人白介素-1受體拮抗劑融合蛋白的構(gòu)建、表達(dá)及生物學(xué)活性鑒定
發(fā)布時(shí)間：2018-08-14 16:03
【摘要】：IL1ra是生物體內(nèi)的一種天然的蛋白質(zhì)分子,與IL-1α、IL-1β同為IL-1家族的成員,其本身無(wú)任何激動(dòng)劑的作用,但能特異性地與IL-1受體結(jié)合,從而拮抗IL-1的各種生物學(xué)效應(yīng)。一系列的證據(jù)證明IL-1在RA發(fā)病的慢性炎癥過(guò)程中具有重要的作用,IL1ra由于可以特異性地抑制IL-1的生物學(xué)功能,因此在RA的治療中備受重視。2001年,由Amgen公司生產(chǎn)的重組人白細(xì)胞介素1受體拮抗劑(rhlL1ra,藥品名稱Kineret)被美國(guó)食品與藥品管理局(FDA)和歐洲EMEA批準(zhǔn)上市,用于治療類風(fēng)濕關(guān)節(jié)炎(RA)。但是,由于ILlra相對(duì)分子質(zhì)量較小,半衰期短,使用過(guò)程中患者皮下注射rhlL1ra時(shí),要達(dá)到需要的血藥濃度和治療效果需要反復(fù)給藥,一般每天注射一次,一個(gè)療程為半年,頻繁用藥加重了患者的身體、心理和經(jīng)濟(jì)負(fù)擔(dān),因此臨床上急需研發(fā)長(zhǎng)效的重組rhIL1rao本研究旨在采用蛋白融合技術(shù)延長(zhǎng)IL1ra在體內(nèi)作用的半衰期,設(shè)計(jì)了以人血清白蛋白(human serum albumin, HSA)為載體蛋白融合IL1ra的融合蛋白。作為血漿的重要成分之一,HSA是許多內(nèi)源因子和外源藥物的載體,正常情況下不易透過(guò)腎小球。在血漿中的半衰期長(zhǎng)達(dá)2周,體內(nèi)分布極廣而且沒(méi)有酶學(xué)和免疫學(xué)活性,因而是一種理想的生物活性蛋白載體。通過(guò)基因工程技術(shù)將HSA基因與IL1ra基因融合,以期獲得既不喪失IL1ra生物活性,且能顯著地延長(zhǎng)其在血漿中半衰期的融合蛋白。這樣的融合蛋白能夠降低給藥頻率,發(fā)揮與IL1ra相似或更好的治療作用。 1.融合基因的構(gòu)建及鑒定 用PCR法從人胎肝cDNA文庫(kù)獲得成熟態(tài)血清白蛋白基因,長(zhǎng)度大約為1.8kb,再將PCR產(chǎn)物克隆入pGEM-T載體中。再經(jīng)PCR獲得不同修飾的HSA、IL1ra基因,構(gòu)建HSA與IL1ra的融合基因,并克隆至pPIC9載體中。用PstI、XhoI、EcoRI酶切鑒定重組質(zhì)粒,再經(jīng)測(cè)序證明已成功構(gòu)建。 2.畢赤酵母轉(zhuǎn)化及PCR法鑒定 制作畢赤酵母GS115(SMD1168)感受態(tài)細(xì)胞,將用SalI線性化的重組表達(dá)載體轉(zhuǎn)化至畢赤酵母GS115(SMD1168)中。轉(zhuǎn)化后鋪于不含組氨酸的RDB平板,30℃培養(yǎng)3天后長(zhǎng)出一批畢赤酵母轉(zhuǎn)化子。以重組酵母的基因組為模板,用5'-AOX1和3'-AOX1為引物進(jìn)行PCR檢測(cè),電泳在2.2kb處有一明顯條帶。結(jié)果顯示HSA/IL1ra融合基因表達(dá)盒已經(jīng)成功的整合至酵母基因組中。 3.畢赤酵母轉(zhuǎn)化子搖瓶誘導(dǎo)表達(dá)及鑒定 隨機(jī)挑選的多個(gè)畢赤酵母轉(zhuǎn)化子,經(jīng)搖瓶培養(yǎng),誘導(dǎo)表達(dá)72h,電泳鑒定在84kD處出現(xiàn)目標(biāo)條帶的轉(zhuǎn)化子并保存。