CN115286705A - 一种黄鳝成纤维细胞因子21重组蛋白及其制备方法和应用 - Google Patents
一种黄鳝成纤维细胞因子21重组蛋白及其制备方法和应用 Download PDFInfo
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Abstract
本发明属于基因工程技术领域,公开了一种黄鳝成纤维细胞因子21重组蛋白及其制备方法和应用,通过从黄鳝肝脏中克隆黄鳝成纤维细胞因子21基因并进行体外表达和分离纯化,获得黄鳝成纤维细胞因子21重组蛋白,该重组蛋白对1型糖尿病模型小鼠的血糖及肝脏、血清等组织中的胆固醇和甘油三酯等含量有积极影响,有望作为药物治疗1型糖尿病。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种黄鳝成纤维细胞因子21重组蛋白及其制备方法和应用。
背景技术
细胞内糖脂代谢的稳态调控是维持机体与细胞基本生命活动的基础,糖脂代谢的紊乱会增加机体患糖尿病、脂肪肝、细胞异常增殖等多种疾病的几率(Jones2016;Lima etal.2015)。机体通过糖原生成、糖原分解、糖酵解和糖异生途径控制葡萄糖代谢,从而严格调节葡萄糖稳态以满足重要器官的能量需求并维持个体健康。肝脏在控制葡萄糖稳态中起主要作用(Han et al.2016)。脂质稳态是通过脂肪生成和脂解的调节来维持的,该过程包含多种关键的酶、转录因子和信号通路(Lee et al.2003),如乙酰辅酶a羧化酶(ACC)、FA合成酶(FAS)和葡萄糖6-磷酸脱氢酶(G6PD)等与脂质代谢相关的酶(Fang et al.2019;Weiet al.2017),甾醇调节元件结合蛋白-1(SREBP1)、PPARα和PPARγ等关键的转录因子(Dutchak et al.2012;Yahagi et al.1999),以及Janus激酶信号转导与转录激活因子(JAK-STAT)和AMP活化蛋白激酶(AMPK)信号通路,通过脂解和脂肪生成参与调节脂质稳态(Hayley et al.2013;Wei et al.2017)。
成纤维细胞生长因子21(FGF21)是FGF亚家族成员之一,由22个多肽组成,在多个器官中合成,可以以旁分泌或内分泌的方式作用于多个靶组织(Fisher and Maratos-Flier 2016;Geng et al.2020)。FGF21通过成纤维细胞生长因子受体(FGFRs)与单程跨膜蛋白(β-Klotho)形成的细胞表面受体发挥作用(Lan et al.2017)。FGF21能增加糖异生和三羧酸循环通量(Badman et al.2007),且具有刺激脂肪酸氧化、酮体产生以及抑制脂肪生成的功能(Inagaki et al.2007;Potthoff et al.2009),因此FGF21被判定为调节糖脂代谢的有效靶点。动物实验也证实,给予外源性重组FGF21可降低肝脏及外周血中三酰甘油水平;FGF21类似物可以降低外周血三酰甘油、总胆固醇水平,并有减轻体质量的作用(Liu etal.,2018)。
尽管人类的重组成纤维细胞因子21蛋白(hFGF21)具有很多的用途,但是人类FGF21(hFGF21)的原生形态不适合临床使用,原因在于其较差的药代动力学特征,使用半衰期短(只有0.5~1.5h)。并且,天然hFGF21还具有在血浆中容易发生蛋白裂解和失活、在溶液中不稳定等缺陷。因此,人们采用各种生物制药工程方法开发hFGF21的类似物和模拟物,期待改善它的生物物理特性和药代动力学特征,以适用于未来临床。故从动物中寻找成纤维细胞因子21基因并制备具有活性的FGF21同源类似物具有重要的科学价值。
发明内容
有鉴于此,本发明通过从黄鳝肝脏中克隆成纤维细胞因子21基因并进行体外表达和分离纯化,获得黄鳝成纤维细胞因子21重组蛋白(rFGF21),从动物模型试验中验证了rFGF21在血糖、体重、血清和肝脏组织中甘油三酯和胆固醇含量方面的活性。
本发明的技术方案具体如下:
本发明首先提供了一种黄鳝成纤维细胞因子21重组蛋白,其氨基酸序列如SEQ IDNO:2所示,编码上述重组蛋白的基因序列如SEQ ID NO:1所示。
本发明还提供了制备上述黄鳝成纤维细胞因子21重组蛋白的方法,包括以下步骤:
