CN109593727B - 6His-Nav1.1融合蛋白稳定表达细胞系及构建 - Google Patents

6His-Nav1.1融合蛋白稳定表达细胞系及构建 Download PDF

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CN109593727B
CN109593727B CN201811608803.XA CN201811608803A CN109593727B CN 109593727 B CN109593727 B CN 109593727B CN 201811608803 A CN201811608803 A CN 201811608803A CN 109593727 B CN109593727 B CN 109593727B
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周爽
赵云峰
吴永宁
李英骥
关立照
郑双佳
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China National Center For Food Safety Risk Assessment
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Abstract

本发明提供一种6His‑Nav1.1融合蛋白稳定表达细胞系及构建，所述的细胞系通过用携带SEQ ID No.3所示序列的重组载体转染HEK293细胞制备得到的。该细胞系能够稳定的表达具有生物活性的6His‑Nav1.1融合蛋白，该蛋白带有的6His标签使Nav1.1更易纯化和富集，为Nav1.1离子通道研究、高通量筛选新药和未知毒素提供了生物识别配基和毒性研究靶点，为神经药物和神经毒素的筛选、分离、鉴定提供了重要的生物亲和材料。同时为生物传感器研制和高通量筛查技术建立提供了核心原料。

Description

6His-Nav1.1融合蛋白稳定表达细胞系及构建
技术领域
本发明涉及基因重组与蛋白表达技术领域，特别是涉及6His-Nav1.1融合蛋白稳定表达细胞系及构建。
背景技术
电压门控钠离子通道(Voltage-gated sodium channels,VGSCs)，是广泛存在于动物的神经系统、心脏、肌肉等组织中可兴奋细胞膜上的膜蛋白，参与动作电位的产生和传导。迄今为止，已在哺乳动物体内分离鉴定出9种亚型：Nav1.1～Nav1.9。其中Nav1.1主要存在于人的神经细胞，是许多神经药物和神经毒素特异性作用的靶点，结合后表现为拮抗剂或激动作用，从而影响动作电位和神经、肌肉间兴奋信号的传导。
作为跨膜蛋白，VGSCs的体外表达难度巨大。VGSCs由α亚基和β亚基组成，其中形成钠离子传输通道的α亚基结构庞大(220～260kD)，包含四个高度同源的重复结构域(I～IV)，每个结构域又含有6个跨膜螺旋(S1～S6)，其中S4螺旋含有较多带的正电荷的氨基酸残基，在S5和S6之间有二条短片段(SS1和SS2)与跨膜孔道的形成有关。
目前，关于有生物活性的VGSCs融合蛋白的体外表达研究极为罕见。Haifa Hallaq等(Cardiovascular Research，2006，72，250-261)报道了绿色荧光蛋白标记心肌钠离子通道蛋白Nav1.5(GFP-Nav1.5)的体外表达。Yan Li Zhang等(Biosensors andBioelectronics，2007，22，1006-1012)报道了在昆虫细胞中表达了6组氨酸标记的人骨骼肌细胞钠离子通道蛋白Nav1.4(6His-Nav1.4)。而对于6His-Nav1.1人神经细胞钠离子通道融合蛋白的体外表达，尚未见报道。
发明内容
基于此，本发明的主要目的是提供一种6His-Nav1.1融合蛋白稳定表达细胞系。
本发明的目的是通过以下技术方案实现的：
一种6His-Nav1.1融合蛋白稳定表达细胞系，所述的细胞系通过用携带SEQ IDNo.3所示序列的重组载体转染HEK293细胞制备得到的。
在其中一些实施例中，构建所述重组载体采用pcDNA3.1/Hygro(+)。
本发明的另一目的是提供一种6His-Nav1.1融合蛋白稳定表达细胞系的构建方法，所述的构建方法包括如下步骤：
(1)构建携带SEQ ID No.3所示序列的重组载体；
(2)用所述重组载体转化EPI400感受态细胞，挑选转化菌落、培养，扩增重组载体；
(3)用所述扩增得到的重组载体转染HEK293细胞，传代培养，筛选，获得6His-Nav1.1融合蛋白稳定表达细胞系。
在其中一些实施例中，步骤(2)中，所述培养的过程中，培养液中添加有illumina公司的CopyCutterTM诱导剂。
在其中一些实施例中，所述诱导剂与培养液的体积比为(1200～800)：1。
在其中一些实施例中，步骤(1)中构建所述重组载体采用pcDNA3.1/Hygro(+)。
在其中一些实施例中，步骤(3)中，所述转染采用的转染试剂为Lipofectamine3000转染试剂或者X-tremeGENE HP DNA Transf.Reag。
本发明的再一目的是提供一种上述的细胞系在神经药物研究中的应用。
本发明的又一目的是提供一种上述的细胞系在神经毒素识别靶点研究中的应用。
本发明的还一目的是提供一种上述的细胞系在生物传感器研究中的应用。
本发明的还一目的是提供一种His-Nav1.1融合蛋白的表达方法，所述的表达方法包括获取上述的6His-Nav1.1融合蛋白稳定表达细胞系，培养，即可。
在其中一些实施例中，所述培养采用的培养基包括添加有终浓度为体积百分比为8～12％的胎牛血清、终浓度为180～220μg/ml潮霉素B的DMEM；所述的培养环境包括35～39℃、4.5～5.5％CO2。
与现有技术相比，本发明具备如下有益效果：
本发明通过用携带SEQ ID No.3所示序列的重组载体转化EPI400感受态细胞并培养转化后的细胞，使得该重组载体得以正确扩增，然后再用扩增后的重组载体转染HEK293细胞，最终经传代培养、筛选获得6His-Nav1.1融合蛋白稳定表达细胞系。该细胞系能够稳定的表达具有生物活性的6His-Nav1.1融合蛋白，该蛋白带有的6His标签使Nav1.1更易纯化和富集，为Nav1.1离子通道研究、高通量筛选新药和未知毒素提供了生物识别配基和毒性研究靶点，为神经药物和神经毒素的筛选、分离、鉴定提供了重要的生物亲和材料。同时为生物传感器研制和高通量筛查技术建立提供了核心原料。
附图说明
图1、实施例1构建的6His-NaV1.1质粒图谱及鉴定结果；
图2、实施例1构建的三批6His-NaV1.1质粒检测结果；
图3、实施例1 6His-NaV1.1细胞PCR图；
图4、实施例1His标签抗体检测6His-NaV1.1细胞Western blot结果；
图5、实施例1 6His-Nav1.1稳转细胞系电生理测定结果；
图6、实施例1获得稳转系的鉴定结果。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
实施例1
本实施例提供一种6His标记的NaV1.1融合蛋白表达细胞系及其构建方法，包括如下步骤：
NaV1.1基因信息：human NaV1.1(SCN1A)，NM_001165963，CDS大小为6030bp。如SEQID No.1所示：
Figure BDA0001924229470000041
GAGCAAACAGTGCTTGTACCACCAGGACCTGACAGCTTCAACTTCTTCACCAGAGAATCTCTTGCGGCTATTGAAAGACGCATTGCAGAAGAAAAGGCAAAGAATCCCAAACCAGACAAAAAAGATGACGACGAAAATGGCCCAAAGCCAAATAGTGACTTGGAAGCTGGAAAGAACCTTCCATTTATTTATGGAGACATTCCTCCAGAGATGGTGTCAGAGCCCCTGGAGGACCTGGACCCCTACTATATCAATAAGAAAACTTTTATAGTATTGAATAAAGGGAAGGCCATCTTCCGGTTCAGTGCCACCTCTGCCCTGTACATTTTAACTCCCTTCAATCCTCTTAGGAAAATAGCTATTAAGATTTTGGTACATTCATTATTCAGCATGCTAATTATGTGCACTATTTTGACAAACTGTGTGTTTATGACAATGAGTAACCCTCCTGATTGGACAAAGAATGTAGAATACACCTTCACAGGAATATATACTTTTGAATCACTTATAAAAATTATTGCAAGGGGATTCTGTTTAGAAGATTTTACTTTCCTTCGGGATCCATGGAACTGGCTCGATTTCACTGTCATTACATTTGCGTACGTCACAGAGTTTGTGGACCTGGGCAATGTCTCGGCATTGAGAACATTCAGAGTTCTCCGAGCATTGAAGACGATTTCAGTCATTCCAGGCCTGAAAACCATTGTGGGAGCCCTGATCCAGTCTGTGAAGAAGCTCTCAGATGTAATGATCCTGACTGTGTTCTGTCTGAGCGTATTTGCTCTAATTGGGCTGCAGCTGTTCATGGGCAACCTGAGGAATAAATGTATACAATGGCCTCCCACCAATGCTTCCTTGGAGGAACATAGTATAGAAAAGAATATAACTGTGAATTATAATGGTACACTTATAAATGAAACTGTCTTTGAGTTTGACTGGAAGTCATATATTCAAGATTCAAGATATCATTATTTCCTGGAGGGTTTTTTAGATGCACTACTATGTGGAAATAGCTCTGATGCAGGCCAATGTCCAGAGGGATATATGTGTGTGAAAGCTGGTAGAAATCCCAATTATGGCTACACAAGCTTTGATACCTTCAGTTGGGCTTTTTTGTCCTTGTTTCGACTAATGACTCAGGACTTCTGGGAAAATCTTTATCAACTGACATTACGTGCTGCTGGGAAAACGTACATGATATTTTTTGTATTGGTCATTTTCTTGGGCTCATTCTACCTAATAAATTTGATCCTGGCTGTGGTGGCCATGGCCTACGAGGAACAGAATCAGGCCACCTTGGAAGAAGCAGAACAGAAAGAGGCCGAATTTCAGCAGATGATTGAACAGCTTAAAAAGCAACAGGAGGCAGCTCAGCAGGCAGCAACGGCAACTGCCTCAGAACATTCCAGAGAGCCCAGTGCAGCAGGCAGGCTCTCAGACAGCTCATCTGAAGCCTCTAAGTTGAGTTCCAAGAGTGCTAAGGAAAGAAGAAATCGGAGGAAGAAAAGAAAACAGAAAGAGCAGTCTGGTGGGGAAGAGAAAGATGAGGATGAATTCCAAAAATCTGAATCTGAGGACAGCATCAGGAGGAAAGGTTTTCGCTTCTCCATTGAAGGGAACCGATTGACATATGAAAAGAGGTACTCCTCCCCACACCAGTCTTTGTTGAGCATCCGTGGCTCCCTATTTTCACCAAGGCGAAATAGCAGAACAAGCCTTTTCAGCTTTAGAGGGCGAGCAAAGGATGTGGGATCTGAGAACGACTTCGCAGATGATGAGCACAGCACCTTTGAGGATAACGAGAGCCGTAGAGATTCCTTGTTTGTGCCCCGACGACACGGAGAGAGACGCAACAGCAACCTGAGTCAGACCAGTAGGTCATCCCGGATGCTGGCAGTGTTTCCAGCGAATGGGAAGATGCACAGCACTGTGGATTGCAATGGTGTGGTTTCCTTGGTTGGTGGACCTTCAGTTCCTACATCGCCTGTTGGACAGCTTCTGCCAGAGGTGATAATAGATAAGCCAGCTACTGATGACAATGGAACAACCACTGAAACTGAAATGAGAAAGAGAAGGTCAAGTTCTTTCCACGTTTCCATGGACTTTCTAGAAGATCCTTCCCAAAGGCAACGAGCAATGAGTATAGCCAGCATTCTAACAAATACAGTAGAAGAACTTGAAGAATCCAGGCAGAAATGCCCACCCTGTTGGTATAAATTTTCCAACATATTCTTAATCTGGGACTGTTCTCCATATTGGTTAAAAGTGAAACATGTTGTCAACCTGGTTGTGATGGACCCATTTGTTGACCTGGCCATCACCATCTGTATTGTCTTAAATACTCTTTTCATGGCCATGGAGCACTATCCAATGACGGACCATTTCAATAATGTGCTTACAGTAGGAAACTTGGTTTTCACTGGGATCTTTACAGCAGAAATGTTTCTGAAAATTATTGCCATGGATCCTTACTATTATTTCCAAGAAGGCTGGAATATCTTTGACGGTTTTATTGTGACGCTTAGCCTGGTAGAACTTGGACTCGCCAATGTGGAAGGATTATCTGTTCTCCGTTCATTTCGATTGCTGCGAGTTTTCAAGTTGGCAAAATCTTGGCCAACGTTAAATATGCTAATAAAGATCATCGGCAATTCCGTGGGGGCTCTGGGAAATTTAACCCTCGTCTTGGCCATCATCGTCTTCATTTTTGCCGTGGTCGGCATGCAGCTCTTTGGTAAAAGCTACAAAGATTGTGTCTGCAAGATCGCCAGTGATTGTCAACTCCCACGCTGGCACATGAATGACTTCTTCCACTCCTTCCTGATTGTGTTCCGCGTGCTGTGTGGGGAGTGGATAGAGACCATGTGGGACTGTATGGAGGTTGCTGGTCAAGCCATGTGCCTTACTGTCTTCATGATGGTCATGGTGATTGGAAACCTAGTGGTCCTGAATCTCTTTCTGGCCTTGCTTCTGAGCTCATTTAGTGCAGACAACCTTGCAGCCACTGATGATGATAATGAAATGAATAATCTCCAAATTGCTGTGGATAGGATGCACAAAGGAGTAGCTTATGTGAAAAGAAAAATATATGAATTTATTCAACAGTCCTTCATTAGGAAACAAAAGATTTTAGATGAAATTAAACCACTTGATGATCTAAACAACAAGAAAGACAGTTGTATGTCCAATCATACAGCAGAAATTGGGAAAGATCTTGACTATCTTAAAGATGTAAATGGAACTACAAGTGGTATAGGAACTGGCAGCAGTGTTGAAAAATACATTATTGATGAAAGTGATTACATGTCATTCATAAACAACCCCAGTCTTACTGTGACTGTACCAATTGCTGTAGGAGAATCTGACTTTGAAAATTTAAACACGGAAGACTTTAGTAGTGAATCGGATCTGGAAGAAAGCAAAGAGAAACTGAATGAAAGCAGTAGCTCATCAGAAGGTAGCACTGTGGACATCGGCGCACCTGTAGAAGAACAGCCCGTAGTGGAACCTGAAGAAACTCTTGAACCAGAAGCTTGTTTCACTGAAGGCTGTGTACAAAGATTCAAGTGTTGTCAAATCAATGTGGAAGAAGGCAGAGGAAAACAATGGTGGAACCTGAGAAGGACGTGTTTCCGAATAGTTGAACATAACTGGTTTGAGACCTTCATTGTTTTCATGATTCTCCTTAGTAGTGGTGCTCTGGCATTTGAAGATATATATATTGATCAGCGAAAGACGATTAAGACGATGTTGGAATATGCTGACAAGGTTTTCACTTACATTTTCATTCTGGAAATGCTTCTAAAATGGGTGGCATATGGCTATCAAACATATTTCACCAATGCCTGGTGTTGGCTGGACTTCTTAATTGTTGATGTTTCATTGGTCAGTTTAACAGCAAATGCCTTGGGTTACTCAGAACTTGGAGCCATCAAATCTCTCAGGACACTAAGAGCTCTGAGACCTCTAAGAGCCTTATCTCGATTTGAAGGGATGAGGGTGGTTGTGAATGCCCTTTTAGGAGCAATTCCATCCATCATGAATGTGCTTCTGGTTTGTCTTATATTCTGGCTAATTTTCAGCATCATGGGCGTAAATTTGTTTGCTGGCAAATTCTACCACTGTATTAACACCACAACTGGTGACAGGTTTGACATCGAAGACGTGAATAATCATACTGATTGCCTAAAACTAATAGAAAGAAATGAGACTGCTCGATGGAAAAATGTGAAAGTAAACTTTGATAATGTAGGATTTGGGTATCTCTCTTTGCTTCAAGTTGCCACATTCAAAGGATGGATGGATATAATGTATGCAGCAGTTGATTCCAGAAATGTGGAACTCCAGCCTAAGTATGAAGAAAGTCTGTACATGTATCTTTACTTTGTTATTTTCATCATCTTTGGGTCCTTCTTCACCTTGAACCTGTTTATTGGTGTCATCATAGATAATTTCAACCAGCAGAAAAAGAAGTTTGGAGGTCAAGACATCTTTATGACAGAAGAACAGAAGAAATACTATAATGCAATGAAAAAATTAGGATCGAAAAAACCGCAAAAGCCTATACCTCGACCAGGAAACAAATTTCAAGGAATGGTCTTTGACTTCGTAACCAGACAAGTTTTTGACATAAGCATCATGATTCTCATCTGTCTTAACATGGTCACAATGATGGTGGAAACAGATGACCAGAGTGAATATGTGACTACCATTTTGTCACGCATCAATCTGGTGTTCATTGTGCTATTTACTGGAGAGTGTGTACTGAAACTCATCTCTCTACGCCATTATTATTTTACCATTGGATGGAATATTTTTGATTTTGTGGTTGTCATTCTCTCCATTGTAGGTATGTTTCTTGCCGAGCTGATAGAAAAGTATTTCGTGTCCCCTACCCTGTTCCGAGTGATCCGTCTTGCTAGGATTGGCCGAATCCTACGTCTGATCAAAGGAGCAAAGGGGATCCGCACGCTGCTCTTTGCTTTGATGATGTCCCTTCCTGCGTTGTTTAACATCGGCCTCCTACTCTTCCTAGTCATGTTCATCTACGCCATCTTTGGGATGTCCAACTTTGCCTATGTTAAGAGGGAAGTTGGGATCGATGACATGTTCAACTTTGAGACCTTTGGCAACAGCATGATCTGCCTATTCCAAATTACAACCTCTGCTGGCTGGGATGGATTGCTAGCACCCATTCTCAACAGTAAGCCACCCGACTGTGACCCTAATAAAGTTAACCCTGGAAGCTCAGTTAAGGGAGACTGTGGGAACCCATCTGTTGGAATTTTCTTTTTTGTCAGTTACATCATCATATCCTTCCTGGTTGTGGTGAACATGTACATCGCGGTCATCCTGGAGAACTTCAGTGTTGCTACTGAAGAAAGTGCAGAGCCTCTGAGTGAGGATGACTTTGAGATGTTCTATGAGGTTTGGGAGAAGTTTGATCCCGATGCAACTCAGTTCATGGAATTTGAAAAATTATCTCAGTTTGCAGCTGCGCTTGAACCGCCTCTCAATCTGCCACAACCAAACAAACTCCAGCTCATTGCCATGGATTTGCCCATGGTGAGTGGTGACCGGATCCACTGTCTTGATATCTTATTTGCTTTTACAAAGCGGGTTCTAGGAGAGAGTGGAGAGATGGATGCTCTACGAATACAGATGGAAGAGCGATTCATGGCTTCCAATCCTTCCAAGGTCTCCTATCAGCCAATCACTACTACTTTAAAACGAAAACAAGAGGAAGTATCTGCTGTCATTATTCAGCGTGCTTACAGACGCCACCTTTTAAAGCGAACTGTAAAACAAGCTTCCTTTACGTACAATAAAAACAAAATCAAAGGTGGGGCTAATCTTCTTATAAAAGAAGACATGATAATTGACAGAATAAATGAAAACTCTATTACAGAAAAAACTGATCTGACCATGTCCACTGCAGCTTGTCCACCTTCCTATGACCGGGTGACAAAGCCAATTGTGGAAAAACATGAGCAAGAAGGCAAAGATGAAAAAGCCAAAGGGAAA
Figure BDA0001924229470000061
1、6His-NaV1.1质粒合成
NaV1.1的CDS的5’端添加KpnI酶切位点(ggtacc)，3’端去除终止密码子TAA，添加6His标签+终止密码子TAA+XhoI酶切位点(catcatcatcatcatcat+taa+ctcgag)。His标签加在Nav1.1蛋白的C端，形成SEQ ID No.2所示的序列。
SEQ ID No.2：
GGTACC
Figure BDA0001924229470000062
