CN109053875A - 突变型igf-1、重组质粒、重组蛋白及应用 - Google Patents
突变型igf-1、重组质粒、重组蛋白及应用 Download PDFInfo
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Abstract
本发明涉及一种突变型IGF‑1、重组质粒、重组蛋白及应用。本发明构建了一种IGF‑1突变体,其与IGF‑1竞争地结合受体IGF‑1R,从而在不影响其他生物行为的情况下,调控肿瘤细胞的某些生物行为。经验证,该IGF‑1突变体可抑制肿瘤细胞的迁移,且不会促进肿瘤细胞的增殖,达到IGF‑1竞争性抑制剂目的。
Description
技术领域
本发明属于分子生物学领域,涉及构建一种突变型IGF-1、重组质粒、重组蛋白及应用。
背景技术
胰岛素样生长因子1(Insulin-Like Growth Factor-1,IGF-1),是由70个氨基酸残基构成的单链多肽,作为一种主要由肝脏细胞合成和分泌的重要生长刺激因子,在人体多种细胞中广泛存在并发挥着重要的生理作用。对IGF-1的研究发现IGF-1与其受体IGF-1R在乳腺癌、子宫癌、前列腺癌、非小细胞肺癌等多种恶性肿瘤中呈现出高水平表达。一旦IGF-1与IGF-1R结合,便诱导IGF-1R自身的特定酪氨酸残基的磷酸化,接着激活一系列下游通路,从而促进肿瘤细胞的增殖、迁移等行为。因此,IGF-1是癌症治疗的靶标之一。
近年来,一些以IGF-1受体为靶点的靶向治疗药物已经被开发出来,包括针对IGF-1R的siRNA和单克隆抗体,以及抑制受体酶活性的激酶抑制剂等。这些研究提供了一种新的思路,即设计一种IGF-1突变体,该突变体作为IGF-1的竞争性抑制剂,可与IGF-1竞争地结合受体IGF-1R,从而可以在不影响其他生物行为的情况下,调控肿瘤细胞的某些生物行为。
发明内容
有鉴于此,本发明的目的之一在于提供突变型IGF-1(Glu37-Phe43-Leu59的突变型IGF-1,即IGF-1成熟肽的第37个氨基酸突变为谷氨酸,第43个氨基酸突变为苯丙氨酸,第59个氨基酸突变为亮氨酸)、利用其构建的重组质粒、诱导表达重组蛋白及应用。
为达到上述目的,本发明提供如下技术方案:
重组质粒的构建方法,包括步骤:
(1)采用重叠延伸PCR(overlap RT-PCR)和定点突变的方法进行PCR扩增,所用引物组合物如下:
IGF-1信号肽引物:
F1:5'-CGCGGATCC GCCACC ATGGGAAAAATCAG-3',如SEQ ID NO.3所示;
R1:5'-AGCGTCTCCGGTCCAGCCGTGGCAG-3',如SEQ ID NO.4所示;
野生型IGF-1成熟肽引物:
F2:5'-GGACCGGAGACGCTCTGCGG-3',如SEQ ID NO.5所示;
R2:5'-CCGGAATTCAGCTGACTTGGCAGGCTTGAGGGGTG-3',如SEQ ID NO.6所示;
突变型IGF-1成熟肽引物:
F3:5'-GGACCGGAGACGCTCTGCG-3',如SEQ ID NO.7所示;
R3:5'-GAAGCCTGTCTGAGGCGCCTCCTCACTGCTGG-3',如SEQ ID NO.8所示;
F4:5'-CCTCAGACAGGCTTCGTGGATGAGTGCT-3',如SEQ ID NO.9所示;
R4:5'-CCGGAATTCAGCTGACTTGGCAGGCTTGAGGGGTGCGCAATACAGCTCC-3',如SEQ IDNO.10所示;
经三轮扩增得到野生型IGF-1,其核苷酸序列如SEQ ID NO.1所示;经四轮扩增得到突变型IGF-1,其核苷酸序列如SEQ ID NO.2所示;扩增条件相同,为95℃预变性5分钟;94℃变性30秒,57℃退火30秒,72℃延伸30秒,共30个循环;最后72℃延伸8分钟。
野生型IGF-1:
ATGGGAAAAATCAGCAGTCTTCCAACCCAATTATTTAAGTGCTGCTTTTGTGATTTCTTGAAGGTGAAGATGCACACCATGTCCTCCTCGCATCTCTTCTACCTGGCGCTGTGCCTGCTCACCTTCACCAGCTCTGCCACGGCTGGACCGGAGACGCTCTGCGGGGCTGAGCTGGTGGATGCTCTTCAGTTCGTGTGTGGAGACAGGGGCTTTTATTTCAACAAGCCCACAGGGTATGGCTCCAGCAGTCGGAGGGCGCCTCAGACAGGCATCGTGGATGAGTGCTGCTTCCGGAGCTGTGATCTAAGGAGGCTGGAGATGTATTGCGCACCCCTCAAGCCTGCCAAGTCAGCT,如SEQ ID NO.1所示;
突变型IGF-1:
ATGGGAAAAATCAGCAGTCTTCCAACCCAATTATTTAAGTGCTGCTTTTGTGATTTCTTGAAGGTGAAGATGCACACCATGTCCTCCTCGCATCTCTTCTACCTGGCGCTGTGCCTGCTCACCTTCACCAGCTCTGCCACGGCTGGACCGGAGACGCTCTGCGGGGCTGAGCTGGTGGATGCTCTTCAGTTCGTGTGTGGAGACAGGGGCTTTTATTTCAACAAGCCCACAGGGTATGGCTCCAGCAGTCGGGAGGCGCCTCAGACAGGCTTCGTGGATGAGTGCTGCTTCCGGAGCTGTGATCTAAGGAGGCTGGAGCTGTATTGCGCACCCCTCAAGCCTGCCAAGTCAGCT,如SEQ ID NO.2所示;
(2)BamH1和EcoR1双酶切,连接,转化,菌落PCR和测序,成功构建野生型IGF-1重组质粒和突变型IGF-1重组质粒。
具体实验过程如下:
双酶切:目的片段双酶切反应体系为50μL:目的片段30μL,10×buffer 5μL,3dH2O13μL,BamH1 1μL,EcoR1 1μL,在37℃恒温水浴中酶切反应30min;载体双酶切反应体系为50μL:载体,10μL,10×buffer 5μL,3dH2O 33μL,BamH1 1μL,EcoR1 1μL,在37℃恒温水浴中酶切反应30min;
连接:反应体系为20μL:目的片段8μL,载体2μL,10×T4buffer 2μL,T4ligase 1μL,3dH2O 7μL,在PCR仪中16℃反应1h,放于4℃冰箱过夜;
转化:①从-80℃冰箱拿出DH5α感受态细胞,将其置于冰上,待其融化后;在超净工作台中,将10μL连接产物或者2μL质粒轻轻加入100μL感受态细胞中,轻轻混匀,冰浴30min;
②水浴锅预热到42℃,将EP管里混匀的连接产物和DH5α感受态细胞,放于水浴锅,42℃水浴1min,水浴完成后,立即放于冰上,冰浴2min;
③向EP管加900μL LB液体空白培养基,混匀,放于摇床,温度为37℃,转速150rpm,摇菌1h;
④摇菌完成后,放入离心机,10000rpm离心1min,上清液留约100μL,弃掉多余的上清液,重悬菌体,将其均匀滴在含25~50μg/mL氨苄青霉素的LB固体培养板上,用涂布棒涂抹均匀;等重悬菌液被完全吸收后,将培养板放在37℃培养箱中倒置培养12-16h。
菌落PCR:
①从条件筛选培养板(前述步骤的LB固体培养板)上挑选大小合适的单菌落,分别溶于10μL灭菌三次蒸馏水中,混匀;
②反应体系为10μL:3dH2O 2μL,2×Taq PCR Mix 5μL,Template(模板)1μL,R-primer(反向引物)1μL,F-primer(正向引物)1μL,扩增条件为95℃预变性5分钟;94℃变性30秒,57℃退火30秒,72℃延伸30秒,共30个循环;最后72℃延伸8分钟;4℃恒温1h;
③PCR产物进行琼脂糖凝胶电泳,选出含目的条带的菌落备用;
④将菌落PCR鉴定成功的样品中,剩下的菌液加入20μL含氨苄的LB液体培养基中,摇菌12-16h;
测序:取1mL样品送往上海生物生工公司测序。
利用上述突变型IGF-1构建质粒、诱导表达所得重组蛋白,其氨基酸序列如SEQ IDNO.11所示。
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSREAPQTGFVDECCFRSCDLRRLELYCAPLKPAKSA,如SEQID NO.11所示。
