CN106397569B - 一种治疗代谢疾病的突变细胞因子融合蛋白 - Google Patents
一种治疗代谢疾病的突变细胞因子融合蛋白 Download PDFInfo
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Abstract
本发明公开了新的突变人细胞因子及其融合蛋白在治疗代谢疾病中的作用，本发明提供的哺乳动物细胞表达突变的人细胞因子为序列表SEQ ID NO:3所示的蛋白质。本发明对野生型人细胞因子基因进行突变和N端改造，所述的融合蛋白含有人细胞因子突变体和人IgG4的Fc段（CH2‑CH3）。本发明提供的哺乳动物细胞表达重组细胞因子融合蛋白具有良好的降血糖活性，特别是所述融合蛋白，还具有稳定性高、半衰期长等优点。并且本发明提供的融合蛋白还能较好的控制血糖的波动，稳定维持24小时血糖处于正常水平。
Description
技术领域
本发明涉及突变的细胞因子，尤其涉及突变的人源细胞因子，本发明还涉及该突变的人源细胞因子在制备治疗代谢疾病药物中的用途，属于突变细胞因子领域。
背景技术
糖尿病是危及全球的一种严重的代谢疾病，目前我国糖尿病患者已达4000万左右，患病率已由十几年前的1%增长至目前的2.5%，并以每年1‰的速度递增。糖尿病已经成为继心脑血管疾病和癌症之后威胁人类健康的第三大杀手。
目前仍缺少治愈糖尿病的方法，临床上对糖尿病以药物控制为主，患者需要终身服药以达到控制血糖水平，减轻糖尿病的症状，延缓并发症的出现的目的。现临床应用的糖尿病药物极易引起一些较严重的副作用，如低血糖、体重增加、心血管毒等副作用。而且由于糖尿病后期多数患者体内胰岛素抵抗现象严重，导致大部分依赖胰岛素通路的药物失效，给糖尿病治疗带来较大困难。因此，探寻更有效，安全的，尤其是可不依赖胰岛素途径独立调节血糖的新型药物一直是糖尿病的治疗研究中现实而紧迫的任务。
成纤维细胞生长因子-21是2000年发现的FGF家族一员，主要在肝脏中表达。经研究发现，FGF21能够高效并持续调节机体糖脂代谢，刺激脂肪细胞的葡萄糖吸收，降低ob /ob和db /db小鼠血浆中葡萄糖和甘油三脂的浓度，并且FGF21可以有效降低糖尿病动物胰岛素水平，改善胰岛素抵抗，不依赖胰岛素进行血糖调节，而且也不会导致类似胰岛素等药物大剂量用药造成的低血糖。更为值得我们注意的是它不同于同家族的其它因子，不会引起细胞的增殖。因此，FGF21有望成为治疗2型糖尿病新型潜力药物。
人免疫球蛋白IgG在体内的半衰期为20d，其稳定性是由于IgG的Fc片段与新生Fc受体（FcRn）结合，避免IgG进入溶酶体中被降解。因此，IgG的Fc片段被用来与活性蛋白连接构成融合蛋白，以提高活性蛋白的体内半衰期，达到长效的目的。
蛋白稳定性的高低直接影响其在宿主细胞中的表达量，为了提高产量，并在不影响蛋白原有活性的基础上，需要将FGF21进行适当的突变，通过与人IgG4的Fc片段连接成融合蛋白的形式来提高FGF21的稳定性，这将加快FGF21的中试生产，对临床具有非常重要的意义。
中国仓鼠卵巢细胞（Chinese hamster ovary cell, CHO cell）属于高等哺乳动物成纤维细胞，是目前重组糖基蛋白生产的首选体系。与原核及酵母等表达系统相比，CHO细胞所具有的优点包括：1）具有准确的翻译后修饰功能，如糖基化、磷酸化、酰基化、二硫键形成等，使其表达的蛋白在分子结构、生物学功能等方面非常接近天然的蛋白；2）具有产物胞外分泌的功能，有利于外源蛋白的分离纯化；3）具有外源基因的高效扩增和表达能力，且基因整合稳定；4）属于半悬浮性细胞，具有贴壁生长特性，耐受剪切力和渗透压的能力较强，亦可进行悬浮的规模性培养，表达水平较高；5）属于成纤维细胞，很少分泌自身的内源性蛋白，利于外源蛋白纯化。
筛选用表达载体结构优化。由于外源基因导入哺乳动物细胞的效率很低，从整合并表达外源基因的工程细胞中筛选出高表达的细胞克隆是一项很费时费力的工作。首先要依靠构建载体内的选择标记采用相应的筛选系统。本专利优化的筛选用表达载体pBMN同时整合了谷氨酰胺合成酶（GS）筛选系统。GS筛选系统有以下两个优点：1）有相对较高的外源基因扩增和表达能力。使用GS体系仅需一轮放大，就能筛选到有效的外源基因表达水平，而较DHFR基因扩增系统筛选效率高出一倍。2）谷氨酰胺合成酶是利用氨与谷氨酸合成谷氨酰胺的，此过程缓解了培养过程中氨积累的问题。
发明内容
本发明的目的之一是提供突变人FGF21及其编码核酸和氨基酸序列。
本发明的目的之二是提供突变人FGF21与人IgG4 Fc段融合蛋白及其编码核酸和氨基酸序列。
本发明的目的之三是构建优化的高通量筛选用表达载体pBMN。
本发明的目的之四是提供含有突变人FGF21基因和其与人IgG4 Fc段连接基因的表达载体以及含有该表达载体的宿主细胞。
本发明目的之五是将该突变人FGF21和其与人IgG4 Fc融合蛋白应用于制备成治疗糖尿病的药物或药物组合物。
本发明上述的内容是通过以下技术方案来实现的：
本发明人通过将含有GS片段的pBMN载体，用Not I和Sal I双酶切连入重组外源基因（FGF21片段），构建出具有双顺反子的真核重组表达载体pBMN，该载体可以通过GS加压筛选方式快速获得稳定高表达重组细胞株。
本发明将人FGF-21基因进行突变得到mhFGF21，相比于突变前，mhFGF21基因在宿主细胞中的表达量显著提高且活性显著增强。
含有该突变的mhFGF21核苷酸序列的表达载体以及含有该表达载体的宿主细胞也当然的包括在本发明的保护范围之内。
为了提高重组mhFGF21的可溶性表达，可以将mhFGF21核苷酸序列与人IgG4 Fc段连接，再将其克隆到表达载体中。其中，优选的，所述的表达载体可以是pBMN，所述的宿主细胞可以是CHO-K1SV；所述融合肽为人IgG4的Fc段。
本发明提供了一种纯化hFGF-21或mhFGF21的方法，包括：将表达载体转染至宿主细胞；筛选稳定细胞系后培养表达目标蛋白，离心收集培养基，对澄清后的上清进行离子交换层析和凝胶过滤层析分离纯化目标蛋白，即得纯度较高的hFGF-21或mhFGF21蛋白。