GAGCAAACAGTGCTTGTACCACCAGGACCTGACAGCTTCAACTTCTTCACCAGAGAATCTCTTGCGGCTATTGAAAGACGCATTGCAGAAGAAAAGGCAAAGAATCCCAAACCAGACAAAAAAGATGACGACGAAAATGGCCCAAAGCCAAATAGTGACTTGGAAGCTGGAAAGAACCTTCCATTTATTTATGGAGACATTCCTCCAGAGATGGTGTCAGAGCCCCTGGAGGACCTGGACCCCTACTATATCAATAAGAAAACTTTTATAGTATTGAATAAAGGGAAGGCCATCTTCCGGTTCAGTGCCACCTCTGCCCTGTACATTTTAACTCCCTTCAATCCTCTTAGGAAAATAGCTATTAAGATTTTGGTACATTCATTATTCAGCATGCTAATTATGTGCACTATTTTGACAAACTGTGTGTTTATGACAATGAGTAACCCTCCTGATTGGACAAAGAATGTAGAATACACCTTCACAGGAATATATACTTTTGAATCACTTATAAAAATTATTGCAAGGGGATTCTGTTTAGAAGATTTTACTTTCCTTCGGGATCCATGGAACTGGCTCGATTTCACTGTCATTACATTTGCGTACGTCACAGAGTTTGTGGACCTGGGCAATGTCTCGGCATTGAGAACATTCAGAGTTCTCCGAGCATTGAAGACGATTTCAGTCATTCCAGGCCTGAAAACCATTGTGGGAGCCCTGATCCAGTCTGTGAAGAAGCTCTCAGATGTAATGATCCTGACTGTGTTCTGTCTGAGCGTATTTGCTCTAATTGGGCTGCAGCTGTTCATGGGCAACCTGAGGAATAAATGTATACAATGGCCTCCCACCAATGCTTCCTTGGAGGAACATAGTATAGAAAAGAATATAACTGTGAATTATAATGGTACACTTATAAATGAAACTGTCTTTGAGTTTGACTGGAAGTCATATATTCAAGATTCAAGATATCATTATTTCCTGGAGGGTTTTTTAGATGCACTACTATGTGGAAATAGCTCTGATGCAGGCCAATGTCCAGAGGGATATATGTGTGTGAAAGCTGGTAGAAATCCCAATTATGGCTACACAAGCTTTGATACCTTCAGTTGGGCTTTTTTGTCCTTGTTTCGACTAATGACTCAGGACTTCTGGGAAAATCTTTATCAACTGACATTACGTGCTGCTGGGAAAACGTACATGATATTTTTTGTATTGGTCATTTTCTTGGGCTCATTCTACCTAATAAATTTGATCCTGGCTGTGGTGGCCATGGCCTACGAGGAACAGAATCAGGCCACCTTGGAAGAAGCAGAACAGAAAGAGGCCGAATTTCAGCAGATGATTGAACAGCTTAAAAAGCAACAGGAGGCAGCTCAGCAGGCAGCAACGGCAACTGCCTCAGAACATTCCAGAGAGCCCAGTGCAGCAGGCAGGCTCTCAGACAGCTCATCTGAAGCCTCTAAGTTGAGTTCCAAGAGTGCTAAGGAAAGAAGAAATCGGAGGAAGAAAAGAAAACAGAAAGAGCAGTCTGGTGGGGAAGAGAAAGATGAGGATGAATTCCAAAAATCTGAATCTGAGGACAGCATCAGGAGGAAAGGTTTTCGCTTCTCCATTGAAGGGAACCGATTGACATATGAAAAGAGGTACTCCTCCCCACACCAGTCTTTGTTGAGCATCCGTGGCTCCCTATTTTCACCAAGGCGAAATAGCAGAACAAGCCTTTTCAGCTTTAGAGGGCGAGCAAAGGATGTGGGATCTGAGAACGACTTCGCAGATGATGAGCACAGCACCTTTGAGGATAACGAGAGCCGTAGAGATTCCTTGTTTGTGCCCCGACGACACGGAGAGAGACGCAACAGCAACCTGAGTCAGACCAGTAGGTCATCCCGGATGCTGGCAGTGTTTCCAGCGAATGGGAAGATGCACAGCACTGTGGATTGCAATGGTGTGGTTTCCTTGGTTGGTGGACCTTCAGTTCCTACATCGCCTGTTGGACAGCTTCTGCCAGAGGTGATAATAGATAAGCCAGCTACTGATGACAATGGAACAACCACTGAAACTGAAATGAGAAAGAGAAGGTCAAGTTCTTTCCACGTTTCCATGGACTTTCTAGAAGATCCTTCCCAAAGGCAACGAGCAATGAGTATAGCCAGCATTCTAACAAATACAGTAGAAGAACTTGAAGAATCCAGGCAGAAATGCCCACCCTGTTGGTATAAATTTTCCAACATATTCTTAATCTGGGACTGTTCTCCATATTGGTTAAAAGTGAAACATGTTGTCAACCTGGTTGTGATGGACCCATTTGTTGACCTGGCCATCACCATCTGTATTGTCTTAAATACTCTTTTCATGGCCATGGAGCACTATCCAATGACGGACCATTTCAATAATGTGCTTACAGTAGGAAACTTGGTTTTCACTGGGATCTTTACAGCAGAAATGTTTCTGAAAATTATTGCCATGGATCCTTACTATTATTTCCAAGAAGGCTGGAATATCTTTGACGGTTTTATTGTGACGCTTAGCCTGGTAGAACTTGGACTCGCCAATGTGGAAGGATTATCTGTTCTCCGTTCATTTCGATTGCTGCGAGTTTTCAAGTTGGCAAAATCTTGGCCAACGTTAAATATGCTAATAAAGATCATCGGCAATTCCGTGGGGGCTCTGGGAAATTTAACCCTCGTCTTGGCCATCATCGTCTTCATTTTTGCCGTGGTCGGCATGCAGCTCTTTGGTAAAAGCTACAAAGATTGTGTCTGCAAGATCGCCAGTGATTGTCAACTCCCACGCTGGCACATGAATGACTTCTTCCACTCCTTCCTGATTGTGTTCCGCGTGCTGTGTGGGGAGTGGATAGAGACCATGTGGGACTGTATGGAGGTTGCTGGTCAAGCCATGTGCCTTACTGTCTTCATGATGGTCATGGTGATTGGAAACCTAGTGGTCCTGAATCTCTTTCTGGCCTTGCTTCTGAGCTCATTTAGTGCAGACAACCTTGCAGCCACTGATGATGATAATGAAATGAATAATCTCCAAATTGCTGTGGATAGGATGCACAAAGGAGTAGCTTATGTGAAAAGAAAAATATATGAATTTATTCAACAGTCCTTCATTAGGAAACAAAAGATTTTAGATGAAATTAAACCACTTGATGATCTAAACAACAAGAAAGACAGTTGTATGTCCAATCATACAGCAGAAATTGGGAAAGATCTTGACTATCTTAAAGATGTAAATGGAACTACAAGTGGTATAGGAACTGGCAGCAGTGTTGAAAAATACATTATTGATGAAAGTGATTACATGTCATTCATAAACAACCCCAGTCTTACTGTGACTGTACCAATTGCTGTAGGAGAATCTGACTTTGAAAATTTAAACACGGAAGACTTTAGTAGTGAATCGGATCTGGAAGAAAGCAAAGAGAAACTGAATGAAAGCAGTAGCTCATCAGAAGGTAGCACTGTGGACATCGGCGCACCTGTAGAAGAACAGCCCGTAGTGGAACCTGAAGAAACTCTTGAACCAGAAGCTTGTTTCACTGAAGGCTGTGTACAAAGATTCAAGTGTTGTCAAATCAATGTGGAAGAAGGCAGAGGAAAACAATGGTGGAACCTGAGAAGGACGTGTTTCCGAATAGTTGAACATAACTGGTTTGAGACCTTCATTGTTTTCATGATTCTCCTTAGTAGTGGTGCTCTGGCATTTGAAGATATATATATTGATCAGCGAAAGACGATTAAGACGATGTTGGAATATGCTGACAAGGTTTTCACTTACATTTTCATTCTGGAAATGCTTCTAAAATGGGTGGCATATGGCTATCAAACATATTTCACCAATGCCTGGTGTTGGCTGGACTTCTTAATTGTTGATGTTTCATTGGTCAGTTTAACAGCAAATGCCTTGGGTTACTCAGAACTTGGAGCCATCAAATCTCTCAGGACACTAAGAGCTCTGAGACCTCTAAGAGCCTTATCTCGATTTGAAGGGATGAGGGTGGTTGTGAATGCCCTTTTAGGAGCAATTCCATCCATCATGAATGTGCTTCTGGTTTGTCTTATATTCTGGCTAATTTTCAGCATCATGGGCGTAAATTTGTTTGCTGGCAAATTCTACCACTGTATTAACACCACAACTGGTGACAGGTTTGACATCGAAGACGTGAATAATCATACTGATTGCCTAAAACTAATAGAAAGAAATGAGACTGCTCGATGGAAAAATGTGAAAGTAAACTTTGATAATGTAGGATTTGGGTATCTCTCTTTGCTTCAAGTTGCCACATTCAAAGGATGGATGGATATAATGTATGCAGCAGTTGATTCCAGAAATGTGGAACTCCAGCCTAAGTATGAAGAAAGTCTGTACATGTATCTTTACTTTGTTATTTTCATCATCTTTGGGTCCTTCTTCACCTTGAACCTGTTTATTGGTGTCATCATAGATAATTTCAACCAGCAGAAAAAGAAGTTTGGAGGTCAAGACATCTTTATGACAGAAGAACAGAAGAAATACTATAATGCAATGAAAAAATTAGGATCGAAAAAACCGCAAAAGCCTATACCTCGACCAGGAAACAAATTTCAAGGAATGGTCTTTGACTTCGTAACCAGACAAGTTTTTGACATAAGCATCATGATTCTCATCTGTCTTAACATGGTCACAATGATGGTGGAAACAGATGACCAGAGTGAATATGTGACTACCATTTTGTCACGCATCAATCTGGTGTTCATTGTGCTATTTACTGGAGAGTGTGTACTGAAACTCATCTCTCTACGCCATTATTATTTTACCATTGGATGGAATATTTTTGATTTTGTGGTTGTCATTCTCTCCATTGTAGGTATGTTTCTTGCCGAGCTGATAGAAAAGTATTTCGTGTCCCCTACCCTGTTCCGAGTGATCCGTCTTGCTAGGATTGGCCGAATCCTACGTCTGATCAAAGGAGCAAAGGGGATCCGCACGCTGCTCTTTGCTTTGATGATGTCCCTTCCTGCGTTGTTTAACATCGGCCTCCTACTCTTCCTAGTCATGTTCATCTACGCCATCTTTGGGATGTCCAACTTTGCCTATGTTAAGAGGGAAGTTGGGATCGATGACATGTTCAACTTTGAGACCTTTGGCAACAGCATGATCTGCCTATTCCAAATTACAACCTCTGCTGGCTGGGATGGATTGCTAGCACCCATTCTCAACAGTAAGCCACCCGACTGTGACCCTAATAAAGTTAACCCTGGAAGCTCAGTTAAGGGAGACTGTGGGAACCCATCTGTTGGAATTTTCTTTTTTGTCAGTTACATCATCATATCCTTCCTGGTTGTGGTGAACATGTACATCGCGGTCATCCTGGAGAACTTCAGTGTTGCTACTGAAGAAAGTGCAGAGCCTCTGAGTGAGGATGACTTTGAGATGTTCTATGAGGTTTGGGAGAAGTTTGATCCCGATGCAACTCAGTTCATGGAATTTGAAAAATTATCTCAGTTTGCAGCTGCGCTTGAACCGCCTCTCAATCTGCCACAACCAAACAAACTCCAGCTCATTGCCATGGATTTGCCCATGGTGAGTGGTGACCGGATCCACTGTCTTGATATCTTATTTGCTTTTACAAAGCGGGTTCTAGGAGAGAGTGGAGAGATGGATGCTCTACGAATACAGATGGAAGAGCGATTCATGGCTTCCAATCCTTCCAAGGTCTCCTATCAGCCAATCACTACTACTTTAAAACGAAAACAAGAGGAAGTATCTGCTGTCATTATTCAGCGTGCTTACAGACGCCACCTTTTAAAGCGAACTGTAAAACAAGCTTCCTTTACGTACAATAAAAACAAAATCAAAGGTGGGGCTAATCTTCTTATAAAAGAAGACATGATAATTGACAGAATAAATGAAAACTCTATTACAGAAAAAACTGATCTGACCATGTCCACTGCAGCTTGTCCACCTTCCTATGACCGGGTGACAAAGCCAATTGTGGAAAAACATGAGCAAGAAGGCAAAGATGAAAAAGCCAAAGGGAAACATCATCATCATCATCAT
