CN113292656A - 用于防治肥胖的中脑星形胶质细胞源性神经营养因子的融合蛋白 - Google Patents
用于防治肥胖的中脑星形胶质细胞源性神经营养因子的融合蛋白 Download PDFInfo
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- CN113292656A CN113292656A CN202010107989.1A CN202010107989A CN113292656A CN 113292656 A CN113292656 A CN 113292656A CN 202010107989 A CN202010107989 A CN 202010107989A CN 113292656 A CN113292656 A CN 113292656A
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Abstract
本发明涉及用于防治肥胖的中脑星形胶质细胞源性神经营养因子的融合蛋白,属于生物医药技术领域。本发明提供了中脑星形胶质细胞源性神经营养因子的融合蛋白,至少含有以下两个片段:中脑星形胶质细胞源性神经营养因子的氨基酸序列和免疫球蛋白Fc段的氨基酸序列。该融合蛋白稳定性好,半衰期长,可用于肥胖、糖尿病及其它代谢性疾病的治疗,具有广阔的应用前景。
Description
技术领域
本发明涉及用于防治肥胖的中脑星形胶质细胞源性神经营养因子的融合蛋白,属于生物医药技术领域。
背景技术
随着社会经济的发展和人们生活方式的改变,肥胖已成为严重的全球性公共卫生问题,是高血压、糖尿病、心脑血管疾病等发生发展的重要危险因素。探寻安全有效的减脂、减肥方法是当今医学研究的热点之一。
肥胖形成的主要原因是长期的能量平衡调节异常,包括能力摄取过多和消耗过少,导致多余的能量以甘油三脂的形式储存在脂肪组织中。脂肪组织可分为白色脂肪组织、棕色脂肪组织及米色脂肪组织。白色脂肪组织主要以甘油三酯的形式储存及释放能量。棕色脂肪组织能有效地将化学能以热能形式消耗。此外,在寒冷、运动等因素刺激下,白色脂肪细胞可发生“白色脂肪棕色化”,生成米色脂肪细胞。研究表明,米色脂肪组织可增加机体产热、提高机体能量代谢,抑制肥胖形成,改善胰岛素抵抗。因此,诱导或促进米色脂肪生成,为肥胖等代谢性疾病的防治提供了新的思路。米色脂肪组织的生成受多种因素的调控,其中分泌因子为一类重要的调节因素。研究表明,多种分泌因子,如利钠肽、BMP7/BMP4、FGF21及Irisin等均可由相应器官分泌进入血液循环,到达脂肪组织,促进米色脂肪细胞的生成。
中脑星形胶质细胞源性神经营养因子(Mesencephalic astrocyte-derivedneurotrophic factor,MANF)是一种新型神经营养因子,也是一种分泌蛋白。近年的研究表明,MANF在肥胖、糖尿病等代谢性疾病中具有重要作用,是一种具有临床应用潜力的蛋白多肽类药物。然而,众所周知,蛋白类药物在胃肠道内易被蛋白酶类降解,仅限于注射给药,而且,由于蛋白多肽类药物血浆半衰期短,为了达到有效治疗浓度,往往需要每天多次反复注射,这无疑给临床使用带来诸多不便。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此,本发明的目的在于提供中脑星形胶质细胞源性神经营养因子的融合蛋白。本发明的另一目的在于提供所述融合蛋白的制备方法和用途。
本发明提供了中脑星形胶质细胞源性神经营养因子的融合蛋白,至少含有以下两个片段:中脑星形胶质细胞源性神经营养因子的氨基酸序列和免疫球蛋白Fc段的氨基酸序列。
