CN109180819A - 一种胰岛素衍生物的制备方法 - Google Patents
一种胰岛素衍生物的制备方法 Download PDFInfo
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- CN109180819A CN109180819A CN201811074273.5A CN201811074273A CN109180819A CN 109180819 A CN109180819 A CN 109180819A CN 201811074273 A CN201811074273 A CN 201811074273A CN 109180819 A CN109180819 A CN 109180819A
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- transferrins
- fusion
- proinsulin
- edta
- nacl
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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Landscapes
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种胰岛素衍生物的制备方法,属于生物医药技术领域。采用本发明的融合体,有效促进胰岛素原蛋白在机体内的活性最大化,本发明还提供一套优化的蛋白体外变性复性反应体系,以使无活性的转铁蛋白‑胰岛素融合蛋白转变为有活性的转铁蛋白‑胰岛素融合蛋白,为转铁蛋白‑胰岛素融合的高效、低成本工业化生产提供一种新的、更为简化的工艺流程。
Description
技术领域
本发明涉及一种胰岛素衍生物的制备方法,属于生物医药技术领域。
背景技术
糖尿病是一类以血糖偏高为主要症状的代谢性疾病,主要分为I型和II型。I型糖尿病(Type1 Diabetes Mellitus,T1DM),又名胰岛素依赖型糖尿病,由自身免疫紊乱导致的胰岛β-细胞缺失所引起,多发生在儿童和青少年。II型糖尿病则由于外周组织对胰岛素的不敏感性(胰岛素抗性),而胰岛β-细胞又分泌不足而引起的一种胰岛素相对不足的一种状况。据世界卫生组织数据显示,全球目前共有糖尿病患者4亿2千万,另外还有1.6亿人处于糖尿病前期。糖尿病已成为严重危害人类健康的重大疾病之一。
胰岛素是由人体胰岛β细胞产生的,用于调节机体碳水化合物和脂肪代谢平衡的重要激素。在未来的20年,全球所需要的胰岛素消费将从目前的120亿美元增加至540亿美元。
人血清转铁蛋白(Human Transferrin,hTF)是一种非血红素结合铁的β-球蛋白,人转铁蛋白具有N端和C端两个同源性极高的结构域,能够与Fe3+紧密结合,由肝脏合成并最终分泌到血浆内。由于转铁蛋白能够通过转铁蛋白受体介导的方式在血清中反复的循环,稳定性很高,而且机体内各组织普遍分布有丰富的转铁蛋白受体,有学者曾提出将转铁蛋白作为一种蛋白标签与胰岛素分子进行融合来生产新的胰岛素衍生物。这种以转铁蛋白融合为基础的胰岛素衍生物至少有以下几个优点:(1)在血液中半衰期长,稳定性高,所以降糖效果好;(2)转铁蛋白受体遍布机体各组织,故其生物活性无组织局限性;(3)转铁蛋白为一天然血清蛋白,不会引起免疫或其它不良反应;(4)作为一种重组蛋白,生产工艺较其它胰岛素衍生物更为简单;(5)胰岛素原转铁蛋白的融合体,能够直接利用转铁蛋白受体介导的方式进入肝脏,并转变为有活性的胰岛素,故不需要在体外进行C肽的切割。因此,转铁蛋白-胰岛素融合蛋白具有很大的临床应用潜力。
转铁蛋白-胰岛素融合蛋白可以通过应用转基因技术,在培养的高等生物细胞中进行生产。然而,利用培养的高等生物细胞进行转铁蛋白-胰岛素融合蛋白生产,其产量相对较低,操作难度较大、成本较高。而大肠杆菌则具有生长快速,生长周期短;易在分子水平上进行改造和修饰;培养基便宜;蛋白产量高的优势。因此,在胰岛素及其衍生物生产工业上,大肠杆菌是目前应用最为广泛的表达系统。
大肠杆菌表达系统虽然广泛应用于胰岛素生产工业上,但存在着严重的技术瓶颈问题。大肠杆菌缺少真核细胞具有的蛋白翻译后的修饰能力,因此表达出来的目标蛋白通常会以不可溶的、无活性的包涵体形式存在,然后对来自包涵体的不溶蛋白进行复杂的变性和复性操作,使目标蛋白变为有活性。采用这样的方式制备得到的胰岛素衍生物活性较低,并且稳定性有待进一步提升。
