人白细胞介素1受体拮抗剂(rhlL1ra,药品名称Kineret)被美国食品与药品管理局(FDA)和欧洲EMEA批准上市,用于治疗类风湿关节炎(RA)。但是,由于ILlra相对分子质量较小,半衰期短,使用过程中患者皮下注射rhlL1ra时,要达到需要的血药浓度和治疗效果需要反复给药,一般每天注射一次,一个疗程为半年,频繁用药加重了患者的身体、心理和经济负担,因此临床上急需研发长效的重组rhIL1rao本研究旨在采用蛋白融合技术延长IL1ra在体内作用的半衰期,设计了以人血清白蛋白(human serum albumin, HSA)为载体蛋白融合IL1ra的融合蛋白。作为血浆的重要成分之一,HSA是许多内源因子和外源药物的载体,正常情况下不易透过肾小球。在血浆中的半衰期长达2周,体内分布极广而且没有酶学和免疫学活性,因而是一种理想的生物活性蛋白载体。通过基因工程技术将HSA基因与IL1ra基因融合,以期获得既不丧失IL1ra生物活性,且能显著地延长其在血浆中半衰期的融合蛋白。这样的融合蛋白能够降低给药频率,发挥与IL1ra相似或更好的治疗作用。 1.融合基因的构建及鉴定 用PCR法从人胎肝cDNA文库获得成熟态血清白蛋白基因,长度大约为1.8kb,再将PCR产物克隆入pGEM-T载体中。再经PCR获得不同修饰的HSA、IL1ra基因,构建HSA与IL1ra的融合基因,并克隆至pPIC9载体中。用PstI、XhoI、EcoRI酶切鉴定重组质粒,再经测序证明已成功构建。 2.毕赤酵母转化及PCR法鉴定 制作毕赤酵母GS115(SMD1168)感受态细胞,将用SalI线性化的重组表达载体转化至毕赤酵母GS115(SMD1168)中。转化后铺于不含组氨酸的RDB平板,30℃培养3天后长出一批毕赤酵母转化子。以重组酵母的基因组为模板,用5'-AOX1和3'-AOX1为引物进行PCR检测,电泳在2.2kb处有一明显条带。结果显示HSA/IL1ra融合基因表达盒已经成功的整合至酵母基因组中。 3.毕赤酵母转化子摇瓶诱导表达及鉴定 随机挑选的多个毕赤酵母转化子,经摇瓶培养,诱导表达72h,电泳鉴定在84kD处出现目标条带的转化子并保存。选取表达量最高的转化子进行发酵培养,取诱导表达12h、24h、48h、72h的发酵液,离心取上清进行SDS-PAGE分析。电泳鉴定发现随着诱导时间的延长,培养基中的分泌蛋白的量增多,在诱导72h时表达量最高,发酵液中HSA-G-IL1ra融合蛋白的浓度约为200mg/L。 4.融合蛋白的纯化 离心收集发酵诱导表达72h的发酵液上清,硫酸铵沉淀,重溶脱盐处理后,经亲和柱、离子交换柱、凝胶柱纯化后得到较纯的融合蛋白。经反相HPLC检测其纯度大于90%。 5. Western Blotting免疫印迹分析 免疫印迹结果分析显示凝胶电泳中的84kD条带既能与兔抗HSA抗血清发生特异性结合,也能与鼠抗人IL1ra单克隆抗体之间发生特异性结合。说明融合蛋白同时具有HSA和IL1ra抗原性。免疫印迹的结果验证了融合蛋白中存在HSA和IL1ra两个结构域。 6.融合蛋白的生物学活性测定 按中国药检所推荐方法用细胞株A375.S2来测定rhIL1ra的生物效价。生物学活性测定结果表明本研究构建表达的HSA/IL1ra融合蛋白具有与ILlra相似的活性,能拮抗IL-1对A375.S2细胞的杀伤作用,并且保护作用呈剂量依赖性。 7.融合蛋白的药代动力学 对小鼠进行皮下注射HSA-G-IL1ra融合蛋白,在不同时间点取血,用ELISA方法对血样进行检测,通过统计学方法得到融合蛋白的药代动力学参数：半衰期为8.125h、是rhIL1ra的17.6倍。清除率为0.182 L/h/kg,是rhIL1ra的0.18倍。证实HSA-G-IL1ra融合蛋白具有长效性。 8.融合蛋白的结构鉴定 通过Western Blotting、N-端氨基酸测序及圆二色谱分析,证实HSA-G-IL1ra融合蛋白具有正确的HSA和ILlra氨基酸序列及结构特征。 综上所述： 本研究成功构建了HSA与ILlra的融合基因,并在毕赤酵母表达体系中获得成功表达；经SDS-PAGE、Western Blotting、N端氨基酸测序及圆二色谱分析等方法证实毕赤酵母表达的HSA/IL1ra融合蛋白具有正确的HSA和ILlra序列及结构；A375.S2细胞IL1杀伤抑制实验证明HSA-G-IL1ra融合蛋白具有与ILlra相同的生物学活性；小鼠的药代动力学实验显示HSA-G-IL1ra融合蛋白比ILlra具有更长的体内的半衰期,降低了清除率,从而证实HSA-G-IL1ra融合蛋白具有长效性。
[Abstract]:IL-1ra is a natural protein molecule in organism. It is a member of the IL-1 family with IL-1a and IL-1beta. It has no agonist effect but can specifically bind to IL-1 receptor and antagonize various biological effects of IL-1. In 2001, recombinant human interleukin-1 receptor antagonist (rhlL1ra, Kineret), produced by Amgen, was approved by the Food and Drug Administration (FDA) and the European EMEA for use in the treatment of rheumatoid arthritis (RA). Because ILlra has small relative molecular weight and short half-life, it is necessary to inject