进一步地,所述中脑星形胶质细胞源性神经营养因子的氨基酸序列如SEQ IDNo.1所示,或者与SEQ ID No.1具有50%以上的同源性且具有相同或相似的生物学活性。
优选地,所述中脑星形胶质细胞源性神经营养因子的氨基酸序列如SEQ ID No.1所示,或者与SEQ ID No.1具有98%以上的同源性且具有相同或相似的生物学活性。
进一步优选地,所述中脑星形胶质细胞源性神经营养因子的氨基酸序列如SEQ IDNo.1所示,或者为来源于人的中脑星形胶质细胞源性神经营养因子的天然序列。
中脑星形胶质细胞源性神经营养因子(MANF)在人到各级动物中均有表达,且其序列在进化上高度保守,人MANF氨基酸序列与小鼠同源性高达98%,与非洲爪蟾蜍具有82%的同源性,与斑马鱼具有72%的同源性,与果蝇具有52%的同源性,与新杆状线虫具有50%的同源性。以下实施例在肥胖小鼠模型上验证药效,因此具体采用鼠源的中脑星形胶质细胞源性神经营养因子来制备融合蛋白,其氨基酸序列如下所示:
除此之外,为制备药物的需要,也可视需要使用其它来源的MANF制备融合蛋白,如人MANF。
进一步地,所述的免疫球蛋白为IgG。
优选地,所述的免疫球蛋白为IgG1。
进一步优选地,所述的免疫球蛋白来源于人或鼠。
进一步地,所述免疫球蛋白Fc段的氨基酸序列如SEQ ID No.2所示,或者与SEQ IDNo.2具有80%以上的同源性且具有相同或相似的功能。
进一步优选地,所述免疫球蛋白Fc段的氨基酸序列如SEQ ID No.2所示,或者为来源于人的IgG1的天然序列。
以下实施例在肥胖小鼠模型上验证药效,因此具体采用鼠源免疫球蛋白IgG1的Fc段来制备融合蛋白,其氨基酸序列如下所示:
除此之外,为制备药物的需要,也可视需要使用其它来源的免疫球蛋白Fc段制备融合蛋白,如人免疫球蛋白IgG1的Fc段,其与SEQ ID No.2具有80%的同源性。
进一步地,所述中脑星形胶质细胞源性神经营养因子的C末端与所述免疫球蛋白Fc段的N末端连接。
优选地,所述中脑星形胶质细胞源性神经营养因子通过连接序列与所述免疫球蛋白Fc段连接,或者两者直接相连。
本发明提供了多核苷酸,其编码所述的融合蛋白。
优选地,所述多核苷酸的序列如SEQ ID No.5所示。
本发明提供了重组载体,其含有所述的多核苷酸。
优选地,所述的载体为proEM。
本发明提供了宿主细胞,其含有所述的重组载体。
优选地,所述的宿主细胞为HEK293细胞。
本发明提供了所述融合蛋白的制备方法,包括如下步骤:培养所述的宿主细胞,使其表达所述的融合蛋白,然后回收所述的融合蛋白,即得。
本发明提供了组合物,它是以所述的融合蛋白为活性成分,加入可接受的辅料或者辅助性成分制备而成的制剂。
优选地,所述的制剂为注射剂。
本发明提供了所述的融合蛋白、所述的组合物在制备抑制体重增长的药品或保健品中的用途。
本发明提供了所述的融合蛋白、所述的组合物在制备抑制胰岛素抵抗的药品或保健品中的用途。
其中,胰岛素抵抗是指各种原因使胰岛素促进葡萄糖摄取和利用的效率下降,此时机体代偿性地分泌过多胰岛素以维持血糖的稳定,引起高胰岛素血症;胰岛素抵抗还易导致代谢综合征和II型糖尿病等疾病。
本发明提供了所述的融合蛋白、所述的组合物在制备治疗和/或预防代谢性疾病的药品或保健品中的用途。
优选地,所述的代谢性疾病为肥胖或II型糖尿病。
本发明利用Fc融合蛋白技术,提供了一种MANF-Fc融合蛋白,显著延长了MANF的体内半衰期,使其能够很好地用于肥胖、糖尿病等代谢性疾病的临床治疗,具有广阔的应用前景。
附图说明
图1为试验例1中MANF-Fc的体内药动学曲线图;