发明内容
为解决上述技术问题,本发明提供一种胰岛素原-转铁蛋白融合体,所述胰岛素原-转铁蛋白融合体融合表达了核苷酸序列如SEQ ID NO.1所示的胰岛素蛋白和核苷酸序列如SEQ ID NO.2所示的转铁蛋白;所述胰岛素蛋白和转铁蛋白之间通过核苷酸序列如SEQID NO.3所示的linker连接。
本发明的第二个目的是提供一种所述胰岛素原-转铁蛋白融合体的编码基因。
本发明的第三个目的是提供一种携带上述编码基因的表达载体。
在本发明的一种实施方式中,所述表达载体为pET系列或pQE系列。
本发明的第四个目的是提供一种表达所述胰岛素原-转铁蛋白融合体的重组菌。
在本发明的一种实施方式中,所述重组菌的宿主细胞为大肠杆菌。
本发明的第五个目的是提供一种所述重组菌的构建方法,包括如下步骤:
(1)基因合成胰岛素原-转铁蛋白融合体的编码基因;
(2)将上述编码基因通过连接酶插入pET28a载体中;
(3)将步骤(2)中插入编码基因的载体导入大肠杆菌BL21感受态细胞中,得到所述的重组菌。
本发明的第六个目的是提供一种所述的胰岛素原-转铁蛋白融合体的变性、复性方法,包括如下步骤:
(1)将包涵体先用洗涤缓冲液1和洗涤缓冲液2各洗涤两次,每次洗涤0.5~1.5h;所述的洗涤缓冲液1为50mM Tris,50mM NaCl,1mM EDTA,1%TritonX100 pH 8.5;所述的洗涤缓冲液2为2M尿素,50mM Tris,50mM NaCl,1mM EDTA,1%TritonX100 pH 8.5;
(2)将步骤(1)洗涤后的包涵体用变性液进行溶解;所述变性液为8M尿素,50mMTris,50mM NaCl,1mM EDTA,50mM DTT,pH 9.5;
(3)将步骤(2)溶解的包涵体依次在复性液1、复性液2、复性液3、复性液4和复性液5中进行梯度透析复性,每级透析10~14h;所述复性液1为6M尿素,50mM TrisHCl,50mMNaCl,1mM EDTA,1.0mM GSH,0.1mM GSSG,pH 9.5;所述复性液2为4M尿素,50mM TrisHCl,50mM NaCl,1mM EDTA,1.0mM GSH,0.1mM
GSSG,pH 9;所述复性液3为2M尿素,50mM TrisHCl,50mM NaCl,1mM EDTA,1.0mMGSH,0.1mM GSSG,pH 8.5;所述复性液4为1M尿素,50mM TrisHCl,50mM NaCl,1mM EDTA,1.0mM GSH,0.1mM GSSG,pH 8.0;所述复性液5为0.01M PBS pH 7.5。
在本发明的一种实施方式中,所述透析是在4℃以下进行透析。
本发明的第七个目的是提供所述的胰岛素原-转铁蛋白融合体在制备降血糖药物或保健品中的应用。
本发明的有益效果是:采用本发明的融合体,有效促进胰岛素原蛋白在机体内的活性最大化,本发明还提供一套优化的蛋白体外变性复性反应体系,以使无活性的转铁蛋白-胰岛素融合蛋白转变为有活性的转铁蛋白-胰岛素融合蛋白,为转铁蛋白-胰岛素融合的高效、低成本工业化生产提供一种新的、更为简化的工艺流程。
附图说明
图1为胰岛素原-转铁蛋白融合基因表达载体示意图;
图2为IPTG诱导ProINS-Tf重组蛋白表达结果;A:10%SDS-PAGE分析ProINS-Tf的表达情况;B:Western blot分析ProINS-Tf的表达情况(M:蛋白marker;lane 1:pET28a/BL21的细胞裂解产物;lane 2:未诱导的pET28a-ProINS-Tf/BL21的细胞裂解产物;lane 3-6分别为1mM IPTG诱导2h、4h、6h和8h的pET28a-ProINS-Tf/BL21的细胞裂解产物);
图3为ProINS-Tf重组蛋白的可溶性分析;M:蛋白marker;lane 1:未诱导pET28a-ProINS-Tf/BL21的细胞裂解产物;lane 2:pET28a-ProINS-Tf/BL21诱导8h的细胞裂解产物;lane 3:pET28a-ProINS-Tf/BL21裂解沉淀;lane 4:pET28a-ProINS-Tf/BL21裂解上清;