本发明还提供了一种纯化mhFGF21-Fc或Fc-mhFGF21的方法，包括：将表达载体转染至宿主细胞；筛选稳定细胞系后培养表达融合蛋白，离心收集培养基，利用ProteinA/G亲和层析，凝胶过滤层析从澄清后上清中分离纯化融合蛋白，即得纯度较高的mhFGF21-Fc或Fc-mhFGF21蛋白。
细胞试验和动物学试验结果表明，本发明突变mhFGF21相比于野生型hFGF21，活性显著提高，能够更加有效降低糖尿病小鼠体内的血糖水平，此外，并且本发明突变体还能较好的控制血糖波动，稳定维持24小时血糖处于正常水平。特别是mhFGF21与Fc连接的融合蛋白，其降糖效果更佳。本发明突变hFGF21或其融合蛋白可作为药物治疗糖尿病，肥胖症或代谢综合症等代谢疾病。
附图说明
图1纯化后的hFGF21、mhFGF21蛋白的SDS-PAGE电泳分析。
图2纯化后的Fc-mhFGF21、mhFGF21-Fc融合蛋白的SDS-PAGE电泳分析。
图3突变蛋白mhFGF21、Fc-mhFGF21和mhFGF21-Fc的体内稳定性的检测。
图4 突变后的mhFGF21、mhFGF21-Fc和Fc-mhFGF21细胞活性检测。
图5 1型糖尿病小鼠注射不同蛋白14d内血糖变化。
图6 1型糖尿病小鼠注射不同蛋白8周后24小时血糖波动。
图7 1型糖尿病小鼠注射不同蛋白8周后糖化血红蛋白变化。
图8 2型糖尿病db/db小鼠注射不同蛋白13d内血糖变化。
图9 2型糖尿病db/db小鼠注射不同蛋白8周后24小时血糖波动。
图10 2型糖尿病db/db小鼠注射不同蛋白8周后糖化血红蛋白变化。
具体实施方案：
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
说明：本发明中涉及的基因的设计、合成和克隆、表达载体的构建、核酸提取、测序和鉴定，以及表达产物的分离和纯化等操作步骤，可按照本领域已知的技术进行(参见分子克隆及CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1 mhFGF21基因的构建
设计4条引物，运用重叠PCR方法构建mhFGF21蛋白基因。
4条引物设计如下：
P1: 5' GGATCCAGCCACCCCATCCCTGACTCCAGTCCTCT 3'
P2: 5' GGATTCCGGGTGGCTCCGGGGGTGCGGGGGG 3'
P3: 5' CCAGGCCTGCCCCCCGCAC CCCCGGAGCCACCC 3'
P4: 5' CGGAATTCTTAGGAAG TGTAGCTGGGGC 3'
1、mhFGF21第一段扩增
运用PCR的方法以含hFGF21基因的质粒为模板，以P1和P2为引物，获取mhFGF21基因第一段。扩增体系为25 µl，循环参数如下：95℃预变性5 min，95 ℃变性30 sec，50 ℃退火1 min，72 ℃延伸1 min，cycle=15，72 ℃终延伸10 min。扩增结束后，取2 µl混合物通过2﹪琼脂糖凝胶电泳观察扩增结果。
2、mhFGF21基因第二段扩增
运用PCR的方法以含hFGF21基因的质粒为模板，以P3和P4为引物，获取mhFGF21基因第二段。扩增体系为25 µl，循环参数如下：95℃预变性5 min，95 ℃变性30sec，48 ℃退火1 min，72 ℃延伸1 min，cycle=15，72 ℃终延伸10 min。扩增结束后，取2 µl混合物通过2﹪琼脂糖凝胶电泳观察扩增结果。
3、全长mhFGF21基因的扩增
运用重叠PCR方法，以mhFGF21第一段和mhFGF21第二段为模板，以P1和P4为引物，扩增全长mhFGF21。扩增体系为25 µl，循环参数如下：95℃预变性5 min，95 ℃变性30sec，46 ℃退火1 min，72 ℃延伸1 min，cycle=15，72 ℃终延伸10 min。扩增结束后，取2 µl混合物通过2﹪琼脂糖凝胶电泳观察扩增结果。
实施例2 Fc基因的构建
参照分子克隆方法，从人血液中用Trizol提取总RNA，反转录cDNA作为模板。以人IgG4的Fc为模板，设计如下引物
P5 5’ GCGGAGTCCAAATATGGTCCCCC 3’
P6 5’ TTAACCCAGAGACAGGGAGA 3’
运用PCR方法，以含cDNA为模板，以P5和P6为引物，获取人IgG4的Fc基因。
实施例3 mhFGF21-Fc基因的构建
设计4条引物，运用重叠PCR方法构建mhFGF21-Fc蛋白基因,将mhFGF21与Fc用(Gly4-Ser)3 linker（序列为5’GGTGGCGGTGGCTCCGGCGGTGGTGGGTCGGGTGGC GGCGGATCT 3’）连接。4条引物设计如下：
P7 5 GGATCC AGCCACCCCATCCCTGACTCCAG 3’
P8 5’ CCACCCGACCCACCACCGCCGGAGCCACCGCCACCGGAAGTGTAGCTGGGGCTTC 3’
P9 5’GGTGGCGGTGGCTCCGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCGGAGTCCAAAT 3’
P10 5’CGCGAATTC TTA ACCCAGAGACAGGGAGA 3’
1、mhFGF21-Fc第一段扩增
运用PCR的方法以含mhFGF21基因的质粒为模板，以P7和P8为引物，获取mhFGF21-Fc基因第一段。扩增体系为25 µl，循环参数如下：95℃预变性5 min，95 ℃变性30sec，48℃退火1 min，72 ℃延伸1 min，cycle=15，72 ℃终延伸10 min。扩增结束后，取2 µl混合物通过2﹪琼脂糖凝胶电泳观察扩增结果。
2、mhFGF21-Fc基因第二段扩增