图2为试验例2中ob/ob小鼠的体重变化曲线图;
图3为试验例2中口服葡萄糖耐量试验和胰岛素耐量试验结果图;
图4为试验例2中ob/ob小鼠白色脂肪棕色化相关基因表达情况图;
图5为试验例3中高脂饮食诱导肥胖小鼠的体重变化曲线图;
图6为试验例3中口服葡萄糖耐量试验和胰岛素耐量试验结果图;
图7为实施例1中琼脂糖凝胶分析MANF-Fc抽提质粒结果图;
图8为实施例1中SDS-PAGE分析MANF-Fc蛋白纯化情况结果图。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1本发明融合蛋白MANF-Fc的制备
全基因合成MANF-Fc基因,其核苷酸序列(SEQ ID No.5)如下:
上述序列的1~6位GAATTC为酶切位点EcoR I,7~17位CCGCCGCCACC为KOZAK序列,1257~1262位TGATAA为终止密码子,1261~1266位AAGCTT为酶切位点HindIII。
然后通过双酶切法将MANF-Fc基因***到表达载体proEM中,并通过酶切法和测序确认最终表达载体的准确性。
接着将MANF-Fc质粒转到DH5α克隆菌株中,具体操作如下:
1、吸取含有MANF-Fc基因的表达质粒80-100ng加入已制备的DH5α感受态细胞中。
2、将加入质粒后的感受态细胞通过热激转化。
3、将转化后的感受态细胞加入LB液体培养基,放入摇床37℃,200rpm震荡培养约30min。
4、将培养后的感受态细胞取出,吸取部分悬液涂在含有氨苄抗性的平板上,放入培养箱,37℃,过夜培养。
5、从新鲜的培养板中挑取单克隆于2-5mL LB培养基,37℃,200rpm培养8h。
6、按1/500比例接种于200mL LB培养基中,37℃,200rpm培养16h。
7、收集培养后的菌液离心,去掉上清。
再通过质粒大抽试剂盒(具体采用Qiagen转染级质粒抽提试剂盒)提取转染级质粒:
1、1%琼脂糖凝胶分析抽提的MANF-Fc质粒,结果见图7,其中Lane M:DNA marker,Lane 1:转染级质粒。
2、转染级质粒DNA质粒质量测定,结果见下表:
检测内容 | 转染级质粒标准 | MANF-Fc质粒检测结果 |
A260/A280 | 1.8-2.0 | 1.89 |
内毒素 | 内毒素<50EU/mg | 鲎试剂检测,<50EU/mg |
无菌检测 | 无菌 | LB平板检测,无菌落 |
之后将质粒通过转染试剂转染到哺乳动物细胞HEK293中进行瞬时表达,具体操作如下:
HEK293细胞复苏与传代
1、提前将水浴锅打开37℃;培养基提前在37℃预热。
2、从液氮罐中取出冻存的HEK293细胞。
3、把冻存细胞立即投入37℃水浴锅中,轻微摇动使其快速融化(约1min),取出。
4、用75%乙醇消毒管外壁,放入生物安全柜内,转移到含10mL培养基的15mL离心管中,离心800rpm,5min。
5、离心后的细胞去掉上清,取少许新鲜培养基重悬细胞,再转移到培养瓶中,加入新鲜培养基轻轻摇动,使细胞分散均匀,取细胞计数及活力检测,使密度控制在3-4×105cells/mL,活力>95%。
6、置于培养箱中110rpm,37℃,5%CO2培养。
7、细胞培养2-3天,密度达到2.0×106cells/mL左右时需对细胞进行传代。
8、从培养瓶中取出部分培养物弃掉,再向培养瓶中补加新鲜培养基,对剩余的细胞培养物稀释(留下的细胞培养物根据细胞密度、培养体积、稀释后密度等而定)。
9、放入培养箱中110rpm,37℃,5%CO2,继续培养。
10、每天需对细胞密度及活力检测,当细胞密度达到2×106cells/mL左右时,要对细胞进行传代。