上述重组蛋白的制备方法,是将突变型IGF-1成熟肽经密码子优化后与pET28a质粒载体重组构建后诱导表达而得。
优选的,具体方法是:将所得重组质粒(pET28a-IGF-1,该质粒是由上海生工生物有限公司制备)转化入大肠杆菌BL21(DE3)中,诱导表达,离心收集菌体,超声波破碎,离心收集上清和沉淀,纯化即可。
是将上述突变型IGF-1重组质粒与pET28a质粒载体重组后转化入大肠杆菌BL21(DE3)中,诱导表达,离心收集菌体,超声波破碎,纯化即可。
优选的,诱导表达条件为:0.6mM IPTG(异丙基硫代半乳糖苷),温度37℃,诱导表达6小时。
优选的,超声波破碎后收集上清和沉淀,将上清和沉淀分别进行SDS-PAGE电泳,检测目的蛋白的存在形式,结果显示目的蛋白存在于沉淀中,弃上清,纯化沉淀。
优选的,纯化条件为:利用Ni-Agarose Resin柱(镍琼脂糖凝胶柱)进行纯化。具体方法是:
①将超声破碎后的沉淀用triton X-100清洗3次,加入8mol/L的尿素作为变性剂溶解蛋白,轻摇2-4h,再采用浓度梯度的方法,即分别6mol/L、4mol/L和2mol/L浓度的尿素作为复性剂,4℃冰箱过夜复性。
②向装填好Ni-Agarose Resin填料的柱中加入适量的蒸馏水将乙醇冲洗干净,重复2-3次,再加入适量的Buffer A(14.6g氯化钠、10mL 1MTris-HCl缓冲液(pH 8.0),蒸馏水至500mL)平衡柱子,平衡结束后即可上样;
③依次以洗脱液A(2.5mL 1M咪唑、47.5mL Buffer A)、B(5mL 1M咪唑、45mLBuffer A)、C(10mL 1M咪唑、40mL Buffer A)、D(12.5mL 1M咪唑、37.5mL Buffer A)、E(4mL1M咪唑、46mL Buffer A)、洗脱镍柱,将洗脱液分别收集起来;
④从收集的洗脱液中取36μL加入7μL 5×SDS-PAGE上样缓冲液后混匀煮沸10min,以15%浓度的胶进行SDS-PAGE电泳,检测纯化效果。
优选的,纯化前后分别利用SDS-PAGE检测蛋白的表达情况和纯化情况。
上述突变型IGF-1、重组质粒、重组蛋白在制备抑制肿瘤细胞迁移和增殖药物中的应用。
所述肿瘤细胞优选为人肝癌Hep.G2细胞。
上述突变型IGF-1、重组质粒、重组蛋白在作为IGF-1竞争性抑制剂中的应用。
本发明的有益效果在于:
本发明构建了一种IGF-1突变体,其与IGF-1竞争地结合受体IGF-1R,从而在不影响其他生物行为的情况下,调控肿瘤细胞的某些生物行为。经验证,该IGF-1突变体可抑制肿瘤细胞的迁移,且不会促进肿瘤细胞的增殖,达到IGF-1竞争性抑制剂目的。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为野生型IGF-1重组质粒和突变型IGF-1重组质粒的构建,其中,A为扩增信号肽的琼脂糖凝胶电泳图,B为扩增野生型IGF-1成熟肽的琼脂糖凝胶电泳图,C为扩增野生型IGF-1目的片段的琼脂糖凝胶电泳图,D为扩增突变型IGF-1成熟肽的琼脂糖凝胶电泳图,E为扩增突变型IGF-1目的片段的琼脂糖凝胶电泳图;F为野生型IFG-1重组质粒的质粒图谱,G为突变型IFG-1重组质粒的质粒图谱,H为野生型IGF-1的测序比对结果,I为突变型IGF-1的测序比对结果;M:Marker表示DNA相对分子质量标准,A中1、2表示IGF-1信号肽的PCR扩增产物,B中1、2表示野生型IGF-1成熟肽的PCR扩增产物,C中1、2表示野生型IGF-1的信号肽和成熟肽的重叠PCR扩增产物,D中1、2表示突变型IGF-1成熟肽的PCR扩增产物,E中1、2表示突变型IGF-1的信号肽和成熟肽的重叠PCR扩增产物;
图2为突变型IGF-1对人肝癌Hep.G2细胞功能的影响,以及同野生型IGF-1的比较,其中,A为突变型IGF-1对人肝癌Hep.G2细胞增殖的影响;B和C为突变型IGF-1对人肝癌Hep.G2细胞迁移的影响;