S1.提取黄鳝肝脏组织总RNA,反转录合成cDNA,采用引物进行PCR扩增,扩增产物经回收、纯化后得到目标基因片段;所述引物的序列如SEQ ID NO:3和SEQ ID NO:4所示;
S2.所述目标基因片段与载体连接,得到表达载体;
S3.将表达载体转化至受体细胞中,再进行诱导表达;
S4.提取纯化目的蛋白。
优选地,所述步骤S2具体为:将目标基因片段与载体pET-28a同时进行EcoRI和Hind III双酶切,经回收、纯化、连接后,构建得到原核表达载体pET-fgf21。
优选地,所述受体细胞为E.coli BL21(DE3)受体细胞。
优选地,所述诱导表达过程具体为:将阳性表达菌株接种至含有卡那霉素的培养基中培养,用IPTG进行诱导表达;进一步,所述阳性表达菌株的获得方法为:将转化后的受体细胞涂布于含卡那霉素平板上进行抗性筛选,并经菌落PCR验证及SDS-PAGE电泳检测获得。
优选地,所述步骤S4具体为:将诱导表达后的菌液离心收集菌体,菌体经超声破碎后离心收集沉淀,将沉淀用平衡缓冲液溶解,收集上清,加至Ni离子亲和柱中,结合后洗脱,即得。
上述黄鳝成纤维细胞因子21重组蛋白在1型糖尿病小鼠中具有降血糖及调节糖脂平衡的作用,且在1型糖尿病小鼠肝脏中具有减缓肝细胞损伤的作用,故所述黄鳝成纤维细胞因子21重组蛋白能够应用于治疗1型糖尿病的药物中,尤其是在抑制和治疗1型糖尿病造成的肝细胞损伤方面有显著的积极作用。
本发明的有益效果为:
1)利用基因工程技术,构建了黄鳝成纤维细胞因子21基因的原核表达载体,通过诱导表达和纯化获得了其重组蛋白rFGF21;
2)本发明制备的重组蛋白rFGF21能够有效调节1型糖尿病小鼠体内的血糖水平,而且首次发现重组蛋白rFGF21能够在减缓肝细胞损伤,故其可作为哺乳动物FGF21类似物应用于糖尿病治疗。
附图说明
图1为实施例1中的SDS-PAGE电泳图,其中,泳道1:空白质粒菌株,泳道2:诱导前的含pET-28-fgf21菌株,泳道3:诱导后的含pET-28-fgf21菌株,泳道4:纯化后的rFGF21蛋白,M:Marker;
图2为实施例2中小鼠肝脏甘油三酯含量与重组蛋白剂量关系图;
图3为实施例2中小鼠肝脏胆固醇含量与重组蛋白剂量关系图;
图4为实施例2中小鼠血清甘油三酯含量与重组蛋白剂量关系图;
图5为实施例2中小鼠血清胆固醇含量与重组蛋白剂量关系图;
图6为实施例2中小鼠血糖含量与重组蛋白剂量关系图;
图7为实施例2中小鼠体重变化与重组蛋白剂量关系图;
图8为实施例2中小鼠肝脏细胞显微切片图,其中,HC:肝细胞,FV:脂肪小泡,V:空泡化,PN:核固缩,PD:实质结构紊乱。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1
制备黄鳝成纤维细胞因子21重组蛋白,具体过程如下:
(1)黄鳝成纤维细胞生长因子FGF21基因的克隆
按照Trizol试剂盒的说明书提取黄鳝肝脏总RNA,利用M-MLV反转录酶合成cDNA,于-80℃低温保存。
根据黄鳝基因设计引物,其具体序列如下:
F21-RC-F:5’-GCCGAATTCATGTGTTTTTCATACCTGG-3’(SEQ ID NO:3);
F21-RC-R:5’-CCGAAGCTTCTTGTCCATTGAAAAACTGAGG-3’(SEQ ID NO:4);
其中,上游引物序列添加了EcoR I酶切位点,下游引物添加了Hind III酶切位点。
进行常规PCR扩增,扩增产物经回收、纯化、连接并测序后,获得了目的基因的开放阅读框序列,其核苷酸序列如SEQ ID NO:1所示。
(2)表达载体的构建
将上述目的基因片段与原核表达载体pET-28a(+)同时进行EcoR I和Hind III双酶切,分别用DNA凝胶回收试剂盒纯化酶切产物,并利用T4 DNA连接酶将扩增产物和载体连接,经测序验证后,成功构建得到原核表达载体pET-fgf21。
(3)重组蛋白rFGF21的表达纯化
将测序正确的pET-fgf21重组质粒转化大肠杆菌BL21(DE3)感受态细胞,涂布于LB平板(含50μg/mL Kana)上,37℃过夜培养。挑取单克隆接种于LB培养基(含50μg/mL Kana)中,37℃、220r/min振荡培养至菌液OD600约为0.6时,加入终浓度为0.1mmol/L的IPTG进行诱导表达,SDS-PAGE凝胶电泳检测FGF21蛋白的表达结果。