图4为IPTG诱导ProINS-NTf重组蛋白表达结果;A:10%SDS-PAGE分析ProINS-NTf的表达情况;B:Western blot分析ProINS-NTf的表达情况(M:蛋白marker;lane 1:pET28a/BL21的细胞裂解产物;lane 2:未诱导的pET28a-ProINS-NTf/BL21的细胞裂解产物;lane3-6分别为1mM IPTG诱导2h、4h、6h和8h的pET28a-ProINS-NTf/BL21的细胞裂解产物);
图5为ProINS-NTf重组蛋白的可溶性分析;M:蛋白marker;lane 1:未诱导pET28a-ProINS-NTf/BL21的细胞裂解产物;lane 2:pET28a-ProINS-NTf/BL21诱导8h的细胞裂解产物;lane 3:pET28a-ProINS-NTf/BL21裂解沉淀;lane 4:pET28a-ProINS-NTf/BL21裂解上清;
图6为ProINS-Tf与ProINS-NTf复性对比;M:蛋白marker;lane 1:未诱导pET28a-ProINS-Tf/BL21的细胞裂解产物;lane 2:未诱导pET28a-ProINS-NTf/BL21的细胞裂解产物;lane 3:pET28a-ProINS-Tf/BL21诱导8h的细胞裂解产物;lane4:pET28a-ProINS-NTf/BL21诱导8h的细胞裂解产物;lane 5:ProINS-Tf包涵体;lane 6:ProINS-NTf包涵体;lane7:复性后可溶ProINS-Tf;lane8:复性后可溶ProINS-NTf;
图7为重组蛋白在1型糖尿病小鼠降血糖效果。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1:胰岛素原-转铁蛋白融合基因的构建
以pET28a为基础载体,我们设计并构建了两种胰岛素原表达质粒:pET28a-Proinsulin-Tf(胰岛素原-转铁蛋白)和pET28a-Proinsulin-NTf(胰岛素原-转铁蛋白氨端)(图1),这两个质粒表达的产物分别是胰岛原-转铁蛋白全长,胰岛原-转铁蛋白氨端的融合蛋白。在胰岛原序列与转铁蛋白序列之间,插入了一段连接序列,其目的是使产生的融合蛋白中胰岛原和转铁蛋白部分可以自由旋转,互不制约二者的空间结构,从而最大限度发挥其生物活性。
为了构建pET28a-Proinsulin-Tf(胰岛素原-转铁蛋白),我们先通过化学合成方法得到一段DNA片段,其中依次包括T7启动子(SEQ ID NO.5)、Lac控制子(SEQ ID NO.6)、胰岛素原基因序列(SEQ ID NO.1)、连接肽序列(SEQ ID NO.3)和人转铁蛋白基因序列(SEQID NO.2)。这个片段分别在5’和3’端带有XbaI和NotI两个内切酶位点,中间序列包含有编译人胰岛原全长(A链+B链+C肽)、4H2连接肽、人转轶蛋白全长的DNA序列。合成以后,该片段通过T4DNA连接酶的作用,直接被插入到pET28a载体的XbaI和NotI酶位点之间。
为了构建pET28a-Proinsulin-NTf,先以上述化学合成得到的DNA片段为模板,通过PCR扩增的方法得到一段不含转铁蛋白碳端的DNA片段,其中依次包括T7启动子(SEQ IDNO.5)、Lac控制子(SEQ ID NO.6)、胰岛素原基因序列(SEQ ID NO.1)、连接肽序列(SEQ IDNO.3)和人转铁蛋白氨端基因序列(SEQ ID NO.4)。然后将该DNA片段,经T4DNA连接酶反应,插入到pET28a载体的XbaI和NotI酶切位点之间。
实施例2:ProINS-Tf和ProINS-NTf重组蛋白的表达及可溶性分析
将重组质粒转化入BL21感受态细胞,涂布含Kan+抗性的LB平板上,37℃倒置培养16h,挑取单菌落,加在含Kan+抗性的LB培养基中,37℃,250rpm/min培养16h。按照1:20的比例,取5ml菌液加入100ml含Kan+抗性和1%葡萄糖的LB培养基,250rpm/min摇3h,使菌液的OD600值为0.6-0.8,此时加入IPTG(终浓度1mM)进行诱导表达,37℃分别诱导表达2h,4h,6h,8h。6000rpm/min离心10min收集菌体,高压均质机破碎菌体,12000rpm/min离心10min,收集上清和沉淀,利用10%SDS-PAGE检测菌体内蛋白的表达水平及对表达蛋白的可溶性进行分析。图2结果显示,IPTG诱导约2小时后,即开始有ProINS-Tf重组蛋白表达,且其表达量有随诱导时间延长而增加的趋势。将诱导表达后的菌体通过高压均质机破碎,分别取上清和沉淀进行10%SDS-PAGE分析,结果发现(图3)ProINS-Tf重组蛋白大部分为不溶蛋白,也即以包涵体的形式存在。