运用PCR的方法以含Fc基因的质粒为模板，以P9和P10为引物，获取mhFGF21-Fc基因第二段。扩增体系为25 µl，循环参数如下：95℃预变性5 min，95 ℃变性30sec，48 ℃退火1 min，72 ℃延伸1 min，cycle=15，72 ℃终延伸10 min。扩增结束后，取2 µl混合物通过2﹪琼脂糖凝胶电泳观察扩增结果。
3、全长mhFGF21-Fc基因的扩增
运用重叠PCR方法，以mhFGF21-Fc第一段和mhFGF21-Fc第二段为模板，以P7和P10为引物，扩增全长mhFGF21-Fc，扩增体系为25 µl，循环参数如下： 95℃预变性5 min，95 ℃变性30sec，46 ℃退火1 min，72 ℃延伸1 min，cycle=15，72 ℃终延伸10 min。扩增结束后，取2 µl混合物通过2﹪琼脂糖凝胶电泳观察扩增结果。
实施例4 Fc-mhFGF21基因的构建
设计4条引物，运用重叠PCR方法构建Fc-mhFGF21蛋白基因，将mhFGF21与Fc用(Gly4-Ser)3 linker（序列为5’GGTGGCGGTGGCTCCGGCGGTGGTGGGTC GGGTGGCGGCGGATCT3’）连接。
4条引物设计如下：
P11 5’ GGATCC AGCGCGGAGTCCAAATATGGT 3’
P12 5’ CCACCCGACCCACCACCGCCGGAGCCACCGCCACCACCCAGAGACAGGGAGAGGCT 3’
P13 5’GCTCCGGCGGTGGTGGGTCGGGTGGCGGCGGATCTCACCCCATCCCTGACTCCAGT 3’
P14 5’CGC GAATTC TTA GGAAGTGTAGCTGGGGCTTCGGCCCT 3’
1、Fc-mhFGF21第一段扩增
运用PCR的方法以含Fc基因的质粒为模板，以P11和P12为引物，获取Fc-mhFGF21基因第一段。扩增体系为25 µl，循环参数如下：95℃预变性5 min，95 ℃变性30sec，50 ℃退火1 min，72 ℃延伸1 min，cycle=15，72 ℃终延伸10 min。扩增结束后，取2 µl混合物通过2﹪琼脂糖凝胶电泳观察扩增结果。
2、Fc-mhFGF21基因第二段扩增
运用PCR的方法以含mhFGF-21基因的质粒为模板，以P13和P14为引物，获取Fc-mhFGF21基因第二段。扩增体系为25 µl，循环参数如下：95℃预变性5 min，95 ℃变性30sec，48 ℃退火1 min，72 ℃延伸1 min，cycle=15，72 ℃终延伸10 min。扩增结束后，取2µl混合物通过2﹪琼脂糖凝胶电泳观察扩增结果。
3、全长Fc-mhFGF21基因的扩增
运用重叠PCR方法，以Fc-mhFGF21第一段和Fc-mhFGF21第二段为模板，以P11和P14为引物，扩增全长mhFGF21-Fc，扩增体系为25 µl，循环参数如下： 95℃预变性5 min，95 ℃变性30sec，46 ℃退火1 min，72 ℃延伸1 min，cycle=15，72 ℃终延伸10 min。扩增结束后，取2 µl混合物通过2﹪琼脂糖凝胶电泳观察扩增结果。
实施例5 mhFGF21、Fc-mhFGF21、mhFGF21-Fc基因表达载体的构建
合成分泌信号肽atggt gttgcagacc caggtcttca tttctctgtt gctctggatctctggcgcct acggg
并在两端分别加入HandIII和BamHI、EcoRI酶切位点并插入T载体，构建T-leader中间过渡载体
将实施例1、3、4终产物经BamHI和EcoRI克隆入T-leader载体上，分别用BamH I与EcoR I双酶切基因克隆产物及T-leader载体，构建得到重组质粒T-leader-FGF21、T-leader-mhFGF21、T-leader-Fc-mhFGF21、T-leader-mhFGF21-Fc
用HindIII和EcoRI酶切重组的T-leader-hFGF21、T-leader-mhFGF21、T-leader-Fc-mhFGF21、T-leader-mhFGF21-Fc载体，将携带有kappaleader信号肽的FGF21基因连入相同酶切位点的pBMN载体，构建pBMN-FGF21重组载体。
将含有GS片段的pBMN载体，重组后的pBMN-FGF21、均用Not I和Sal I双酶切，选择大片段相连，构建出具有双顺反子的真核重组表达载体，命名为pBMN-screen-hFGF21、pBMN-screen- mhFGF21、pBMN-screen-Fc-mhFGF21、pBMN-screen-mhFGF21-Fc。
实施例6稳定高表达重组细胞株的筛选
1、细胞转染
CHO-K1SV细胞的生长条件为高糖DMEM培养基，10% FBS，8mM L-Glutamine，青霉素100 ug/ml，链霉素100μg/ml，37℃、5% CO2饱和湿度条件下培养。
取生长状况良好的CHO-K1SV细胞用不含青霉素和链霉素的上述培养基按照每孔2×105个细胞接种于六孔板，待生长在六孔板的细胞汇合度达80%左右，用LipofectamineTM2000转染试剂分别将pBMN-hFGF21、pBMN-mhFGF21、pBMN-Fc-mhFGF21、pBMN- mhFGF21-Fc载体导入CHO-K1SV细胞，转染方法参照Invitrogen公司试剂盒说明书：首先，取纯化后且不含内毒素的4μg重组质粒（pBMN-hFGF21、pBMN-mhFGF21、pBMN-Fc-mhFGF21、pBMN-mhFGF21 -Fc）加入250 μl的无血清的培养基Opti-MEM中，混匀，室温孵育5 min，记为A液；同时，取10μl LipofectamineTM 2000加入250 μl的无血清的培养基Opti-MEM中，室温孵育5 min，记为B液；然后，将A液注入B液中，室温孵育20 min；最后，弃去六孔板中的培养基，用无血清的培养基Opti-MEM洗涤细胞两次，将转染液缓慢加至细胞表面，放在细胞培养箱培养6 h后，弃去转染液，更换新鲜的不含抗生素的完全培养基于37℃、5% CO2饱和湿度的条件下继续培养，应用荧光显微镜进行观察。