取200μL检测表达的原菌液加入200mL LB培养基(含50μg/mL Kana)中,扩大培养至菌液OD600约为0.6时,加入终浓度为0.1mmol/L的IPTG诱导培养。
诱导培养后的菌液4℃、10000r/min离心10min收集菌体,冰上超声破碎菌体5min后,用8mol/L的尿素溶解包涵体。上清经过0.45μm滤膜过滤后加入Ni-NTAHis·Bind Resin亲和柱(七海生物,中国上海)结合1h以上,用含20mmol/L咪唑的Tris-HCl缓冲液去除杂蛋白,用含100mmol/L咪唑的Tris-HCl缓冲液洗脱rFGF21蛋白。将纯化后的rFGF21蛋白置于含不同浓度尿素的Tris-HCl缓冲液中进行梯度透析,直至去除rFGF21蛋白中的尿素。BCA试剂盒(Biosharp,中国北京)检测蛋白浓度,利用SDS-PAGE凝胶电泳检测FGF21蛋白的纯化结果。经氨基酸测序分析,FGF21蛋白的氨基酸序列如SEQ ID NO:2所示。
SDS-PAGE电泳结果如图1所示,表明本实施例成功实现了rFGF21重组蛋白的体外表达与纯化。
实施例2
昆明种雄性小鼠,平均体重21±1.7g,由湖北中医高专实验动物中心提供,饲养在26℃有空调的房内,自然光照,自由采食和饮水,适应性喂养7d后用于建模。
1型糖尿病小鼠模型的建立参考文献方法(Li et al.2012),造模前小鼠禁食12h以上,将STZ(Solarbio,中国上海)用pH为4.3的0.1mol/L的柠檬酸钠/柠檬酸缓冲溶液溶解,对小鼠连续5天腹腔注射诱导1型糖尿病,剂量为50mg/kg体质量,对照组小鼠注射等体积的柠檬酸钠/柠檬酸缓冲溶液。最后1次注射7天后小鼠尾静脉采血并使用血糖仪(鱼跃,中国上海)检测血糖浓度,连续检测3日,血糖水平均高于16.7mmoL/L的小鼠被认定为糖尿病造模成功。
将建模成功的小鼠根据血糖水平与体重随机分为3组,每组5只,分别腹腔注射100μL 0.9%生理盐水(T1DM组),100μL rFGF21蛋白(0.125mg/Kg体质量)(0.125mg/kg rFGF21组),100μL rFGF21蛋白(0.75mg/Kg体质量)(0.75mg/kg rFGF21组),并随机选取5只健康小鼠,腹腔注射100μL 0.9%生理盐水(对照组),连续注射15天,每天一次。最后一次注射24h后,对小鼠采取摘除眼球法采血,采出的全血4000r/min离心10min分离血清。采血结束后颈椎脱臼处死小鼠,采集小鼠肝脏和血清一起保存于-80℃进行后续的甘油三酯和胆固醇的测定。
①小鼠肝脏和血清中甘油三酯与胆固醇含量检测
按照生产说明书,使用总胆固醇(TC)含量检测试剂盒(Solarbio,中国北京)提取小鼠血清及肝脏的总胆固醇并测定各样品在500nm处吸光值,根据标准曲线计算各样品中TC含量。
根据制造商的说明,使用甘油三酯(TG)含量检测试剂盒(Solarbio,中国北京)提取小鼠血清及肝脏的甘油三酯并测定各样品在420nm处吸光值,通过与标准品(1mg/mL)在420nm处吸光值的比值计算各样品中TG含量。
结果如图2~图5所示:经腹腔重复注射所述重组蛋白15d后,可显著降低T1DM小鼠血清和肝脏组织总胆固醇、甘油三酯的含量,即所述重组蛋白具有调节糖脂代谢平衡的作用。
②小鼠血糖含量测定及体重变化检测
每次对T1DM小鼠注射重组蛋白rFGF21前,记录小鼠体重并尾静脉采血,用血糖仪(鱼跃,中国上海)检测血糖浓度。
结果表明:T1DM组小鼠血糖显著高于对照组,与T1DM组小鼠相比,rFGF21注射组小鼠的血糖在1-3d显著降低,在6-9d回升至与T1DM组小鼠相同水平,然后又于12-15d显著降低(图6)。T1DM组小鼠的体重变化值显著低于对照组,0.75mg/kg rFGF21注射组小鼠的体重变化值在3d与15d时显著低于T1DM组与0.125mg/kg rFGF21注射组,除此之外,rFGF21注射组与T1DM组小鼠的体重变化值没有显著差异(图7)。
③注射重组蛋白rFGF21对小鼠肝脏细胞的保护作用分析