選取表達(dá)量最高的轉(zhuǎn)化子進(jìn)行發(fā)酵培養(yǎng),取誘導(dǎo)表達(dá)12h、24h、48h、72h的發(fā)酵液,離心取上清進(jìn)行SDS-PAGE分析。電泳鑒定發(fā)現(xiàn)隨著誘導(dǎo)時(shí)間的延長(zhǎng),培養(yǎng)基中的分泌蛋白的量增多,在誘導(dǎo)72h時(shí)表達(dá)量最高,發(fā)酵液中HSA-G-IL1ra融合蛋白的濃度約為200mg/L。 4.融合蛋白的純化 離心收集發(fā)酵誘導(dǎo)表達(dá)72h的發(fā)酵液上清,硫酸銨沉淀,重溶脫鹽處理后,經(jīng)親和柱、離子交換柱、凝膠柱純化后得到較純的融合蛋白。經(jīng)反相HPLC檢測(cè)其純度大于90%。 5. Western Blotting免疫印跡分析 免疫印跡結(jié)果分析顯示凝膠電泳中的84kD條帶既能與兔抗HSA抗血清發(fā)生特異性結(jié)合,也能與鼠抗人IL1ra單克隆抗體之間發(fā)生特異性結(jié)合。說(shuō)明融合蛋白同時(shí)具有HSA和IL1ra抗原性。免疫印跡的結(jié)果驗(yàn)證了融合蛋白中存在HSA和IL1ra兩個(gè)結(jié)構(gòu)域。 6.融合蛋白的生物學(xué)活性測(cè)定 按中國(guó)藥檢所推薦方法用細(xì)胞株A375.S2來(lái)測(cè)定rhIL1ra的生物效價(jià)。生物學(xué)活性測(cè)定結(jié)果表明本研究構(gòu)建表達(dá)的HSA/IL1ra融合蛋白具有與ILlra相似的活性,能拮抗IL-1對(duì)A375.S2細(xì)胞的殺傷作用,并且保護(hù)作用呈劑量依賴性。 7.融合蛋白的藥代動(dòng)力學(xué) 對(duì)小鼠進(jìn)行皮下注射HSA-G-IL1ra融合蛋白,在不同時(shí)間點(diǎn)取血,用ELISA方法對(duì)血樣進(jìn)行檢測(cè),通過(guò)統(tǒng)計(jì)學(xué)方法得到融合蛋白的藥代動(dòng)力學(xué)參數(shù)：半衰期為8.125h、是rhIL1ra的17.6倍。清除率為0.182 L/h/kg,是rhIL1ra的0.18倍。證實(shí)HSA-G-IL1ra融合蛋白具有長(zhǎng)效性。 8.融合蛋白的結(jié)構(gòu)鑒定 通過(guò)Western Blotting、N-端氨基酸測(cè)序及圓二色譜分析,證實(shí)HSA-G-IL1ra融合蛋白具有正確的HSA和ILlra氨基酸序列及結(jié)構(gòu)特征。 綜上所述： 本研究成功構(gòu)建了HSA與ILlra的融合基因,并在畢赤酵母表達(dá)體系中獲得成功表達(dá)；經(jīng)SDS-PAGE、Western Blotting、N端氨基酸測(cè)序及圓二色譜分析等方法證實(shí)畢赤酵母表達(dá)的HSA/IL1ra融合蛋白具有正確的HSA和ILlra序列及結(jié)構(gòu)；A375.S2細(xì)胞IL1殺傷抑制實(shí)驗(yàn)證明HSA-G-IL1ra融合蛋白具有與ILlra相同的生物學(xué)活性；小鼠的藥代動(dòng)力學(xué)實(shí)驗(yàn)顯示HSA-G-IL1ra融合蛋白比ILlra具有更長(zhǎng)的體內(nèi)的半衰期,降低了清除率,從而證實(shí)HSA-G-IL1ra融合蛋白具有長(zhǎng)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392.1
本文編號(hào)：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392.1
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