MANF-Fc质粒转染HEK293细胞
1、转染前1天将HEK293细胞悬浮培养至500mL,接种密度为1×106cells/mL,置于培养箱中110rpm,37℃,5%CO2培养。
2、转染当天使细胞密度控制在1-1.5×106cells/mL左右。
3、DNA-转染试剂混合物:向转染缓冲液中加入DNA和转染试剂混匀,37℃温育。
4、将DNA-转染试剂混合物加入待转染细胞中,放入培养箱中110rpm,37℃,5%CO2培养。
5、收集:转染后约4-6天,取出细胞培养物,离心,收集上清或细胞。
再通过亲和层析纯化MANF-Fc蛋白,具体操作如下:取转染培养5天后的细胞培养液离心,上清用0.22um滤器过滤,在4℃环境下透析至缓冲液1×PBS,pH7.4中,透析结束后再用Protein A柱纯化,结果见图8,其中Lane M:SDS-PAGE Protein Marker,Lane 1:离心后上清,Lane 2:上清同Protein A孵育后流出液,Lane 3-6:0.1M甘氨酸洗脱组分。经Protein A亲和层析纯化,目标蛋白MANF-Fc主要存在于洗脱组分Lane 3-4中,收集上述目标蛋白,并将其透析到1×PBS,pH7.8中,透析结束后用0.22um滤器过滤,并分装冻于-80℃。
MANF-Fc蛋白稳定性测试(冻融实验):取一支冻于-80℃的MANF-Fc蛋白,放置于冰水浴中5-10min待其缓慢融化,融化后放置于4℃冰箱内0.5h,无异常现象,说明蛋白冻融实验是正常的。
以下通过试验例证明本发明的有益效果。试验例中使用的MANF-Fc即根据实施例1制备得到的融合蛋白。
试验例1 MANF-Fc体内半衰期检测
C57BL/6小鼠皮下注射给予MANF-Fc(0.3mg/kg),分别于给药后的第1,4,7,10,13,17,21,25天尾静脉采血,采用Western blot的方法检测血清中MANF的表达量,检测结果见图1。
单独的MANF在体内的半衰期极短,注射后采用Western blot法在血清中几乎无法检测到MANF的存在。然而图1的结果显示,一次性给予MANF-Fc融合蛋白后,血清中的MANF浓度逐渐升高,到第7天的时候达到峰值,随后缓慢降低,一直持续到给药后的第25天,血清中仍然能检测到较高浓度的MANF,表明MANF-Fc融合蛋白的血浆半衰期明显增加。
试验例2 MANF-Fc对ob/ob肥胖小鼠的治疗效果
8周龄ob/ob瘦素缺陷型小鼠分为2组,分别皮下注射给予MANF-Fc融合蛋白和对照的Fc蛋白(0.3mg/kg),每周给药一次,一共给予3次。给药后每天监测小鼠体重,结果见图2。于末次给药后采用口服葡萄糖耐量试验和胰岛素耐量试验考察模型小鼠的胰岛素敏感性,结果见图3。上述小鼠处死后,取皮下脂肪组织检测棕色化指标UCP-1、Cidea、Dio2、Pgc-1α的表达,结果见图4。
结果显示,每周一次皮下注射给予MANF-Fc融合蛋白能够有效地控制ob/ob小鼠的体重增长,显著降低胰岛素抵抗,并能促进白色脂肪棕色化。
试验例3 MANF-Fc对高脂饮食(HFD)诱导肥胖小鼠的治疗效果
C57BL/6小鼠高脂饮食(HFD)喂养12周后,随机分为2组,分别皮下注射给予MANF-Fc融合蛋白和对照的Fc蛋白(0.3mg/kg),每周给药一次,一共给予3次。给药后每天监测小鼠体重,结果见图5。于末次给药后采用口服葡萄糖耐量试验和胰岛素耐量试验考察模型小鼠的胰岛素敏感性,结果见图6。
结果显示,每周一次皮下注射给予MANF-Fc融合蛋白能够有效地控制高脂饮食诱导肥胖小鼠的体重增长,显著降低胰岛素抵抗。