将小鼠肝脏用苯甲醛固定后包埋在石蜡中。矢状切片用苏木精-伊红(H&E)试剂盒(Solarbio,中国北京)染色,然后在正置显微镜下观察细胞损伤程度。结果表明,健康小鼠的肝脏细胞排列整齐,建模成功的T1DM小鼠肝脏细胞排列紊乱,脂肪小泡数量增加,且出现细胞质固缩与细胞质空泡化现象。注射不同浓度rFGF21蛋白后,肝细胞损伤程度呈剂量依赖性降低,0.75mg/kg rFGF21注射组的小鼠肝脏细胞恢复至正常水平,接近有序排列,表现为几乎没有观察到细胞空泡化现象,而0.125mg/kg注射组则仍有部分细胞排列紊乱、脂肪小泡数量有所增加,细胞质空泡化现象也有少量存在(图8)。这些结果表明了重组蛋白rFGF21的注射有利于减缓1型糖尿病对肝组织细胞的损伤作用。
以上所述本发明的具体实施方式,并不构成对本发明保护范围的限定。任何根据本发明的技术构思所做出的各种其他相应的改变与变形,均应包含在本发明权利要求的保护范围内。
SEQUENCE LISTING
<110> 长江大学
<120> 一种黄鳝成纤维细胞因子21重组蛋白及其制备方法和应用
<130> 2021.12.23
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1143
<212> DNA
<213> 黄鳝
<400> 1
caaaaaaaaa gataaacacc ataaactcaa aacaacgagt catttctgcc cgtaattgct 60
tgttgtaatt tacttactag atgatgcaga tcgatgtgtt tttcatacct ggaagcctgg 120
atgggaaaga aaaacatgac ggcttactgt cagcaaaagg tcagagaagt gcatctctac 180
acagataacc acagaagagg gatgtatctg caaatgactt tggatgggac agtgtcagga 240
agtgatgttc agacccctta cagtgtgctg gagctgaaag cagttaaacc aggccacata 300
gtcatcaagg gacagttatc atctttgttt ctctgtgtgg acaacacggg ccatttgagg 360
ggccagagtc actacacaga ggccgactgc agcttcaaag aactgctgct ggtggatgga 420
tacacacgtt ttctttcctc atcccatgga tttcctgtgt ctctggcaac aaaacagtca 480
ccagagcaac actcagtccc cttcattcga ttcttaccac ttaggaatac cctgatagtg 540
gagggtgtat ctgaacaacc accagacaat cagaaatatt tcagcgtgga ctctgatgac 600
cttctcggaa tgattcatag gcctatggtc agtcctcagt tttcaatgga caagtagcac 660
tacacagagg ccgactgcag cttcatttga tggtatcaag gaaaagctcc aggaaatcag 720
ccaggccatt tctgctacct tcaccccaaa ctaaatcctc tctatcctgt ctgatcttaa 780
tccattccaa ctttttccat tccttctttt ttcacatgca cacacagagc acctcccaaa 840
atgaagccag tgcttaaatg atgcactttt ttgcaacaaa atggtgggac ttttattttt 900
tctcccaagc aaacaccttg cacaagcaca tgaaagcaaa gaaatacacg gacatacatg 960
ctcagcacac acatgcttca ctcagtgctt ggccaactgc ttacattttg tgtaacagtt 1020
gtacgtgttg tgcctgaacc tgtgtaatga tgcttaactg atgaaaactg tgctttgatg 1080
atatacagct caataaacat ctgactgttt atactaaaaa aaaaaaaaaa aaaaaaaaaa 1140