阳性细胞的筛选
经转染的细胞培养36 h后，显微镜下观察荧光率能够达到80%及为最佳转染结果，此后进行细胞的加压筛选，将经转染的细胞设为实验组，不做转染的细胞设为阴性对照组。弃去完全培养基，用无菌PBS冲洗细胞3次，换成加压培养基。加压培养基的主要成分为：不含谷氨酰胺的IMDM培养基，10%FBS，50 mol/l MSX，5 mol/l L-Proline。加压5d后，实验组假阳性细胞大量死亡，更换新鲜的加压培养基继续培养，15d后镜下观察：实验组细胞每孔都形成了多个单克隆，而对照组细胞基本死亡。将每孔的细胞进行扩培后冻存保种。
实施例7 hFGF21、mhFGF21、Fc-mhFGF21、mhFGF21-Fc突变蛋白的制备
分选后得到的混合克隆体待生长完全后，按照常规方法消化后传代，并以血球计数板对消化后的细胞进行计数，各混合克隆体以105个/ml的相同浓度传至含2 0ml无血清培养基三角瓶，培养3 d后，取上清，1500 rpm，5 min，弃沉淀，用上清保存在4℃备用。
实施例8 hFGF21、mhFGF21蛋白的纯化
收集培养基，4℃，8000rpm离心15min，取上清，上清过0.45μm滤膜后进行5 kD超滤膜浓缩，并置换于IEX buffer A（20mM Tris、10 mM NaCl，pH8.0）中，缓冲液置换后经AKTApurifier 100系统，与5倍柱体积IEX buffer A平衡好的Capto Q柱（装于XK16/20空柱，柱高10cm，流速300cm/h）完全结合后，用3-4倍柱体积IEX buffer A冲洗；当紫外曲线达到稳定的基线时，利用IEX buffer A和IEX buffer B（20mM Tris、1M NaCl，pH8.0）混合液洗脱，15%和10%IEX bufferB液冲洗杂蛋白，20% IEX bufferB液洗脱目标蛋白，收集各洗脱峰，并进行15%SDS-PAGE电泳分析。
利用5kD的中空纤维柱将离子交换洗脱目标峰进行浓缩，浓缩后蛋白通过Superloop进入AKTA purifier 100系统，用2倍柱体积PBS（pH7.0）平衡好的Superdex 75分子筛柱（装于XK16/70空柱，柱高60cm，流速1mL/min）进行分离纯化，收集各流穿峰，并进行15%SDS-PAGE电泳分析。结果显示纯化后蛋白纯度在95%以上，如图1所示，泳道1为蛋白质标准分子量Marker，泳道2和3分别为纯化后的mhFGF21和hFGF21蛋白。
实施例9 Fc-mhFGF21、mhFGF21-Fc融合蛋白的纯化
收集培养基，4℃，8000rpm离心15min，取上清，上清液过0.45μm滤膜澄清后，通过泵进入AKTA purifier 100系统，与2-3倍柱体积Binding buffer （20mM Na3PO4、150 mMNaCl，pH7.4）平衡好的Protein A柱（装于XK16/20空柱，柱高10cm，流速100cm/h）完全结合后，用4-5倍柱体积的Binding buffer冲洗杂蛋白；当紫外曲线达到稳定的基线时，再用2-3倍柱体积Elution buffer（100mM柠檬酸钠，pH3.0）洗脱目的蛋白，把结合在填料上的融合蛋白洗脱下来并收集到试管（其中含有1M Tris，pH 9.0）中。
把Superdex 200凝胶过滤柱（装于XK16/70空柱，柱高60cm，流速1mL/min）接到AKTA purifier 100系统中，先用2倍柱体积的蒸馏水替换其保护液（20%乙醇），再用2倍柱体积的Desalting buffer（20mM Na3PO4、150 mM NaCl，pH7.0）平衡柱子，然后将亲和层析洗脱液通过Superloop进样。收集各洗脱峰，并进行15% SDS-PAGE电泳分析。结果显示经纯化过后，蛋白纯度在95%以上，如图2所示，泳道1为蛋白质标准分子量Marker，泳道3和4分别为纯化前的mhFGF21-Fc和Fc-mhFGF21蛋白，泳道2和5分别为纯化后的mhFGF21-Fc和Fc-mhFGF21蛋白。
实施例10本发明突变蛋白mhFGF21、Fc-mhFGF21和mhFGF21-Fc的体内稳定性的检测
取体重约400g的雄性大鼠（购置于上海斯莱克实验动物有限责任公司，动物质量合格证号SCXK（沪）2012-0005）16只，随机分为4组，每组4只。分别皮下注射实施例6制备的突变蛋白mhFGF21、Fc-mhFGF21、mhFGF21-Fc和野生型hFGF21，剂量30nmol/kg，在给药前（0h），给药后的1h、3h、5h、7h、24 h，于耳缘静脉采血250μL左右，12000r/m离心10min，取上清。
用稀释好的不同浓度的mhFGF21、Fc-mhFGF21、mhFGF21-Fc和hFGF21蛋白分别建立蛋白浓度含量的标准曲线，各种稀释好的血清包被酶标板，应用 ELISA间接法测定各血清中目标蛋白的含量，统计学分析并计算出mhFGF21、Fc-mhFGF21、mhFGF21-Fc和hFGF21的体内半衰期。体内半衰期t1/2=0.301*（t2-t1）/log（OD1/OD2），其中OD1和OD2分别表示t1和t2时取出血清所对应酶标板上的平均光吸收值。
结果如图3所示，经公式计算出突变蛋白mhFGF21、Fc-mhFGF21、mhFGF21-Fc和野生型hFGF21的体内半衰期分别约为52min、469min、442min和36min。说明了hFGF21蛋白经突变改造后体内半衰期显著增加。
实施例11重组蛋白的细胞活性检测
葡萄糖浓度的检测：处理的细胞培养24h后，用葡萄糖氧化酶-过氧化物酶法（GOD-POD法）（Accorsi PA，2005）检测培养基中残留的葡萄糖含量。取培养基上清液2 ml加入到200 ml葡萄糖检测液中，每孔葡萄糖至少重复检测3次，37 ℃反应5～10分钟后，在500nm波长下测OD值。