Figure BDA0001924229470000081
CTCGAG
SEQ ID No.2中，5’端GGTACC为KpnI酶切位点，
Figure BDA0001924229470000082
为起始密码，3’端CATCATCATCATCATCAT为6His标签，
Figure BDA0001924229470000083
为终止密码，CTCGAG为XhoI酶切位点。SEQ IDNo.2去除KpnI酶切位点GGTACC、XhoI酶切位点CTCGAG之后的序列记作SEQ ID No.3，SEQ IDNo.3如下所示：
Figure BDA0001924229470000091
GAGCAAACAGTGCTTGTACCACCAGGACCTGACAGCTTCAACTTCTTCACCAGAGAATCTCTTGCGGCTATTGAAAGACGCATTGCAGAAGAAAAGGCAAAGAATCCCAAACCAGACAAAAAAGATGACGACGAAAATGGCCCAAAGCCAAATAGTGACTTGGAAGCTGGAAAGAACCTTCCATTTATTTATGGAGACATTCCTCCAGAGATGGTGTCAGAGCCCCTGGAGGACCTGGACCCCTACTATATCAATAAGAAAACTTTTATAGTATTGAATAAAGGGAAGGCCATCTTCCGGTTCAGTGCCACCTCTGCCCTGTACATTTTAACTCCCTTCAATCCTCTTAGGAAAATAGCTATTAAGATTTTGGTACATTCATTATTCAGCATGCTAATTATGTGCACTATTTTGACAAACTGTGTGTTTATGACAATGAGTAACCCTCCTGATTGGACAAAGAATGTAGAATACACCTTCACAGGAATATATACTTTTGAATCACTTATAAAAATTATTGCAAGGGGATTCTGTTTAGAAGATTTTACTTTCCTTCGGGATCCATGGAACTGGCTCGATTTCACTGTCATTACATTTGCGTACGTCACAGAGTTTGTGGACCTGGGCAATGTCTCGGCATTGAGAACATTCAGAGTTCTCCGAGCATTGAAGACGATTTCAGTCATTCCAGGCCTGAAAACCATTGTGGGAGCCCTGATCCAGTCTGTGAAGAAGCTCTCAGATGTAATGATCCTGACTGTGTTCTGTCTGAGCGTATTTGCTCTAATTGGGCTGCAGCTGTTCATGGGCAACCTGAGGAATAAATGTATACAATGGCCTCCCACCAATGCTTCCTTGGAGGAACATAGTATAGAAAAGAATATAACTGTGAATTATAATGGTACACTTATAAATGAAACTGTCTTTGAGTTTGACTGGAAGTCATATATTCAAGATTCAAGATATCATTATTTCCTGGAGGGTTTTTTAGATGCACTACTATGTGGAAATAGCTCTGATGCAGGCCAATGTCCAGAGGGATATATGTGTGTGAAAGCTGGTAGAAATCCCAATTATGGCTACACAAGCTTTGATACCTTCAGTTGGGCTTTTTTGTCCTTGTTTCGACTAATGACTCAGGACTTCTGGGAAAATCTTTATCAACTGACATTACGTGCTGCTGGGAAAACGTACATGATATTTTTTGTATTGGTCATTTTCTTGGGCTCATTCTACCTAATAAATTTGATCCTGGCTGTGGTGGCCATGGCCTACGAGGAACAGAATCAGGCCACCTTGGAAGAAGCAGAACAGAAAGAGGCCGAATTTCAGCAGATGATTGAACAGCTTAAAAAGCAACAGGAGGCAGCTCAGCAGGCAGCAACGGCAACTGCCTCAGAACATTCCAGAGAGCCCAGTGCAGCAGGCAGGCTCTCAGACAGCTCATCTGAAGCCTCTAAGTTGAGTTCCAAGAGTGCTAAGGAAAGAAGAAATCGGAGGAAGAAAAGAAAACAGAAAGAGCAGTCTGGTGGGGAAGAGAAAGATGAGGATGAATTCCAAAAATCTGAATCTGAGGACAGCATCAGGAGGAAAGGTTTTCGCTTCTCCATTGAAGGGAACCGATTGACATATGAAAAGAGGTACTCCTCCCCACACCAGTCTTTGTTGAGCATCCGTGGCTCCCTATTTTCACCAAGGCGAAATAGCAGAACAAGCCTTTTCAGCTTTAGAGGGCGAGCAAAGGATGTGGGATCTGAGAACGACTTCGCAGATGATGAGCACAGCACCTTTGAGGATAACGAGAGCCGTAGAGATTCCTTGTTTGTGCCCCGACGACACGGAGAGAGACGCAACAGCAACCTGAGTCAGACCAGTAGGTCATCCCGGATGCTGGCAGTGTTTCCAGCGAATGGGAAGATGCACAGCACTGTGGATTGCAATGGTGTGGTTTCCTTGGTTGGTGGACCTTCAGTTCCTACATCGCCTGTTGGACAGCTTCTGCCAGAGGTGATAATAGATAAGCCAGCTACTGATGACAATGGAACAACCACTGAAACTGAAATGAGAAAGAGAAGGTCAAGTTCTTTCCACGTTTCCATGGACTTTCTAGAAGATCCTTCCCAAAGGCAACGAGCAATGAGTATAGCCAGCATTCTAACAAATACAGTAGAAGAACTTGAAGAATCCAGGCAGAAATGCCCACCCTGTTGGTATAAATTTTCCAACATATTCTTAATCTGGGACTGTTCTCCATATTGGTTAAAAGTGAAACATGTTGTCAACCTGGTTGTGATGGACCCATTTGTTGACCTGGCCATCACCATCTGTATTGTCTTAAATACTCTTTTCATGGCCATGGAGCACTATCCAATGACGGACCATTTCAATAATGTGCTTACAGTAGGAAACTTGGTTTTCACTGGGATCTTTACAGCAGAAATGTTTCTGAAAATTATTGCCATGGATCCTTACTATTATTTCCAAGAAGGCTGGAATATCTTTGACGGTTTTATTGTGACGCTTAGCCTGGTAGAACTTGGACTCGCCAATGTGGAAGGATTATCTGTTCTCCGTTCATTTCGATTGCTGCGAGTTTTCAAGTTGGCAAAATCTTGGCCAACGTTAAATATGCTAATAAAGATCATCGGCAATTCCGTGGGGGCTCTGGGAAATTTAACCCTCGTCTTGGCCATCATCGTCTTCATTTTTGCCGTGGTCGGCATGCAGCTCTTTGGTAAAAGCTACAAAGATTGTGTCTGCAAGATCGCCAGTGATTGTCAACTCCCACGCTGGCACATGAATGACTTCTTCCACTCCTTCCTGATTGTGTTCCGCGTGCTGTGTGGGGAGTGGATAGAGACCATGTGGGACTGTATGGAGGTTGCTGGTCAAGCCATGTGCCTTACTGTCTTCATGATGGTCATGGTGATTGGAAACCTAGTGGTCCTGAATCTCTTTCTGGCCTTGCTTCTGAGCTCATTTAGTGCAGACAACCTTGCAGCCACTGATGATGATAATGAAATGAATAATCTCCAAATTGCTGTGGATAGGATGCACAAAGGAGTAGCTTATGTGAAAAGAAAAATATATGAATTTATTCAACAGTCCTTCATTAGGAAACAAAAGATTTTAGATGAAATTAAACCACTTGATGATCTAAACAACAAGAAAGACAGTTGTATGTCCAATCATACAGCAGAAATTGGGAAAGATCTTGACTATCTTAAAGATGTAAATGGAACTACAAGTGGTATAGGAACTGGCAGCAGTGTTGAAAAATACATTATTGATGAAAGTGATTACATGTCATTCATAAACAACCCCAGTCTTACTGTGACTGTACCAATTGCTGTAGGAGAATCTGACTTTGAAAATTTAAACACGGAAGACTTTAGTAGTGAATCGGATCTGGAAGAAAGCAAAGAGAAACTGAATGAAAGCAGTAGCTCATCAGAAGGTAGCACTGTGGACATCGGCGCACCTGTAGAAGAACAGCCCGTAGTGGAACCTGAAGAAACTCTTGAACCAGAAGCTTGTTTCACTGAAGGCTGTGTACAAAGATTCAAGTGTTGTCAAATCAATGTGGAAGAAGGCAGAGGAAAACAATGGTGGAACCTGAGAAGGACGTGTTTCCGAATAGTTGAACATAACTGGTTTGAGACCTTCATTGTTTTCATGATTCTCCTTAGTAGTGGTGCTCTGGCATTTGAAGATATATATATTGATCAGCGAAAGACGATTAAGACGATGTTGGAATATGCTGACAAGGTTTTCACTTACATTTTCATTCTGGAAATGCTTCTAAAATGGGTGGCATATGGCTATCAAACATATTTCACCAATGCCTGGTGTTGGCTGGACTTCTTAATTGTTGATGTTTCATTGGTCAGTTTAACAGCAAATGCCTTGGGTTACTCAGAACTTGGAGCCATCAAATCTCTCAGGACACTAAGAGCTCTGAGACCTCTAAGAGCCTTATCTCGATTTGAAGGGATGAGGGTGGTTGTGAATGCCCTTTTAGGAGCAATTCCATCCATCATGAATGTGCTTCTGGTTTGTCTTATATTCTGGCTAATTTTCAGCATCATGGGCGTAAATTTGTTTGCTGGCAAATTCTACCACTGTATTAACACCACAACTGGTGACAGGTTTGACATCGAAGACGTGAATAATCATACTGATTGCCTAAAACTAATAGAAAGAAATGAGACTGCTCGATGGAAAAATGTGAAAGTAAACTTTGATAATGTAGGATTTGGGTATCTCTCTTTGCTTCAAGTTGCCACATTCAAAGGATGGATGGATATAATGTATGCAGCAGTTGATTCCAGAAATGTGGAACTCCAGCCTAAGTATGAAGAAAGTCTGTACATGTATCTTTACTTTGTTATTTTCATCATCTTTGGGTCCTTCTTCACCTTGAACCTGTTTATTGGTGTCATCATAGATAATTTCAACCAGCAGAAAAAGAAGTTTGGAGGTCAAGACATCTTTATGACAGAAGAACAGAAGAAATACTATAATGCAATGAAAAAATTAGGATCGAAAAAACCGCAAAAGCCTATACCTCGACCAGGAAACAAATTTCAAGGAATGGTCTTTGACTTCGTAACCAGACAAGTTTTTGACATAAGCATCATGATTCTCATCTGTCTTAACATGGTCACAATGATGGTGGAAACAGATGACCAGAGTGAATATGTGACTACCATTTTGTCACGCATCAATCTGGTGTTCATTGTGCTATTTACTGGAGAGTGTGTACTGAAACTCATCTCTCTACGCCATTATTATTTTACCATTGGATGGAATATTTTTGATTTTGTGGTTGTCATTCTCTCCATTGTAGGTATGTTTCTTGCCGAGCTGATAGAAAAGTATTTCGTGTCCCCTACCCTGTTCCGAGTGATCCGTCTTGCTAGGATTGGCCGAATCCTACGTCTGATCAAAGGAGCAAAGGGGATCCGCACGCTGCTCTTTGCTTTGATGATGTCCCTTCCTGCGTTGTTTAACATCGGCCTCCTACTCTTCCTAGTCATGTTCATCTACGCCATCTTTGGGATGTCCAACTTTGCCTATGTTAAGAGGGAAGTTGGGATCGATGACATGTTCAACTTTGAGACCTTTGGCAACAGCATGATCTGCCTATTCCAAATTACAACCTCTGCTGGCTGGGATGGATTGCTAGCACCCATTCTCAACAGTAAGCCACCCGACTGTGACCCTAATAAAGTTAACCCTGGAAGCTCAGTTAAGGGAGACTGTGGGAACCCATCTGTTGGAATTTTCTTTTTTGTCAGTTACATCATCATATCCTTCCTGGTTGTGGTGAACATGTACATCGCGGTCATCCTGGAGAACTTCAGTGTTGCTACTGAAGAAAGTGCAGAGCCTCTGAGTGAGGATGACTTTGAGATGTTCTATGAGGTTTGGGAGAAGTTTGATCCCGATGCAACTCAGTTCATGGAATTTGAAAAATTATCTCAGTTTGCAGCTGCGCTTGAACCGCCTCTCAATCTGCCACAACCAAACAAACTCCAGCTCATTGCCATGGATTTGCCCATGGTGAGTGGTGACCGGATCCACTGTCTTGATATCTTATTTGCTTTTACAAAGCGGGTTCTAGGAGAGAGTGGAGAGATGGATGCTCTACGAATACAGATGGAAGAGCGATTCATGGCTTCCAATCCTTCCAAGGTCTCCTATCAGCCAATCACTACTACTTTAAAACGAAAACAAGAGGAAGTATCTGCTGTCATTATTCAGCGTGCTTACAGACGCCACCTTTTAAAGCGAACTGTAAAACAAGCTTCCTTTACGTACAATAAAAACAAAATCAAAGGTGGGGCTAATCTTCTTATAAAAGAAGACATGATAATTGACAGAATAAATGAAAACTCTATTACAGAAAAAACTGATCTGACCATGTCCACTGCAGCTTGTCCACCTTCCTATGACCGGGTGACAAAGCCAATTGTGGAAAAACATGAGCAAGAAGGCAAAGATGAAAAAGCCAAAGGGAAACATCATCATCATCATCAT
Figure BDA0001924229470000111
载体为pcDNA3.1/Hygro(+)，Amp(氨苄霉素抗性)抗性。构建质粒请参见图1。
2、6His-NaV1.1质粒扩增
转化EPI400感受态细胞：取1μl 6His-NaV1.1质粒加入50μl EPI400感受态细胞中，弹拨混匀，冰浴30min。42℃热休克90sec，冰浴1min～2min，加入500μl LB培养基，37℃、150rpm振荡培养1h，获得菌液。取100μl菌液铺于含100μg/ml Amp的琼脂板上，37℃培养至转化菌落出现。
50ml离心管中加入5ml含100μg/ml Amp的LB培养基，从长有转化菌落的琼脂板上挑取单个菌落，放入管中，37℃、200rpm，振荡摇培过夜。
将过夜培养的菌液按1:10的体积比转接到新鲜的LB培养基中，加入100μg/mlAmp，同时按1000:1的体积比加入诱导剂(induction solution)(采用illumina公司的CopyCutterTM诱导剂)，混合均匀后，37℃摇菌4h。该步骤中，培养液与诱导剂的体积比为(1200～800)：1范围内均可。
从菌液中提取质粒，检测浓度与纯度，经酶切及测序鉴定后，待用。图2是对三批扩增质粒鉴定结果，其中第二批浓度高、纯度好，选择其进行后续转染实验。
3、6His-NaV1.1质粒转染
传HEK293细胞至6cm皿，第二天使用Lipofectamine 3000转染试剂转染6His-NaV1.1质粒。转染时：6His-NaV1.1质粒与转染试剂比例为1μg:2μl。转染次日换液，转染后约48h传代。
提取传代后细胞的蛋白及RNA，用于Western blot和PCR检测，结果分别参见图3、图4。据图可知，传代后的细胞能够表达6His-NaV1.1蛋白。
传代后细胞加200μg/ml HygromycinB(潮霉素B)进行筛选，每隔3天～4天换液，约2周～3周后，待转染组细胞形成明显克隆时传代，提蛋白及RNA，检测6His-NaV1.1表达情况，并用膜片钳测试电生理活性。
该步骤中，采用的转染试剂也可以为Roche公司的X-tremeGENE HP DNATransf.Reag。
4、稳转系筛选
确定有生物活性的6His-NaV1.1表达后，继续筛选单克隆：采用有限稀释法将细胞传至96孔板中继续培养，每孔约1.5个细胞，用含Hygromycin B的培养基筛选约2周，挑取单克隆孔的细胞扩大培养，至细胞量足够多时验证NaV1.1的表达情况，获得6His-Nav1.1融合蛋白稳定表达细胞系。结果参见图5、图6。图5是转染后细胞电生理检测结果，可获得800pA的电信号，证明转染后细胞表达的6His-Nav1.1具有生物功能。图6是稳转系的鉴定结果，其中Nav1.1-His pool是筛选出的稳转细胞系。
实施例2、6His-Nav1.1融合蛋白的表达
实施例1筛选出稳定表达6His-Nav1.1融合蛋白的细胞系后，采用HEK293细胞常规培养及传代方法即可。即：
培养基为：添加有终浓度为10％的FBS(胎牛血清)、终浓度为200μg/mlHygromycin B(潮霉素B)的DMEM。
培养条件为：在37℃、5％CO2培养箱中培养；细胞长到80％左右需要传代。
传代步骤包括：培养基于37℃水浴加热，至培养基温度达到或超过室温；吸去培养皿中原有的培养基，用PBS清洗一遍，加入0.25％胰酶消化约1min，加入培养基终止消化，轻轻吹打使细胞从培养皿表面脱落，同时细胞悬液中无肉眼可见细胞团块；将细胞悬液转移至15ml无菌离心管中，1000rpm，离心5分钟；弃掉上清，用1ml新鲜培养基重悬细胞；按约1:5的比例将细胞传至新的培养皿中，放入培养箱中培养。
对比例1
本对比例是实施例1的对比例，相对于实施例1的主要差别包括选用的感受态细胞为Top10感受态细胞，具体步骤如下：按说明书操作，取1μl6His-NaV1.1质粒加入100μlTop10感受态细胞中，弹拨混匀，冰浴30min。42℃热休克90sec，冰浴2min～3min，加入900μlLB培养基，37℃、150rpm振荡培养45min，获得菌液。取100μl菌液铺于含相应抗生素的琼脂板上，37℃培养至转化菌落出现。其余操作同实施例1。
结果，扩增的质粒序列不正确，无法构建6His-Nav1.1融合蛋白稳定表达细胞系。
对比例2
本对比例是实施例1的对比例，相对于实施例1的主要差别包括选用的感受态细胞为DH5α感受态细胞，具体步骤如下：按说明书操作，取1μl6His-NaV1.1质粒加入100μl DH5α感受态细胞中，弹拨混匀，冰浴30min。42℃热休克90sec，冰浴2min～3min，加入900μl LB培养基，37℃、150rpm振荡培养45min，获得菌液。取100μl菌液铺于含相应抗生素的琼脂板上，37℃培养至转化菌落出现。其余操作同实施例1。
结果，扩增的质粒序列不正确，6His-Nav1.1融合蛋白稳定表达细胞系。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 国家食品安全风险评估中心
<120> 6His-Nav1.1融合蛋白稳定表达细胞系及构建
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6030
<212> DNA
<213> Homo sapiens
<400> 1
atggagcaaa cagtgcttgt accaccagga cctgacagct tcaacttctt caccagagaa 60
tctcttgcgg ctattgaaag acgcattgca gaagaaaagg caaagaatcc caaaccagac 120
aaaaaagatg acgacgaaaa tggcccaaag ccaaatagtg acttggaagc tggaaagaac 180
cttccattta tttatggaga cattcctcca gagatggtgt cagagcccct ggaggacctg 240
gacccctact atatcaataa gaaaactttt atagtattga ataaagggaa ggccatcttc 300
cggttcagtg ccacctctgc cctgtacatt ttaactccct tcaatcctct taggaaaata 360
gctattaaga ttttggtaca ttcattattc agcatgctaa ttatgtgcac tattttgaca 420
aactgtgtgt ttatgacaat gagtaaccct cctgattgga caaagaatgt agaatacacc 480
ttcacaggaa tatatacttt tgaatcactt ataaaaatta ttgcaagggg attctgttta 540
gaagatttta ctttccttcg ggatccatgg aactggctcg atttcactgt cattacattt 600
gcgtacgtca cagagtttgt ggacctgggc aatgtctcgg cattgagaac attcagagtt 660