图3为重组蛋白突变型IGF-1的获得,其中,A为突变型IGF-1诱导表达,B为纯化后SDS-PAGE跑胶结果。
具体实施方式
下面将结合附图,对本发明的优选实施例进行详细的描述。
实施例1
参考图1,重组质粒的构建方法,包括步骤:
(1)采用重叠延伸PCR(overlap RT-PCR)和定点突变的方法进行PCR扩增,所用引物组合物如下:
IGF-1信号肽引物:
F1:5'-CGCGGATCC GCCACC ATGGGAAAAATCAG-3',如SEQ ID NO.3所示;
R1:5'-AGCGTCTCCGGTCCAGCCGTGGCAG-3',如SEQ ID NO.4所示;
野生型IGF-1成熟肽引物:
F2:5'-GGACCGGAGACGCTCTGCGG-3',如SEQ ID NO.5所示;
R2:5'-CCGGAATTCAGCTGACTTGGCAGGCTTGAGGGGTG-3',如SEQ ID NO.6所示;
突变型IGF-1成熟肽引物:
F3:5'-GGACCGGAGACGCTCTGCG-3',如SEQ ID NO.7所示;
R3:5'-GAAGCCTGTCTGAGGCGCCTCCTCACTGCTGG-3',如SEQ ID NO.8所示;
F4:5'-CCTCAGACAGGCTTCGTGGATGAGTGCT-3',如SEQ ID NO.9所示;
R4:5'-CCGGAATTCAGCTGACTTGGCAGGCTTGAGGGGTGCGCAATACAGCTCC-3',如SEQ IDNO.10所示;
经三轮扩增得到野生型IGF-1,其核苷酸序列如SEQ ID NO.1所示;经四轮扩增得到突变型IGF-1,其核苷酸序列如SEQ ID NO.2所示;扩增条件相同,为95℃预变性5分钟;94℃变性30秒,57℃退火30秒,72℃延伸30秒,共30个循环;最后72℃延伸8分钟。
野生型IGF-1:
ATGGGAAAAATCAGCAGTCTTCCAACCCAATTATTTAAGTGCTGCTTTTGTGATTTCTTGAAGGTGAAGATGCACACCATGTCCTCCTCGCATCTCTTCTACCTGGCGCTGTGCCTGCTCACCTTCACCAGCTCTGCCACGGCTGGACCGGAGACGCTCTGCGGGGCTGAGCTGGTGGATGCTCTTCAGTTCGTGTGTGGAGACAGGGGCTTTTATTTCAACAAGCCCACAGGGTATGGCTCCAGCAGTCGGAGGGCGCCTCAGACAGGCATCGTGGATGAGTGCTGCTTCCGGAGCTGTGATCTAAGGAGGCTGGAGATGTATTGCGCACCCCTCAAGCCTGCCAAGTCAGCT,如SEQ ID NO.1所示;
突变型IGF-1:
ATGGGAAAAATCAGCAGTCTTCCAACCCAATTATTTAAGTGCTGCTTTTGTGATTTCTTGAAGGTGAAGATGCACACCATGTCCTCCTCGCATCTCTTCTACCTGGCGCTGTGCCTGCTCACCTTCACCAGCTCTGCCACGGCTGGACCGGAGACGCTCTGCGGGGCTGAGCTGGTGGATGCTCTTCAGTTCGTGTGTGGAGACAGGGGCTTTTATTTCAACAAGCCCACAGGGTATGGCTCCAGCAGTCGGGAGGCGCCTCAGACAGGCTTCGTGGATGAGTGCTGCTTCCGGAGCTGTGATCTAAGGAGGCTGGAGCTGTATTGCGCACCCCTCAAGCCTGCCAAGTCAGCT,如SEQ ID NO.2所示;
(2)BamH1和EcoR1双酶切,连接,转化,菌落PCR和测序,成功构建野生型IGF-1重组质粒和突变型IGF-1重组质粒。
具体实验过程如下:
双酶切:目的片段双酶切反应体系为50μL:目的片段30μL,10×buffer 5μL,3dH2O13μL,BamH1 1μL,EcoR1 1μL,在37℃恒温水浴中酶切反应30min;载体双酶切反应体系为50μL:载体,10μL,10×buffer 5μL,3dH2O 33μL,BamH1 1μL,EcoR1 1μL,在37℃恒温水浴中酶切反应30min;所述目的片段为上述经重叠延伸PCR扩增所得基因片段,所述载体为框架质粒pcDNA3.1-EGFP-FLAG,是将pcDNA3.1-FLAG质粒上的FLAG标签切下,连在pcDNA3.1-EGFP质粒上;