计算培养液中残留的葡萄糖浓度，公式为：
葡萄糖浓度(mmol/L)=OD样品/OD标准×5.55 mmol/L
计算细胞对葡萄糖的消耗率，公式为：
细胞葡萄糖消耗率（%）=[（C空白葡萄糖-C给药葡萄糖）/ C空白葡萄糖] ×100 %
HepG2细胞（中国医学科学院基础所细胞库）饥饿12h后，分别用不同浓度（10、100、1000nmol/L）的hFGF21、mhFGF21、Fc-mhFGF1和mhFGF21-Fc刺激细胞24h，用GOD-POD法检测培养基中残留的葡萄糖含量。
数据分析结果显示，不同浓度时经mhFGF21蛋白刺激的HepG2细胞葡萄糖吸收都高于hFGF21蛋白刺激的HepG2细胞葡萄糖吸收，且在中、高浓度刺激的细胞葡萄糖吸收显著高于hFGF21蛋白刺激的HepG2细胞葡萄糖吸收(**p<0.01)，说明mhFGF21蛋白活性优于hFGF21蛋白。而相对于mhFGF21，Fc-mhFGF21和mhFGF21-Fc蛋白体外细胞活性也显著提高，如图4所示。
实施例12 本发明突变蛋白mhFGF21、Fc-mhFGF21和mhFGF21-Fc的体内药效试验
1、实验蛋白在1型糖尿病动物模型上的活性检测
1型糖尿病动物模型制备：昆明小鼠（购置于上海斯莱克实验动物有限责任公司，动物质量合格证号SCXK（沪）2012-0005）适应性饲喂至体重达到35 g左右时开始实验。实验开始前，需将小鼠禁食不禁水12 h。第一次按30mg/kg剂量，腹腔注射 STZ，间隔3日按20mg/kg 剂量，腹腔第二次注射 STZ；对照组只注射柠檬酸 -柠檬酸钠(pH 4.4)缓冲液。注射3周后，选取空腹血糖浓度>16.65 mmol/L小鼠进行实验。实验过程中动物自由进食、饮水．
分组，给药及指标检测：选取50只成模的血糖接近均值糖尿病模型小鼠，分成5组，每组10只，每天给予实验组（Vehicle组、mhFGF21组、Fc-mhFGF21组和mhFGF21-Fc组）相应的受试物或溶剂一次，皮下注射，剂量0.5mg/kg/天，连续观察14d内各实验组小鼠的血糖变化情况。给药8周后，检测小鼠24小时内的血糖变化，每隔一小时检测一次，并取血样测定糖化血红蛋白，所得实验数据进行统计学分析。具体实验结果如下图5，6和7。
结果显示各给药组小鼠血糖变化趋势一致，但hFGF21组小鼠血糖较mhFGF21组、mhFGF21-Fc组和Fc-mhFGF21组小鼠血糖下降缓慢，其中Fc-mhFGF21组小鼠长期降糖效果最佳。24小时血糖波动和糖化血红蛋白变化结果也表明，突变后蛋白mhFGF21、Fc-mhFGF21和mhFGF21-Fc控制血糖能力都明显优于hFGF21，其中Fc-mhFGF21体内生物学活性最为明显。
2、实验蛋白在2型糖尿病动物模型上活性检测
分组、给药及指标检测：2型糖尿病db/db模型小鼠（上海斯莱克实验动物有限责任公司，动物质量合格证号SCXK（沪）2012-0005，选取50只成模的血糖接近均值的糖尿病模型小鼠，分成5组，每组10只，每天给予实验组（Vehicle组、mhFGF21组、Fc-mhFGF21组和mhFGF21-Fc组）相应的受试物或溶剂一次，皮下注射，剂量0.5mg/kg/天，连续注射7d后停药5d，观察13d内各实验组小鼠的血糖变化情况。给药8周后，检测小鼠24小时内的血糖变化，每隔一小时检测一次，并取血样测定糖化血红蛋白，所得实验数据进行统计学分析。具体实验结果如图8，9和10。
结果显示各给药组小鼠血糖变化趋势一致，但hFGF21组小鼠血糖较mhFGF21组、mhFGF21-Fc组和Fc-mhFGF21组小鼠血糖下降缓慢，回升快，其中Fc-mhFGF21组小鼠长期降糖效果和停药后血糖控制能力最佳。24小时血糖波动和糖化血红蛋白变化结果也表明，突变后蛋白mhFGF21、Fc-mhFGF21和mhFGF21-Fc控制血糖能力都明显优于hFGF21，其中Fc-mhFGF21体内生物学活性最为明显。
序列表
<110> 深圳红石科创生物科技发展有限公司
<120> 一种治疗代谢疾病的突变细胞生长因子融合蛋白
<210> SEQ ID NO: 1
<211> 549
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<400> SEQ ID NO: 1
agccacccca tccctgactc cagtcctctc ctgcaattcg ggggccaagt ccggcagcgg 60
tacctctaca cagatgatgc ccagcagaca gaagcccacc tggagatcag ggaggatggg 120
acggtggggg gcgctgctga ccagagcccc gaaagtctcc tgcagctgaa agccttgaag 180
ccgggagtta ttcaaatctt gggagtcaag acatccaggt tcctgtgcca gcggccagat 240
ggggccctgt atggatcgct ccactttgac cctgaggcct gcagcttccg ggagctgctt 300
cttgaggacg gatacaatgt ttaccagtcc gaagcccacg gcctcccgct gcacctgcca 360
gggaacaagt ccccacaccg ggaccctgca ccccgaggac cagctcgctt cctgccacta 420
ccaggcctgc cccccgcacc cccggagcca cccggaatcc tggcccccca gccccccgat 480
gtgggctcct cggaccctct gagcatggtg ggaccttccc agggccgaag ccccagctac 540
acttcctga 549
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<400> SEQ ID NO: 2