与ProINS-Tf重组蛋白透导表达实验对比,可以发现ProINS-NTf重组蛋白的表达时间相接近,但其产量明显高于ProINS-Tf重组蛋白(图4)。我们推测,这可能与ProINS-NTf融合基因相对较小,更易于被转录翻译有关。
对ProINS-NTf融合蛋白可溶性分析结果发现,与ProINS-Tf重组蛋白一样,大部分ProINS-NTf蛋白也是以不溶的包涵体形式存在(图5)。
实施例3:ProINS-Tf及ProINS-NTf融合蛋白的变性复性
图3和图5的结果显示ProINS-Tf和ProINS-NTf重组蛋白均以不可溶的包涵体形式存在表达于大肠杆菌中。为了得到可溶的ProINS-Tf和ProINS-NTf重组蛋白,我们将包涵体先用洗涤缓冲液1(50mM Tris,50mM NaCl,1mM EDTA,1%TritonX100PH8.5)和洗涤缓冲液2(2M尿素,50mM Tris,50mM NaCl,1mM EDTA,1%TritonX100PH8.5)各洗涤两次,每次1h,洗涤后的包涵体用变性液(8M尿素,50mM Tris,50mM NaCl,1mM EDTA,50mM DTT,PH9.5)溶解。
下一步是对将溶解的包涵体蛋白进行变性复性处理。首先,将包涵体液置于透析袋,在复性液1(6M尿素,50mM TrisHCl,50mM NaCl,1mM EDTA,1.0mM GSH,0.1mM GSSG,PH9.5),复性液2(4M尿素,50mM TrisHCl,50mM NaCl,1mM EDTA,1.0mM GSH,0.1mM GSSG,PH9),复性液3(2M尿素,50mM TrisHCl,50mM NaCl,1mM EDTA,1.0mM GSH,0.1mM GSSG,PH8.5),复性液4(1M尿素,50mM TrisHCl,50mM NaCl,1mM EDTA,1.0mM GSH,0.1mM GSSG,PH8.0),复性液5(0.01M PBS PH7.5)中梯度透析复性,每级12h,透析过程在4℃下进行。将最后一步复性的蛋白12000rpm/min离心20min,上清即为复性后的可溶蛋白。
对包涵体进行洗涤,变性和复性。分别取等比例的包涵体,变性产物以及复性产物后进行10%SDS-PAGE分析。图6结果显示,包涵体经过洗涤后纯度有所增加,去除了大多数的杂蛋白,但是除了目的蛋白外在55kd附近依旧有着一条较明显的蛋白带,结合图2中的Western blot结果猜想可能是由于目的蛋白在表达过程中发生了部分的降解。通过ImageJ对条带灰度值分析,大约有50%重组蛋白通过复性转变成了可溶蛋白。
实施例4:ProINS-Tf和ProINS-NTf的降糖活性检验
为了检测ProINS-Tf和ProINS-NTf的降糖活性,我们利用链脲佐菌素(STZ)诱导胰岛细胞凋亡的方式构建了1型糖尿病的小鼠模型。然而向1型糖尿病的小鼠分别注射了PBS(n=5),人胰岛素(67.5mg/dL,n=5)和纯化后的ProINS-Tf和ProINS-NTf重组蛋白(67.5mg/dL,n=5),检测其血糖的变化情况。如图6所示,胰岛素在注射后快速起效,60分钟后血糖就下降到了65mg/dL左右,随后血糖急速上升,120分钟血糖就接近于初始血糖水平。ProINS-Tf(67.5mg/dL)注射后缓慢起效,血糖下降更加平缓,注射1小时内血糖变化不明显,240分钟左右血糖下降到120mg/dL,之后缓慢的上升,300分钟后血糖依旧维持在200mg/dL以下。而注射同样剂量的ProINS-NTf则没有对小鼠血糖产生任何明显的影响。这些结果说明ProINS-Tf(胰岛素原-转铁蛋白融合蛋白)具有长效降血糖的活性,而ProINS-NTf(胰岛素原-转铁蛋白氨端融合蛋白)不具备降糖的生物学活性。我们推测,这可能是由于转铁蛋白与其受体的结合需要转铁蛋白N端和C端结构域的协同参与,因此仅仅转铁蛋白的N端结构域并不能够高效的结合转铁蛋白受体,从而将胰岛素原带进肝脏转变成具有活性的胰岛素。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
<110> 苏州康宇生物科技有限公司
<120> 一种胰岛素衍生物的制备方法
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 258