连接:反应体系为20μL:目的片段8μL,载体2μL,10×T4buffer 2μL,T4ligase 1μL,3dH2O 7μL,在PCR仪中16℃反应1h,放于4℃冰箱过夜;
转化:①从-80℃冰箱拿出DH5α感受态细胞,将其置于冰上,待其融化后;在超净工作台中,将10μL连接产物或者2μL质粒轻轻加入100μL感受态细胞中,轻轻混匀,冰浴30min;
②水浴锅预热到42℃,将EP管里混匀的连接产物和DH5α感受态细胞,放于水浴锅,42℃水浴1min,水浴完成后,立即放于冰上,冰浴2min;
③向EP管加900μL LB液体空白培养基,混匀,放于摇床,温度为37℃,转速150rpm,摇菌1h;
④摇菌完成后,放入离心机,10000rpm离心1min,上清液留约100μL,弃掉多余的上清液,重悬菌体,将其均匀滴在含25~50μg/mL氨苄青霉素的LB固体培养板上,用涂布棒涂抹均匀;等重悬菌液被完全吸收后,将培养板放在37℃培养箱中倒置培养12-16h。
菌落PCR:
①从条件筛选培养板上挑选大小合适的单菌落,分别溶于10μL灭菌三次蒸馏水中,混匀;
②反应体系为10μL:3dH2O 2μL,2×Taq PCR Mix 5μL,Template(模板)1μL,R-primer(反向引物)1μL,F-primer(正向引物)1μL,扩增条件为95℃预变性5分钟;94℃变性30秒,57℃退火30秒,72℃延伸30秒,共30个循环;最后72℃延伸8分钟;4℃恒温1h;
野生型IGF1引物对
正向引物:5'-CGCGGATCC GCCACC ATGGGAAAAATCAG-3',如SEQ ID NO.3所示;
反向引物:5'-CCGGAATTCAGCTGACTTGGCAGGCTTGAGGGGTG-3',如SEQ ID NO.6所示;
突变型IGF1引物对
正向引物:5'-CGCGGATCC GCCACC ATGGGAAAAATCAG-3',如SEQ ID NO.3所示;
反向引物:
5'-CCGGAATTCAGCTGACTTGGCAGGCTTGAGGGGTGCGCAATACAGCTCC-3',如SEQ IDNO.10所示;
③PCR产物进行琼脂糖凝胶电泳,选出含目的条带的菌落备用;
④将菌落PCR鉴定成功的样品中,剩下的菌液加入20μL含氨苄的LB液体培养基中,摇菌12-16h;
测序:取1mL样品送往上海生物生工公司测序。
实施例2
野生型IGF-1重组质粒和突变型IGF-1重组质粒对人肝癌Hep.G2功能的影响(图2)
1、人肝癌Hep.G2细胞的培养
使用含体积百分数10%胎牛血清的高糖DMEM培养基,37℃、饱和湿度、体积分数为0.05的CO2孵育箱中孵育。
2、人肝癌Hep.G2细胞增殖
将人肝癌Hep.G2细胞接种到6孔板,过夜(12-16h)培养,将空质粒、野生型IGF-1重组质粒以及突变型IGF-1重组质粒分别转染到人肝癌Hep.G2细胞中,6小时后换新的培养基,48小时后MTT检测细胞增殖情况。
检测结果表明:野生型IGF-1重组质粒会促进人肝癌Hep.G2细胞的增殖,而突变型IGF-1重组质粒几乎不会促进人肝癌Hep.G2细胞的增殖。
3、人肝癌Hep.G2细胞迁移
将人肝癌Hep.G2接种到6孔板,过夜培养,将空质粒、野生型IGF-1重组质粒以及突变型IGF-1重组质粒分别转染到人肝癌Hep.G2细胞中,48小时后对细胞进行划痕,划痕后将细胞培养基换成无血清的条件培养基,对孔板中的细胞每隔12小时换新的培养基,并拍照记录细胞在0小时、12小时、24小时的细胞情况。
结果发现:突变型IGF-1重组质粒能抑制人肝癌Hep.G2细胞的迁移能力。
由以上实验结果可以得出结论:突变型IGF-1能抑制人肝癌Hep.G2细胞的迁移且不会促进人肝癌Hep.G2细胞的增殖,该突变体有成为一种新的抑癌药物的潜力。
实施例3
突变型IGF-1重组蛋白的制备:
将所得重组质粒(pET28a-IGF-1,该质粒是由上海生工生物有限公司制备)转化入大肠杆菌BL21(DE3)中,0.6mM IPTG,在37℃时,诱导表达6小时;离心收集菌体后,超声波破碎,离心收集上清和沉淀,SDS-PAGE检测蛋白的表达情况。如图3中A所示,检测到大量表达的突变型IGF-1蛋白条带,表明成功诱导表达了重组蛋白。最后利用Ni-Agarose Resin柱对突变型IGF-1进行纯化,SDS-PAGE检测蛋白的纯化情况。如图3中B所示除了目的条带以外,几乎不存在其它杂带,表明突变型IGF-1纯化成功。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
序列表
<110> 重庆大学
<120> 突变型IGF-1、重组质粒、重组蛋白及应用
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 354
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgggaaaaa tcagcagtct tccaacccaa ttatttaagt gctgcttttg tgatttcttg 60
aaggtgaaga tgcacaccat gtcctcctcg catctcttct acctggcgct gtgcctgctc 120
accttcacca gctctgccac ggctggaccg gagacgctct gcggggctga gctggtggat 180
gctcttcagt tcgtgtgtgg agacaggggc ttttatttca acaagcccac agggtatggc 240
tccagcagtc ggagggcgcc tcagacaggc atcgtggatg agtgctgctt ccggagctgt 300
gatctaagga ggctggagat gtattgcgca cccctcaagc ctgccaagtc agct 354
<210> 2
<211> 354
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgggaaaaa tcagcagtct tccaacccaa ttatttaagt gctgcttttg tgatttcttg 60
aaggtgaaga tgcacaccat gtcctcctcg catctcttct acctggcgct gtgcctgctc 120
accttcacca gctctgccac ggctggaccg gagacgctct gcggggctga gctggtggat 180
gctcttcagt tcgtgtgtgg agacaggggc ttttatttca acaagcccac agggtatggc 240
tccagcagtc gggaggcgcc tcagacaggc ttcgtggatg agtgctgctt ccggagctgt 300
gatctaagga ggctggagct gtattgcgca cccctcaagc ctgccaagtc agct 354
<210> 3
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cgcggatccg ccaccatggg aaaaatcag 29
<210> 4
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
agcgtctccg gtccagccgt ggcag 25
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ggaccggaga cgctctgcgg 20
<210> 6
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ccggaattca gctgacttgg caggcttgag gggtg 35
<210> 7