ctccgagcat tgaagacgat ttcagtcatt ccaggcctga aaaccattgt gggagccctg 720
atccagtctg tgaagaagct ctcagatgta atgatcctga ctgtgttctg tctgagcgta 780
tttgctctaa ttgggctgca gctgttcatg ggcaacctga ggaataaatg tatacaatgg 840
cctcccacca atgcttcctt ggaggaacat agtatagaaa agaatataac tgtgaattat 900
aatggtacac ttataaatga aactgtcttt gagtttgact ggaagtcata tattcaagat 960
tcaagatatc attatttcct ggagggtttt ttagatgcac tactatgtgg aaatagctct 1020
gatgcaggcc aatgtccaga gggatatatg tgtgtgaaag ctggtagaaa tcccaattat 1080
ggctacacaa gctttgatac cttcagttgg gcttttttgt ccttgtttcg actaatgact 1140
caggacttct gggaaaatct ttatcaactg acattacgtg ctgctgggaa aacgtacatg 1200
atattttttg tattggtcat tttcttgggc tcattctacc taataaattt gatcctggct 1260
gtggtggcca tggcctacga ggaacagaat caggccacct tggaagaagc agaacagaaa 1320
gaggccgaat ttcagcagat gattgaacag cttaaaaagc aacaggaggc agctcagcag 1380
gcagcaacgg caactgcctc agaacattcc agagagccca gtgcagcagg caggctctca 1440
gacagctcat ctgaagcctc taagttgagt tccaagagtg ctaaggaaag aagaaatcgg 1500
aggaagaaaa gaaaacagaa agagcagtct ggtggggaag agaaagatga ggatgaattc 1560
caaaaatctg aatctgagga cagcatcagg aggaaaggtt ttcgcttctc cattgaaggg 1620
aaccgattga catatgaaaa gaggtactcc tccccacacc agtctttgtt gagcatccgt 1680
ggctccctat tttcaccaag gcgaaatagc agaacaagcc ttttcagctt tagagggcga 1740
gcaaaggatg tgggatctga gaacgacttc gcagatgatg agcacagcac ctttgaggat 1800
aacgagagcc gtagagattc cttgtttgtg ccccgacgac acggagagag acgcaacagc 1860
aacctgagtc agaccagtag gtcatcccgg atgctggcag tgtttccagc gaatgggaag 1920
atgcacagca ctgtggattg caatggtgtg gtttccttgg ttggtggacc ttcagttcct 1980
acatcgcctg ttggacagct tctgccagag gtgataatag ataagccagc tactgatgac 2040
aatggaacaa ccactgaaac tgaaatgaga aagagaaggt caagttcttt ccacgtttcc 2100
atggactttc tagaagatcc ttcccaaagg caacgagcaa tgagtatagc cagcattcta 2160
acaaatacag tagaagaact tgaagaatcc aggcagaaat gcccaccctg ttggtataaa 2220
ttttccaaca tattcttaat ctgggactgt tctccatatt ggttaaaagt gaaacatgtt 2280
gtcaacctgg ttgtgatgga cccatttgtt gacctggcca tcaccatctg tattgtctta 2340
aatactcttt tcatggccat ggagcactat ccaatgacgg accatttcaa taatgtgctt 2400
acagtaggaa acttggtttt cactgggatc tttacagcag aaatgtttct gaaaattatt 2460
gccatggatc cttactatta tttccaagaa ggctggaata tctttgacgg ttttattgtg 2520
acgcttagcc tggtagaact tggactcgcc aatgtggaag gattatctgt tctccgttca 2580
tttcgattgc tgcgagtttt caagttggca aaatcttggc caacgttaaa tatgctaata 2640
aagatcatcg gcaattccgt gggggctctg ggaaatttaa ccctcgtctt ggccatcatc 2700
gtcttcattt ttgccgtggt cggcatgcag ctctttggta aaagctacaa agattgtgtc 2760
tgcaagatcg ccagtgattg tcaactccca cgctggcaca tgaatgactt cttccactcc 2820
ttcctgattg tgttccgcgt gctgtgtggg gagtggatag agaccatgtg ggactgtatg 2880
gaggttgctg gtcaagccat gtgccttact gtcttcatga tggtcatggt gattggaaac 2940
ctagtggtcc tgaatctctt tctggccttg cttctgagct catttagtgc agacaacctt 3000
gcagccactg atgatgataa tgaaatgaat aatctccaaa ttgctgtgga taggatgcac 3060
aaaggagtag cttatgtgaa aagaaaaata tatgaattta ttcaacagtc cttcattagg 3120
aaacaaaaga ttttagatga aattaaacca cttgatgatc taaacaacaa gaaagacagt 3180
tgtatgtcca atcatacagc agaaattggg aaagatcttg actatcttaa agatgtaaat 3240
ggaactacaa gtggtatagg aactggcagc agtgttgaaa aatacattat tgatgaaagt 3300
gattacatgt cattcataaa caaccccagt cttactgtga ctgtaccaat tgctgtagga 3360
gaatctgact ttgaaaattt aaacacggaa gactttagta gtgaatcgga tctggaagaa 3420
agcaaagaga aactgaatga aagcagtagc tcatcagaag gtagcactgt ggacatcggc 3480
gcacctgtag aagaacagcc cgtagtggaa cctgaagaaa ctcttgaacc agaagcttgt 3540
ttcactgaag gctgtgtaca aagattcaag tgttgtcaaa tcaatgtgga agaaggcaga 3600
ggaaaacaat ggtggaacct gagaaggacg tgtttccgaa tagttgaaca taactggttt 3660
gagaccttca ttgttttcat gattctcctt agtagtggtg ctctggcatt tgaagatata 3720
tatattgatc agcgaaagac gattaagacg atgttggaat atgctgacaa ggttttcact 3780
tacattttca ttctggaaat gcttctaaaa tgggtggcat atggctatca aacatatttc 3840
accaatgcct ggtgttggct ggacttctta attgttgatg tttcattggt cagtttaaca 3900
gcaaatgcct tgggttactc agaacttgga gccatcaaat ctctcaggac actaagagct 3960
ctgagacctc taagagcctt atctcgattt gaagggatga gggtggttgt gaatgccctt 4020
ttaggagcaa ttccatccat catgaatgtg cttctggttt gtcttatatt ctggctaatt 4080
ttcagcatca tgggcgtaaa tttgtttgct ggcaaattct accactgtat taacaccaca 4140
actggtgaca ggtttgacat cgaagacgtg aataatcata ctgattgcct aaaactaata 4200
gaaagaaatg agactgctcg atggaaaaat gtgaaagtaa actttgataa tgtaggattt 4260
gggtatctct ctttgcttca agttgccaca ttcaaaggat ggatggatat aatgtatgca 4320
gcagttgatt ccagaaatgt ggaactccag cctaagtatg aagaaagtct gtacatgtat 4380
ctttactttg ttattttcat catctttggg tccttcttca ccttgaacct gtttattggt 4440
gtcatcatag ataatttcaa ccagcagaaa aagaagtttg gaggtcaaga catctttatg 4500
acagaagaac agaagaaata ctataatgca atgaaaaaat taggatcgaa aaaaccgcaa 4560
aagcctatac ctcgaccagg aaacaaattt caaggaatgg tctttgactt cgtaaccaga 4620
caagtttttg acataagcat catgattctc atctgtctta acatggtcac aatgatggtg 4680
gaaacagatg accagagtga atatgtgact accattttgt cacgcatcaa tctggtgttc 4740
attgtgctat ttactggaga gtgtgtactg aaactcatct ctctacgcca ttattatttt 4800
accattggat ggaatatttt tgattttgtg gttgtcattc tctccattgt aggtatgttt 4860