需要说明的是,本说明书中描述的具体特征、结构、材料或者特点可以在任一个或多个实施例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例以及不同实施例的特征进行结合和组合。
序列表
<110> 四川大学华西医院
<120> 用于防治肥胖的中脑星形胶质细胞源性神经营养因子的融合蛋白
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Claims (14)
1.中脑星形胶质细胞源性神经营养因子的融合蛋白,其特征是:至少含有以下两个片段:中脑星形胶质细胞源性神经营养因子的氨基酸序列和免疫球蛋白Fc段的氨基酸序列。
2.如权利要求1所述的融合蛋白,其特征是:所述中脑星形胶质细胞源性神经营养因子的氨基酸序列如SEQ ID No.1所示,或者与SEQ ID No.1具有50%以上的同源性且具有相同或相似的生物学活性;优选地,所述中脑星形胶质细胞源性神经营养因子的氨基酸序列如SEQ ID No.1所示,或者与SEQ ID No.1具有98%以上的同源性且具有相同或相似的生物学活性;进一步优选地,所述中脑星形胶质细胞源性神经营养因子的氨基酸序列如SEQ IDNo.1所示,或者为来源于人的中脑星形胶质细胞源性神经营养因子的天然序列。
3.如权利要求1所述的融合蛋白,其特征是:所述的免疫球蛋白为IgG;优选地,所述的免疫球蛋白为IgG1;进一步优选地,所述的免疫球蛋白来源于人或鼠。
4.如权利要求1所述的融合蛋白,其特征是:所述免疫球蛋白Fc段的氨基酸序列如SEQID No.2所示,或者与SEQ ID No.2具有80%以上的同源性且具有相同或相似的功能;进一步优选地,所述免疫球蛋白Fc段的氨基酸序列如SEQ ID No.2所示,或者为来源于人的IgG1的天然序列。
5.如权利要求1~4任意一项所述的融合蛋白,其特征是:所述中脑星形胶质细胞源性神经营养因子的C末端与所述免疫球蛋白Fc段的N末端连接;优选地,所述中脑星形胶质细胞源性神经营养因子通过连接序列与所述免疫球蛋白Fc段连接,或者两者直接相连;进一步优选地,所述的连接序列如SEQ ID No.3所示。
6.如权利要求1~5任意一项所述的融合蛋白,其特征是:所述融合蛋白的氨基酸序列如SEQ ID No.4所示。
7.多核苷酸,其特征是:编码权利要求1~6任意一项所述的融合蛋白;优选地,所述多核苷酸的序列如SEQ ID No.5所示。
8.重组载体,其特征是:含有权利要求7所述的多核苷酸;优选地,所述的载体为proEM。
9.宿主细胞,其特征是:含有权利要求8所述的重组载体;优选地,所述的宿主细胞为HEK293细胞。
10.权利要求1~6任意一项所述融合蛋白的制备方法,其特征是:包括如下步骤:培养权利要求9所述的宿主细胞,使其表达所述的融合蛋白,然后回收所述的融合蛋白,即得。
11.组合物,其特征是:它是以权利要求1~6任意一项所述的融合蛋白为活性成分,加入可接受的辅料或者辅助性成分制备而成的制剂;优选地,所述的制剂为注射剂。
12.权利要求1~6任意一项所述的融合蛋白、权利要求11所述的组合物在制备抑制体重增长的药品或保健品中的用途。
13.权利要求1~6任意一项所述的融合蛋白、权利要求11所述的组合物在制备抑制胰岛素抵抗的药品或保健品中的用途。
14.权利要求1~6任意一项所述的融合蛋白、权利要求11所述的组合物在制备治疗和/或预防代谢性疾病的药品或保健品中的用途;优选地,所述的代谢性疾病为肥胖或II型糖尿病。
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