agccacccca tccctgactc cagtcctctc ctgcaattcg ggggccaagt ccggcagcgg 60
tacctctaca cagatgatgc ccagcagaca gaagcccacc tggagatcag ggaggatggg 120
acggtggggg gcgctgctga ccagagcccc gaaagtctcc tgcagctgaa agccttgaag 180
ccgggagtta ttcaaatctt gggagtcaag acatccaggt tcctgtgcca gcggccagat 240
ggggccctgt atggatcgct ccactttgac cctgaggcct gcagcttccg ggagctgctt 300
cttgaggacg gatacaatgt ttaccagtcc gaagcccacg gcctcccgct gcacctgcca 360
gggaacaagt ccccacaccg ggaccctgca ccccgaggac cagctcgctt cctgccacta 420
ccaggcctgc cccccgcacc cccggagcca cccggaatcc tggcccccca gccccccgat 480
gtgggctcct cggaccctct gagcatggtg ggaccttccc agggccgaag ccccagctac 540
acttccggtg gcggtggctc cggcggtggt gggtcgggtg gcggcggatc tgcggagtcc 600
aaatatggtc ccccatgccc accctgccca gcacctgagt tcctgggggg accatcagtc 660
ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 720
tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 780
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 840
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 900
tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 960
gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 1020
aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1080
tgggaaagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1140
gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 1200
aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 1260
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<210> SEQ ID NO: 3
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<212> DNA
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agcgcggagt ccaaatatgg tcccccatgc ccaccctgcc cagcacctga gttcctgggg 60
ggaccatcag tcttcctgtt ccccccaaaa cccaaggaca ctctcatgat ctcccggacc 120
cctgaggtca cgtgcgtggt ggtggacgtg agccaggaag accccgaggt ccagttcaac 180
tggtacgtgg atggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagttc 240
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaacggc 300
aaggagtaca agtgcaaggt ctccaacaaa ggcctcccgt cctccatcga gaaaaccatc 360
tccaaagcca aagggcagcc ccgagagcca caggtgtaca ccctgccccc atcccaggag 420
gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta ccccagcgac 480
atcgccgtgg agtgggaaag caatgggcag ccggagaaca actacaagac cacgcctccc 540
gtgctggact ccgacggctc cttcttcctc tacagcaggc taaccgtgga caagagcagg 600
tggcaggagg ggaatgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 660
acacagaaga gcctctccct gtctctgggt ggtggcggtg gctccggcgg tggtgggtcg 720
ggtggcggcg gatctcaccc catccctgac tccagtcctc tcctgcaatt cgggggccaa 780
gtccggcagc ggtacctcta cacagatgat gcccagcaga cagaagccca cctggagatc 840