<212> DNA
<213> (人工序列)
<400> 1
tttgtgaacc aacacctgtg cggctcacac ctggtggaag ctctctacct agtgtgcggg 60
gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120
caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180
tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240
ctggagaact actgcaac 258
<210> 2
<211> 2037
<212> DNA
<213> (人工序列)
<400> 2
gtccctgata aaactgtgag atggtgtgca gtgtcggagc atgaggccac taagtgccag 60
agtttccgcg accatatgaa aagcgtcatt ccatccgatg gtcccagtgt tgcttgtgtg 120
aagaaagcct cctaccttga ttgcatcagg gccattgcgg caaacgaagc ggatgctgtg 180
acactggatg caggtttggt gtatgatgct tacctggctc ccaataacct gaagcctgtg 240
gtggcagagt tctatgggtc aaaagaggat ccacagactt tctattatgc tgttgctgtg 300
gtgaagaagg atagtggctt ccagatgaac cagcttcgag gcaagaagtc ctgccacacg 360
ggtctaggca ggtccgctgg gtggaacatc cccataggct tactttactg tgacttacct 420
gagccacgta aacctcttga gaaagcagtg gccaatttct tctcgggcag ctgtgcccct 480
tgtgcggatg ggacggactt cccccagctg tgtcaactgt gtccagggtg tggctgctcc 540
acccttaacc aatacttcgg ctactcggga gccttcaagt gtctgaagga tggtgctggg 600
gatgtggcct ttgtcaagca ctcgactata tttgagaact tggcaaacaa ggctgacagg 660
gaccagtatg agctgctttg cctggacaac acccggaagc cggtagatga atacaaggac 720
tgccacttgg cccaggtccc ttctcatacc gtcgtggccc gaagtatggg cggcaaggag 780
gacttgatct gggagcttct caaccaggcc caggaacatt ttggcaaaga caaatcaaaa 840
gaattccaac tattcagctc tcctcatggg aaggacctgc tgtttaagga ctctgcccac 900
gggtttttaa aagtcccccc caggatggat gccaagatgt acctgggcta tgagtatgtc 960
actgccatcc ggaatctacg ggaaggcaca tgcccagaag ccccaacaga tgaatgcaag 1020
cctgtgaagt ggtgtgcgct gagccaccac gagaggctca agtgtgatga gtggagtgtt 1080
aacagtgtag ggaaaataga gtgtgtatca gcagagacca ccgaagactg catcgccaag 1140
atcatgaatg gagaagctga tgccatgagc ttggatggag ggtttgtcta catagcgggc 1200
aagtgtggtc tggtgcctgt cttggcagaa aactacaata agagcgataa ttgtgaggat 1260
acaccagagg cagggtattt tgctgtagca gtggtgaaga aatcagcttc tgacctcacc 1320