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggaccggaga cgctctgcg 19
<210> 8
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gaagcctgtc tgaggcgcct cctcactgct gg 32
<210> 9
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cctcagacag gcttcgtgga tgagtgct 28
<210> 10
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccggaattca gctgacttgg caggcttgag gggtgcgcaa tacagctcc 49
<210> 11
<211> 70
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe
1 5 10 15
Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly
20 25 30
Ser Ser Ser Arg Glu Ala Pro Gln Thr Gly Phe Val Asp Glu Cys Cys
35 40 45
Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Leu Tyr Cys Ala Pro Leu
50 55 60
Lys Pro Ala Lys Ser Ala
65 70
Claims (9)
1.突变型IGF-1,其特征在于,其核苷酸序列如SEQ ID NO.2所示。
2.利用权利要求1所述突变型IGF-1构建的重组质粒。
3.权利要求2所述重组质粒的构建方法,其特征在于,包括步骤:
(1)采用重叠延伸PCR和定点突变的方法进行PCR扩增,所用引物组合物如下:
IGF-1信号肽所用引物:
F1:5'-CGCGGATCC GCCACC ATGGGAAAAATCAG-3',如SEQ ID NO.3所示;
R1:5'-AGCGTCTCCGGTCCAGCCGTGGCAG-3',如SEQ ID NO.4所示;
突变型IGF-1成熟肽所用引物:
F3:5'-GGACCGGAGACGCTCTGCG-3',如SEQ ID NO.7所示;
R3:5'-GAAGCCTGTCTGAGGCGCCTCCTCACTGCTGG-3',如SEQ ID NO.8所示;
F4:5'-CCTCAGACAGGCTTCGTGGATGAGTGCT-3',如SEQ ID NO.9所示;
R4:5'-CCGGAATTCAGCTGACTTGGCAGGCTTGAGGGGTGCGCAATACAGCTCC-3',如SEQ ID NO.10所示;
扩增条件为95℃预变性5分钟;94℃变性30秒,57℃退火30秒,72℃延伸30秒,共30个循环;最后72℃延伸8分钟;进行四轮扩增,得到权利要求1所述的突变型IGF-1;
(2)BamH1和EcoR1双酶切,连接,转化,菌落PCR和测序,成功构建权利要求2所述的重组质粒。
4.利用权利要求1所述突变型IGF-1构建质粒、诱导表达所得重组蛋白,其特征在于,其氨基酸序列如SEQ ID NO.11所示。
5.权利要求4所述重组蛋白的制备方法,其特征在于,是将突变型IGF-1成熟肽经密码子优化后与pET28a质粒载体重组构建后诱导表达而得。
6.根据权利要求5所述的制备方法,其特征在于,具体方法是:将所得重组质粒转化入大肠杆菌中,诱导表达,离心收集菌体,超声波破碎,离心收集上清和沉淀,纯化即可。
7.权利要求1所述突变型IGF-1、权利要求2所述重组质粒、权利要求4所述重组蛋白在制备抑制肿瘤细胞迁移和增殖药物中的应用。
8.根据权利要求7所述的应用,所述肿瘤细胞为人肝癌Hep.G2细胞。
9.权利要求1所述突变型IGF-1、权利要求2所述重组质粒、权利要求4所述重组蛋白在作为IGF-1竞争性抑制剂中的应用。
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