agggaggatg ggacggtggg gggcgctgct gaccagagcc ccgaaagtct cctgcagctg 900
aaagccttga agccgggagt tattcaaatc ttgggagtca agacatccag gttcctgtgc 960
cagcggccag atggggccct gtatggatcg ctccactttg accctgaggc ctgcagcttc 1020
cgggagctgc ttcttgagga cggatacaat gtttaccagt ccgaagccca cggcctcccg 1080
ctgcacctgc cagggaacaa gtccccacac cgggaccctg caccccgagg accagctcgc 1140
ttcctgccac taccaggcct gccccccgca cccccggagc cacccggaat cctggccccc 1200
cagccccccg atgtgggctc ctcggaccct ctgagcatgg tgggaccttc ccagggccga 1260
agccccagct acacttcctg a 1281
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<212> PRT
<213> 人工序列
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<400> SEQ ID NO: 4
Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg 20
Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly 40
Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys 60
Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp 80
Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu 100
Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro 120
Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu 140
Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp 160
Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr 180
Thr Ser *** 182
<210> SEQ ID NO: 5
<211> 426
<212> PRT
<213> 人工序列
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<400> SEQ ID NO: 5
Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg 20
Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly 40
Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys 60
Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp 80
Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu 100
Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro 120
Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu 140
Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp 160
Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr 180
Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser 200
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 220
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 240
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 260
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 280
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 300
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 320
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 340
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 360
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 380
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly 400