tgggacaatc tgaaaggcaa gaagtcctgc catacggcag ttggcagaac cgctggctgg 1380
aacatcccca tgggcctgct ctacaataag atcaaccact gcagatttga tgaatttttc 1440
agtgaaggtt gtgcccctgg gtctaagaaa gactccagtc tctgtaagct gtgtatgggc 1500
tcaggcctaa acctgtgtga acccaacaac aaagagggat actacggcta cacaggcgct 1560
ttcaggtgtc tggttgagaa gggagatgtg gcctttgtga aacaccagac tgtcccacag 1620
aacactgggg gaaaaaaccc tgatccatgg gctaagaatc tgaatgaaaa agactatgag 1680
ttgctgtgcc ttgatggtac caggaaacct gtggaggagt atgcgaactg ccacctggcc 1740
agagccccga atcacgctgt ggtcacacgg aaagataagg aagcttgcgt ccacaagata 1800
ttacgtcaac agcagcacct atttggaagc aacgtaactg actgctcggg caacttttgt 1860
ttgttccggt cggaaaccaa ggaccttctg ttcagagatg acacagtatg tttggccaaa 1920
cttcatgaca gaaacacata tgaaaaatac ttaggagaag aatatgtcaa ggctgttggt 1980
aacctgagaa aatgctccac ctcatcactc ctggaagcct gcactttccg tagacct 2037
<210> 3
<211> 138
<212> DNA
<213> (人工序列)
<400> 3
gcagaggctg ctgctaaaga ggctgctgca aaagaggctg ctgcaaaaga ggctgctgca 60
aaagctgcag aggctgctgc taaagaggct gctgcaaaag aggctgctgc aaaagaggct 120
gctgcaaaag ctctcgag 138
<210> 4
<211> 1014
<212> DNA
<213> (人工序列)
<400> 4
gtccctgata aaactgtgag atggtgtgca gtgtcggagc atgaggccac taagtgccag 60
agtttccgcg accatatgaa aagcgtcatt ccatccgatg gtcccagtgt tgcttgtgtg 120
aagaaagcct cctaccttga ttgcatcagg gccattgcgg caaacgaagc ggatgctgtg 180
acactggatg caggtttggt gtatgatgct tacctggctc ccaataacct gaagcctgtg 240
gtggcagagt tctatgggtc aaaagaggat ccacagactt tctattatgc tgttgctgtg 300
gtgaagaagg atagtggctt ccagatgaac cagcttcgag gcaagaagtc ctgccacacg 360
ggtctaggca ggtccgctgg gtggaacatc cccataggct tactttactg tgacttacct 420
gagccacgta aacctcttga gaaagcagtg gccaatttct tctcgggcag ctgtgcccct 480
tgtgcggatg ggacggactt cccccagctg tgtcaactgt gtccagggtg tggctgctcc 540
acccttaacc aatacttcgg ctactcggga gccttcaagt gtctgaagga tggtgctggg 600
gatgtggcct ttgtcaagca ctcgactata tttgagaact tggcaaacaa ggctgacagg 660