rhlll1ra subcutaneously in order to achieve the desired blood concentration and therapeutic effect. It is usually injected once a day for half a year. Frequent drug use aggravates the physical, psychological and economic burden of patients. Therefore, it is urgent to develop a long-term clinical effect. Recombinant rhIL-1rao The aim of this study was to prolong the half-life of IL-1ra in vivo by using protein fusion technique. The fusion protein of IL-1ra fused with human serum albumin (HSA) was designed. As one of the important components of plasma, HSA is the carrier of many endogenous factors and exogenous drugs, and it is difficult to penetrate normally. The half-life of HSA gene in plasma is as long as 2 weeks. It is widely distributed in vivo and has no enzymatic and immunological activities. Therefore, HSA gene is an ideal carrier of bioactive proteins. Fusion protein. Such fusion protein can reduce the frequency of administration and play a similar or better therapeutic role to IL1ra.
Construction and identification of 1. fusion genes
The mature serum albumin gene was obtained from human fetal liver cDNA library by PCR. The length of the mature serum albumin gene was about 1.8 kb. The PCR products were cloned into pGEM-T vector. The fusion genes of HSA and IL1ra were obtained by PCR and cloned into pPIC9 vector. Successful construction.
2. Pichia pastoris transformation and PCR identification
Pichia pastoris GS115 (SMD1168) receptive cells were prepared and transformed into Pichia pastoris GS115 (SMD1168) by SalI linearized recombinant expression vector. After transformation, a batch of Pichia pastoris transformants were grown on the RDB plate without histidine. The recombinant yeast genome was used as template and 5'-AOX1 and 3'-AOX1 as primers for PCR. The results showed that the HSA/IL1ra fusion gene expression cassette had been successfully integrated into the yeast genome.
Expression and identification of 3. Pichia pastoris transformants in shake flask
Several Pichia pastoris transformants were cultured in shaking flask for 72 hours. The transformants with the highest expression level were identified by electrophoresis and preserved at 84 kD. The fermentation broth with the highest expression level was obtained for 12, 24, 48 and 72 hours and the supernatant was centrifuged for SDS-PAGE analysis. The expression of HSA-G-IL1ra fusion protein was the highest at 72 h after induction. The concentration of HSA-G-IL1ra fusion protein in fermentation broth was about 200 mg/L.