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 420
Leu Ser Leu Ser Leu Gly *** 426
<210> SEQ ID NO: 6
<211> 426
<212> PRT
<213> 人工序列
<220>
<223>
<400> SEQ ID NO: 6
Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly 20
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 40
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn 60
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 80
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 100
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 120
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu 140
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 160
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 180
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 200
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 220
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 240
Gly Gly Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln 260
Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile 280
Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu 300
Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys 320
Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe 340
Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro 360
Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg 380
Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro 400
Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg 420
Ser Pro Ser Tyr Thr Ser *** 426
Claims (13)
1.一种突变细胞因子mhFGF21，其氨基酸序列如序列表中SEQ ID NO: 4所示。
2.编码权利要求 1 所述突变细胞因子mhFGF21的基因，其核酸序列如序列表中SEQ IDNO:1所示。
3.融合蛋白mhFGF21-Fc，其序列如序列表中SEQ ID NO:5所示。
4.融合蛋白Fc-mhFGF21，其序列如序列表中SEQ ID NO:6所示。
5.编码权利要求3所述的融合蛋白mhFGF21-Fc的基因，其核酸序列如序列表中SEQ IDNO:2所示。
6.编码权利要求4所述的融合蛋白Fc-mhFGF21的基因，其核酸序列如序列表中SEQ IDNO:3所示。
7.含有权利要求2或权利要求5或权利要求6所述的基因的表达载体，所述表达载体为pBMN载体。
8.含有权利要求2或权利要求5或权利要求6所述的基因的宿主细胞，所述宿主细胞是中国仓鼠卵巢细胞。
9.一种制备权利要求1所述的mhFGF21突变细胞因子的方法，包括：将编码mhFGF21突变细胞因子的核苷酸与表达载体相连接，得到重组表达载体；将该重组表达载体转化宿主细胞；培养重组宿主细胞并诱导表达mhFGF21，收集培养基，纯化，即得。
10.一种制备权利要求3所述的融合蛋白mhFGF21-Fc的方法，包括：将编码融合蛋白mhFGF21-Fc的核苷酸与表达载体相连接，得到重组表达载体；将该重组表达载体转化宿主细胞；培养宿主细胞表达mhFGF21-Fc融合蛋白，收集培养基，纯化，即得。
11.一种制备权利要求4所述的融合蛋白Fc-mhFGF21的方法，包括：将编码融合蛋白Fc-mhFGF21的核苷酸与表达载体相连接，得到重组表达载体；将该重组表达载体转化宿主细胞；培养宿主细胞表达Fc- mhFGF21融合蛋白，收集培养基，纯化，即得。
12.一种治疗代谢疾病的药物组合物，包括：治疗上有效量的权利要求1所述的突变细胞因子或权利要求3所述的融合蛋白或权利要求4所述的融合蛋白，以及药学上可接受的载体或辅料。
13.权利要求1所述的突变细胞因子或权利要求3所述的融合蛋白或权利要求4所述的融合蛋白在制备治疗代谢疾病药物中的用途，所述的代谢疾病为糖尿病、肥胖症或代谢综合症。
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| CN111909246B (zh) * | 2019-05-08 | 2024-01-19 | 中国科学院脑科学与智能技术卓越创新中心 | 高效感染支持细胞的aav突变体 |
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