cttgccgagc tgatagaaaa gtatttcgtg tcccctaccc tgttccgagt gatccgtctt 4920
gctaggattg gccgaatcct acgtctgatc aaaggagcaa aggggatccg cacgctgctc 4980
tttgctttga tgatgtccct tcctgcgttg tttaacatcg gcctcctact cttcctagtc 5040
atgttcatct acgccatctt tgggatgtcc aactttgcct atgttaagag ggaagttggg 5100
atcgatgaca tgttcaactt tgagaccttt ggcaacagca tgatctgcct attccaaatt 5160
acaacctctg ctggctggga tggattgcta gcacccattc tcaacagtaa gccacccgac 5220
tgtgacccta ataaagttaa ccctggaagc tcagttaagg gagactgtgg gaacccatct 5280
gttggaattt tcttttttgt cagttacatc atcatatcct tcctggttgt ggtgaacatg 5340
tacatcgcgg tcatcctgga gaacttcagt gttgctactg aagaaagtgc agagcctctg 5400
agtgaggatg actttgagat gttctatgag gtttgggaga agtttgatcc cgatgcaact 5460
cagttcatgg aatttgaaaa attatctcag tttgcagctg cgcttgaacc gcctctcaat 5520
ctgccacaac caaacaaact ccagctcatt gccatggatt tgcccatggt gagtggtgac 5580
cggatccact gtcttgatat cttatttgct tttacaaagc gggttctagg agagagtgga 5640
gagatggatg ctctacgaat acagatggaa gagcgattca tggcttccaa tccttccaag 5700
gtctcctatc agccaatcac tactacttta aaacgaaaac aagaggaagt atctgctgtc 5760
attattcagc gtgcttacag acgccacctt ttaaagcgaa ctgtaaaaca agcttccttt 5820
acgtacaata aaaacaaaat caaaggtggg gctaatcttc ttataaaaga agacatgata 5880
attgacagaa taaatgaaaa ctctattaca gaaaaaactg atctgaccat gtccactgca 5940
gcttgtccac cttcctatga ccgggtgaca aagccaattg tggaaaaaca tgagcaagaa 6000
ggcaaagatg aaaaagccaa agggaaataa 6030
<210> 2
<211> 6060
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggtaccatgg agcaaacagt gcttgtacca ccaggacctg acagcttcaa cttcttcacc 60
agagaatctc ttgcggctat tgaaagacgc attgcagaag aaaaggcaaa gaatcccaaa 120
ccagacaaaa aagatgacga cgaaaatggc ccaaagccaa atagtgactt ggaagctgga 180
aagaaccttc catttattta tggagacatt cctccagaga tggtgtcaga gcccctggag 240
gacctggacc cctactatat caataagaaa acttttatag tattgaataa agggaaggcc 300
atcttccggt tcagtgccac ctctgccctg tacattttaa ctcccttcaa tcctcttagg 360
aaaatagcta ttaagatttt ggtacattca ttattcagca tgctaattat gtgcactatt 420
ttgacaaact gtgtgtttat gacaatgagt aaccctcctg attggacaaa gaatgtagaa 480
tacaccttca caggaatata tacttttgaa tcacttataa aaattattgc aaggggattc 540
tgtttagaag attttacttt ccttcgggat ccatggaact ggctcgattt cactgtcatt 600
acatttgcgt acgtcacaga gtttgtggac ctgggcaatg tctcggcatt gagaacattc 660
agagttctcc gagcattgaa gacgatttca gtcattccag gcctgaaaac cattgtggga 720
gccctgatcc agtctgtgaa gaagctctca gatgtaatga tcctgactgt gttctgtctg 780
agcgtatttg ctctaattgg gctgcagctg ttcatgggca acctgaggaa taaatgtata 840
caatggcctc ccaccaatgc ttccttggag gaacatagta tagaaaagaa tataactgtg 900
aattataatg gtacacttat aaatgaaact gtctttgagt ttgactggaa gtcatatatt 960
caagattcaa gatatcatta tttcctggag ggttttttag atgcactact atgtggaaat 1020
agctctgatg caggccaatg tccagaggga tatatgtgtg tgaaagctgg tagaaatccc 1080
aattatggct acacaagctt tgataccttc agttgggctt ttttgtcctt gtttcgacta 1140
atgactcagg acttctggga aaatctttat caactgacat tacgtgctgc tgggaaaacg 1200
tacatgatat tttttgtatt ggtcattttc ttgggctcat tctacctaat aaatttgatc 1260
ctggctgtgg tggccatggc ctacgaggaa cagaatcagg ccaccttgga agaagcagaa 1320
cagaaagagg ccgaatttca gcagatgatt gaacagctta aaaagcaaca ggaggcagct 1380
cagcaggcag caacggcaac tgcctcagaa cattccagag agcccagtgc agcaggcagg 1440
ctctcagaca gctcatctga agcctctaag ttgagttcca agagtgctaa ggaaagaaga 1500
aatcggagga agaaaagaaa acagaaagag cagtctggtg gggaagagaa agatgaggat 1560
gaattccaaa aatctgaatc tgaggacagc atcaggagga aaggttttcg cttctccatt 1620
gaagggaacc gattgacata tgaaaagagg tactcctccc cacaccagtc tttgttgagc 1680
atccgtggct ccctattttc accaaggcga aatagcagaa caagcctttt cagctttaga 1740
gggcgagcaa aggatgtggg atctgagaac gacttcgcag atgatgagca cagcaccttt 1800
gaggataacg agagccgtag agattccttg tttgtgcccc gacgacacgg agagagacgc 1860
aacagcaacc tgagtcagac cagtaggtca tcccggatgc tggcagtgtt tccagcgaat 1920
gggaagatgc acagcactgt ggattgcaat ggtgtggttt ccttggttgg tggaccttca 1980
gttcctacat cgcctgttgg acagcttctg ccagaggtga taatagataa gccagctact 2040
gatgacaatg gaacaaccac tgaaactgaa atgagaaaga gaaggtcaag ttctttccac 2100
gtttccatgg actttctaga agatccttcc caaaggcaac gagcaatgag tatagccagc 2160
attctaacaa atacagtaga agaacttgaa gaatccaggc agaaatgccc accctgttgg 2220
tataaatttt ccaacatatt cttaatctgg gactgttctc catattggtt aaaagtgaaa 2280
catgttgtca acctggttgt gatggaccca tttgttgacc tggccatcac catctgtatt 2340
gtcttaaata ctcttttcat ggccatggag cactatccaa tgacggacca tttcaataat 2400
gtgcttacag taggaaactt ggttttcact gggatcttta cagcagaaat gtttctgaaa 2460
attattgcca tggatcctta ctattatttc caagaaggct ggaatatctt tgacggtttt 2520
attgtgacgc ttagcctggt agaacttgga ctcgccaatg tggaaggatt atctgttctc 2580
cgttcatttc gattgctgcg agttttcaag ttggcaaaat cttggccaac gttaaatatg 2640
ctaataaaga tcatcggcaa ttccgtgggg gctctgggaa atttaaccct cgtcttggcc 2700
atcatcgtct tcatttttgc cgtggtcggc atgcagctct ttggtaaaag ctacaaagat 2760
tgtgtctgca agatcgccag tgattgtcaa ctcccacgct ggcacatgaa tgacttcttc 2820
cactccttcc tgattgtgtt ccgcgtgctg tgtggggagt ggatagagac catgtgggac 2880
tgtatggagg ttgctggtca agccatgtgc cttactgtct tcatgatggt catggtgatt 2940
ggaaacctag tggtcctgaa tctctttctg gccttgcttc tgagctcatt tagtgcagac 3000