4. purification of fusion protein
The supernatant of fermentation broth induced by centrifugal fermentation for 72 hours was collected, precipitated by ammonium sulfate, and desalted by re-dissolution. After purification by affinity column, ion exchange column and gel column, the purity of the fusion protein was over 90%.
5. Western Blotting Western blot analysis
Western blot analysis showed that the 84 kD band in gel electrophoresis could bind specifically to rabbit anti-HSA serum and mouse anti-human IL 1Ra monoclonal antibody, indicating that the fusion protein possessed both HSA and IL 1Ra antigenicity. Domain.
Determination of biological activity of 6. fusion protein
The bioavailability of rhIL1ra was determined by the cell line A375.S2 recommended by the Chinese Pharmaceutical Inspection Institute. The results of bioassay showed that the HSA/IL1ra fusion protein constructed in this study had similar activity to ILlra and could antagonize the killing effect of IL-1 on A375.S2 cells in a dose-dependent manner.
7. pharmacokinetics of fusion protein
The mice were subcutaneously injected with HSA-G-IL1ra fusion protein, and the blood samples were taken at different time points. The pharmacokinetic parameters of HSA-G-IL1ra fusion protein were obtained by ELISA. The half-life of HSA-G-IL1ra fusion protein was 8.125 h, 17.6 times that of rhIL-1ra. The clearance rate was 0.182 L/h/kg, 0.18 times that of rhIL-1ra fusion protein. Long term.
Structural identification of 8. fusion protein
Western Blotting, N-terminal amino acid sequencing and circular dichroism analysis confirmed that the fusion protein of HSA-G-IL1ra had correct sequence and structural characteristics of HSA and ILlra amino acids.
In summary:
The fusion gene of HSA and ILlra was successfully constructed and successfully expressed in Pichia pastoris expression system. The fusion protein of HSA/IL1ra expressed in Pichia pastoris was confirmed to have the correct sequence and structure of HSA and ILlra by SDS-PAGE, Western Blotting, N-terminal amino acid sequencing and circular dichroism analysis. Inhibitory test showed that HSA-G-IL1ra fusion protein had the same biological activity as ILlra, and the pharmacokinetic test in mice showed that HSA-G-IL1ra fusion protein had longer half-life in vivo and lower clearance rate than ILlra, thus confirming the long-term efficacy of HSA-G-IL1ra fusion protein.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.1
本文编号：2183389
[Abstract]:IL-1ra is a natural protein molecule in organism. It is a member of the IL-1 family with IL-1a and IL-1beta. It has no agonist effect but can specifically bind to IL-1 receptor and antagonize various biological effects of IL-1. In 2001, recombinant human interleukin-1 receptor antagonist (rhlL1ra, Kineret), produced by Amgen, was approved by the Food and Drug Administration (FDA) and the European EMEA for use in the treatment of rheumatoid arthritis (RA). Because ILlra has small relative molecular weight and short half-life, it is necessary to inject rhlll1ra subcutaneously in order to achieve the desired blood concentration and therapeutic effect. It is usually injected once a day for half a year. Frequent drug use aggravates the physical, psychological and economic burden of patients. Therefore, it is urgent to develop a long-term clinical effect. Recombinant rhIL-1rao The aim of this study was to prolong the half-life of IL-1ra in vivo by using protein fusion technique. The fusion protein of IL-1ra fused with human serum albumin (HSA) was designed. As one of the important components of plasma, HSA is the carrier of many endogenous factors and exogenous drugs, and it is difficult to penetrate normally. The half-life of HSA gene in plasma is as long as 2 weeks. It is widely distributed in vivo and has no enzymatic and immunological activities. Therefore, HSA gene is an ideal carrier of bioactive proteins. Fusion protein. Such fusion protein can reduce the frequency of administration and play a similar or better therapeutic role to IL1ra.