gaccagtatg agctgctttg cctggacaac acccggaagc cggtagatga atacaaggac 720
tgccacttgg cccaggtccc ttctcatacc gtcgtggccc gaagtatggg cggcaaggag 780
gacttgatct gggagcttct caaccaggcc caggaacatt ttggcaaaga caaatcaaaa 840
gaattccaac tattcagctc tcctcatggg aaggacctgc tgtttaagga ctctgcccac 900
gggtttttaa aagtcccccc caggatggat gccaagatgt acctgggcta tgagtatgtc 960
actgccatcc ggaatctacg ggaaggcaca tgcccagaag ccccaacaga tgaa 1014
<210> 5
<211> 17
<212> DNA
<213> (人工序列)
<400> 5
taatacgact cactata 17
<210> 6
<211> 25
<212> DNA
<213> (人工序列)
<400> 6
ggaattgtga gcggataaca attcc 25
Claims (10)
1.一种胰岛素原-转铁蛋白融合体,其特征在于,所述胰岛素原-转铁蛋白融合体融合表达了核苷酸序列如SEQ ID NO.1所示的胰岛素蛋白和核苷酸序列如SEQ ID NO.2所示的转铁蛋白;所述胰岛素蛋白和转铁蛋白之间通过核苷酸序列如SEQ ID NO.3所示的linker连接。
2.一种权利要求1所述的胰岛素原-转铁蛋白融合体的编码基因。
3.一种携带权利要求2所述的编码基因的表达载体。
4.根据权利要求3所述的表达载体,其特征在于,所述表达载体为pET系列或pQE系列。
5.一种表达权利要求1所述的胰岛素原-转铁蛋白融合体的重组菌。
6.根据权利要求6所述的重组菌,其特征在于,所述重组菌的宿主细胞为大肠杆菌。
7.一种权利要求6所述的重组菌的构建方法,其特征在于,包括如下步骤:
(1)基因合成胰岛素原-转铁蛋白融合体的编码基因;
(2)将上述编码基因通过连接酶插入pET28a载体中;
(3)将步骤(2)中插入编码基因的载体导入大肠杆菌BL21感受态细胞中,得到所述的重组菌。
8.一种权利要求1所述的胰岛素原-转铁蛋白融合体的变性、复性方法,其特征在于,包括如下步骤:
(1)将包涵体先用洗涤缓冲液1和洗涤缓冲液2各洗涤两次,每次洗涤0.5~1.5h;所述的洗涤缓冲液1为50mM Tris,50mM NaCl,1mM EDTA,1%TritonX100pH8.5;所述的洗涤缓冲液2为2M尿素,50mM Tris,50mM NaCl,1mM EDTA,1%TritonX100pH8.5;
(2)将步骤(1)洗涤后的包涵体用变性液进行溶解;所述变性液为8M尿素,50mM Tris,50mM NaCl,1mM EDTA,50mM DTT,pH9.5;
(3)将步骤(2)溶解的包涵体依次在复性液1、复性液2、复性液3、复性液4和复性液5中进行梯度透析复性,每级透析10~14h;所述复性液1为6M尿素,50mM TrisHCl,50mM NaCl,1mM EDTA,1.0mM GSH,0.1mM GSSG,pH9.5;所述复性液2为4M尿素,50mM TrisHCl,50mMNaCl,1mM EDTA,1.0mM GSH,0.1mM GSSG,pH9;所述复性液3为2M尿素,50mM TrisHCl,50mMNaCl,1mM EDTA,1.0mM GSH,0.1mM GSSG,pH8.5;所述复性液4为1M尿素,50mM TrisHCl,50mM NaCl,1mM EDTA,1.0mM GSH,0.1mM GSSG,pH8.0;所述复性液5为0.01M PBS pH7.5。
9.根据权利要求8所述的方法,其特征在于,所述透析是在4℃以下进行透析。
10.权利要求1所述的胰岛素原-转铁蛋白融合体在制备降血糖药物或保健品中的应用。
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CN104902929A (zh) * | 2012-05-21 | 2015-09-09 | 沈维强 | 使用蛋白质前体作为前药的方法 |
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