aaa 1143
<210> 2
<211> 187
<212> PRT
<213> 人工序列
<400> 2
Met Cys Phe Ser Tyr Leu Glu Ala Trp Met Gly Lys Lys Asn Met Thr
1 5 10 15
Ala Tyr Cys Gln Gln Lys Val Arg Glu Val His Leu Tyr Thr Asp Asn
20 25 30
His Arg Arg Gly Met Tyr Leu Gln Met Thr Leu Asp Gly Thr Val Ser
35 40 45
Gly Ser Asp Val Gln Thr Pro Tyr Ser Val Leu Glu Leu Lys Ala Val
50 55 60
Lys Pro Gly His Ile Val Ile Lys Gly Gln Leu Ser Ser Leu Phe Leu
65 70 75 80
Cys Val Asp Asn Thr Gly His Leu Arg Gly Gln Ser His Tyr Thr Glu
85 90 95
Ala Asp Cys Ser Phe Lys Glu Leu Leu Leu Val Asp Gly Tyr Thr Arg
100 105 110
Phe Leu Ser Ser Ser His Gly Phe Pro Val Ser Leu Ala Thr Lys Gln
115 120 125
Ser Pro Glu Gln His Ser Val Pro Phe Ile Arg Phe Leu Pro Leu Arg
130 135 140
Asn Thr Leu Ile Val Glu Gly Val Ser Glu Gln Pro Pro Asp Asn Gln
145 150 155 160
Lys Tyr Phe Ser Val Asp Ser Asp Asp Leu Leu Gly Met Ile His Arg
165 170 175
Pro Met Val Ser Pro Gln Phe Ser Met Asp Lys
180 185
<210> 3
<211> 28
<212> DNA
<213> 人工序列
<400> 3
gccgaattca tgtgtttttc atacctgg 28
<210> 4
<211> 31
<212> DNA
<213> 人工序列
<400> 4
ccgaagcttc ttgtccattg aaaaactgag g 31
Claims (8)
1.一种黄鳝成纤维细胞因子21重组蛋白,其特征在于,其氨基酸序列如SEQ ID NO:2所示。
2.编码权利要求1所述黄鳝成纤维细胞因子21重组蛋白的核酸分子,其特征在于,其核苷酸序列如SEQ ID NO:1所示。
3.制备权利要求1所述黄鳝成纤维细胞因子21重组蛋白的方法,其特征在于,包括以下步骤:
S1.提取黄鳝肝脏组织总RNA,反转录合成cDNA,采用引物进行PCR扩增,扩增产物经回收、纯化后得到目标基因片段,所述引物的序列如SEQ ID NO:3和SEQ ID NO:4所示;
S2.所述目标基因片段与载体连接,得到表达载体;
S3.将表达载体转化至受体细胞中,再进行诱导表达;
S4.提取纯化目的蛋白。
4.根据权利要求3所述的方法,其特征在于,步骤S2具体为:将目标基因片段与载体pET-28a同时进行EcoRI和Hind III双酶切,经回收、纯化、连接后,构建得到原核表达载体pET-fgf21。
5.根据权利要求3所述的方法,其特征在于,所述受体细胞为E.coli BL21(DE3)受体细胞。
6.根据权利要求3所述的方法,其特征在于,步骤S4具体为:将诱导表达后的菌液离心收集菌体,菌体经超声破碎后离心收集沉淀,将沉淀用平衡缓冲液溶解,收集上清,加至Ni离子亲和柱中,结合后洗脱,即得目的蛋白。
7.权利要求1所述黄鳝成纤维细胞因子21重组蛋白在制备治疗1型糖尿病药物中的应用。
8.根据权利要求7所述的应用,其特征在于,所述黄鳝成纤维细胞因子21重组蛋白在制备抑制1型糖尿病造成的肝细胞损伤的药物中的应用。
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