Construction and identification of 1. fusion genes
The mature serum albumin gene was obtained from human fetal liver cDNA library by PCR. The length of the mature serum albumin gene was about 1.8 kb. The PCR products were cloned into pGEM-T vector. The fusion genes of HSA and IL1ra were obtained by PCR and cloned into pPIC9 vector. Successful construction.
2. Pichia pastoris transformation and PCR identification
Pichia pastoris GS115 (SMD1168) receptive cells were prepared and transformed into Pichia pastoris GS115 (SMD1168) by SalI linearized recombinant expression vector. After transformation, a batch of Pichia pastoris transformants were grown on the RDB plate without histidine. The recombinant yeast genome was used as template and 5'-AOX1 and 3'-AOX1 as primers for PCR. The results showed that the HSA/IL1ra fusion gene expression cassette had been successfully integrated into the yeast genome.
Expression and identification of 3. Pichia pastoris transformants in shake flask
Several Pichia pastoris transformants were cultured in shaking flask for 72 hours. The transformants with the highest expression level were identified by electrophoresis and preserved at 84 kD. The fermentation broth with the highest expression level was obtained for 12, 24, 48 and 72 hours and the supernatant was centrifuged for SDS-PAGE analysis. The expression of HSA-G-IL1ra fusion protein was the highest at 72 h after induction. The concentration of HSA-G-IL1ra fusion protein in fermentation broth was about 200 mg/L.
4. purification of fusion protein
The supernatant of fermentation broth induced by centrifugal fermentation for 72 hours was collected, precipitated by ammonium sulfate, and desalted by re-dissolution. After purification by affinity column, ion exchange column and gel column, the purity of the fusion protein was over 90%.
5. Western Blotting Western blot analysis
Western blot analysis showed that the 84 kD band in gel electrophoresis could bind specifically to rabbit anti-HSA serum and mouse anti-human IL 1Ra monoclonal antibody, indicating that the fusion protein possessed both HSA and IL 1Ra antigenicity. Domain.
Determination of biological activity of 6. fusion protein
The bioavailability of rhIL1ra was determined by the cell line A375.S2 recommended by the Chinese Pharmaceutical Inspection Institute. The results of bioassay showed that the HSA/IL1ra fusion protein constructed in this study had similar activity to ILlra and could antagonize the killing effect of IL-1 on A375.S2 cells in a dose-dependent manner.
7. pharmacokinetics of fusion protein
The mice were subcutaneously injected with HSA-G-IL1ra fusion protein, and the blood samples were taken at different time points. The pharmacokinetic parameters of HSA-G-IL1ra fusion protein were obtained by ELISA. The half-life of HSA-G-IL1ra fusion protein was 8.125 h, 17.6 times that of rhIL-1ra. The clearance rate was 0.182 L/h/kg, 0.18 times that of rhIL-1ra fusion protein. Long term.
Structural identification of 8. fusion protein
Western Blotting, N-terminal amino acid sequencing and circular dichroism analysis confirmed that the fusion protein of HSA-G-IL1ra had correct sequence and structural characteristics of HSA and ILlra amino acids.
In summary:
The fusion gene of HSA and ILlra was successfully constructed and successfully expressed in Pichia pastoris expression system. The fusion protein of HSA/IL1ra expressed in Pichia pastoris was confirmed to have the correct sequence and structure of HSA and ILlra by SDS-PAGE, Western Blotting, N-terminal amino acid sequencing and circular dichroism analysis. Inhibitory test showed that HSA-G-IL1ra fusion protein had the same biological activity as ILlra, and the pharmacokinetic test in mice showed that HSA-G-IL1ra fusion protein had longer half-life in vivo and lower clearance rate than ILlra, thus confirming the long-term efficacy of HSA-G-IL1ra fusion protein.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.1
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