aaccttgcag ccactgatga tgataatgaa atgaataatc tccaaattgc tgtggatagg 3060
atgcacaaag gagtagctta tgtgaaaaga aaaatatatg aatttattca acagtccttc 3120
attaggaaac aaaagatttt agatgaaatt aaaccacttg atgatctaaa caacaagaaa 3180
gacagttgta tgtccaatca tacagcagaa attgggaaag atcttgacta tcttaaagat 3240
gtaaatggaa ctacaagtgg tataggaact ggcagcagtg ttgaaaaata cattattgat 3300
gaaagtgatt acatgtcatt cataaacaac cccagtctta ctgtgactgt accaattgct 3360
gtaggagaat ctgactttga aaatttaaac acggaagact ttagtagtga atcggatctg 3420
gaagaaagca aagagaaact gaatgaaagc agtagctcat cagaaggtag cactgtggac 3480
atcggcgcac ctgtagaaga acagcccgta gtggaacctg aagaaactct tgaaccagaa 3540
gcttgtttca ctgaaggctg tgtacaaaga ttcaagtgtt gtcaaatcaa tgtggaagaa 3600
ggcagaggaa aacaatggtg gaacctgaga aggacgtgtt tccgaatagt tgaacataac 3660
tggtttgaga ccttcattgt tttcatgatt ctccttagta gtggtgctct ggcatttgaa 3720
gatatatata ttgatcagcg aaagacgatt aagacgatgt tggaatatgc tgacaaggtt 3780
ttcacttaca ttttcattct ggaaatgctt ctaaaatggg tggcatatgg ctatcaaaca 3840
tatttcacca atgcctggtg ttggctggac ttcttaattg ttgatgtttc attggtcagt 3900
ttaacagcaa atgccttggg ttactcagaa cttggagcca tcaaatctct caggacacta 3960
agagctctga gacctctaag agccttatct cgatttgaag ggatgagggt ggttgtgaat 4020
gcccttttag gagcaattcc atccatcatg aatgtgcttc tggtttgtct tatattctgg 4080
ctaattttca gcatcatggg cgtaaatttg tttgctggca aattctacca ctgtattaac 4140
accacaactg gtgacaggtt tgacatcgaa gacgtgaata atcatactga ttgcctaaaa 4200
ctaatagaaa gaaatgagac tgctcgatgg aaaaatgtga aagtaaactt tgataatgta 4260
ggatttgggt atctctcttt gcttcaagtt gccacattca aaggatggat ggatataatg 4320
tatgcagcag ttgattccag aaatgtggaa ctccagccta agtatgaaga aagtctgtac 4380
atgtatcttt actttgttat tttcatcatc tttgggtcct tcttcacctt gaacctgttt 4440
attggtgtca tcatagataa tttcaaccag cagaaaaaga agtttggagg tcaagacatc 4500
tttatgacag aagaacagaa gaaatactat aatgcaatga aaaaattagg atcgaaaaaa 4560
ccgcaaaagc ctatacctcg accaggaaac aaatttcaag gaatggtctt tgacttcgta 4620
accagacaag tttttgacat aagcatcatg attctcatct gtcttaacat ggtcacaatg 4680
atggtggaaa cagatgacca gagtgaatat gtgactacca ttttgtcacg catcaatctg 4740
gtgttcattg tgctatttac tggagagtgt gtactgaaac tcatctctct acgccattat 4800
tattttacca ttggatggaa tatttttgat tttgtggttg tcattctctc cattgtaggt 4860
atgtttcttg ccgagctgat agaaaagtat ttcgtgtccc ctaccctgtt ccgagtgatc 4920
cgtcttgcta ggattggccg aatcctacgt ctgatcaaag gagcaaaggg gatccgcacg 4980
ctgctctttg ctttgatgat gtcccttcct gcgttgttta acatcggcct cctactcttc 5040
ctagtcatgt tcatctacgc catctttggg atgtccaact ttgcctatgt taagagggaa 5100
gttgggatcg atgacatgtt caactttgag acctttggca acagcatgat ctgcctattc 5160
caaattacaa cctctgctgg ctgggatgga ttgctagcac ccattctcaa cagtaagcca 5220
cccgactgtg accctaataa agttaaccct ggaagctcag ttaagggaga ctgtgggaac 5280
ccatctgttg gaattttctt ttttgtcagt tacatcatca tatccttcct ggttgtggtg 5340
aacatgtaca tcgcggtcat cctggagaac ttcagtgttg ctactgaaga aagtgcagag 5400
cctctgagtg aggatgactt tgagatgttc tatgaggttt gggagaagtt tgatcccgat 5460
gcaactcagt tcatggaatt tgaaaaatta tctcagtttg cagctgcgct tgaaccgcct 5520
ctcaatctgc cacaaccaaa caaactccag ctcattgcca tggatttgcc catggtgagt 5580
ggtgaccgga tccactgtct tgatatctta tttgctttta caaagcgggt tctaggagag 5640
agtggagaga tggatgctct acgaatacag atggaagagc gattcatggc ttccaatcct 5700
tccaaggtct cctatcagcc aatcactact actttaaaac gaaaacaaga ggaagtatct 5760
gctgtcatta ttcagcgtgc ttacagacgc caccttttaa agcgaactgt aaaacaagct 5820
tcctttacgt acaataaaaa caaaatcaaa ggtggggcta atcttcttat aaaagaagac 5880
atgataattg acagaataaa tgaaaactct attacagaaa aaactgatct gaccatgtcc 5940
actgcagctt gtccaccttc ctatgaccgg gtgacaaagc caattgtgga aaaacatgag 6000
caagaaggca aagatgaaaa agccaaaggg aaacatcatc atcatcatca ttaactcgag 6060
<210> 3
<211> 6048
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggagcaaa cagtgcttgt accaccagga cctgacagct tcaacttctt caccagagaa 60
tctcttgcgg ctattgaaag acgcattgca gaagaaaagg caaagaatcc caaaccagac 120
aaaaaagatg acgacgaaaa tggcccaaag ccaaatagtg acttggaagc tggaaagaac 180
cttccattta tttatggaga cattcctcca gagatggtgt cagagcccct ggaggacctg 240
gacccctact atatcaataa gaaaactttt atagtattga ataaagggaa ggccatcttc 300
cggttcagtg ccacctctgc cctgtacatt ttaactccct tcaatcctct taggaaaata 360
gctattaaga ttttggtaca ttcattattc agcatgctaa ttatgtgcac tattttgaca 420
aactgtgtgt ttatgacaat gagtaaccct cctgattgga caaagaatgt agaatacacc 480
ttcacaggaa tatatacttt tgaatcactt ataaaaatta ttgcaagggg attctgttta 540
gaagatttta ctttccttcg ggatccatgg aactggctcg atttcactgt cattacattt 600
gcgtacgtca cagagtttgt ggacctgggc aatgtctcgg cattgagaac attcagagtt 660
ctccgagcat tgaagacgat ttcagtcatt ccaggcctga aaaccattgt gggagccctg 720
atccagtctg tgaagaagct ctcagatgta atgatcctga ctgtgttctg tctgagcgta 780
tttgctctaa ttgggctgca gctgttcatg ggcaacctga ggaataaatg tatacaatgg 840
cctcccacca atgcttcctt ggaggaacat agtatagaaa agaatataac tgtgaattat 900
aatggtacac ttataaatga aactgtcttt gagtttgact ggaagtcata tattcaagat 960
tcaagatatc attatttcct ggagggtttt ttagatgcac tactatgtgg aaatagctct 1020
gatgcaggcc aatgtccaga gggatatatg tgtgtgaaag ctggtagaaa tcccaattat 1080
ggctacacaa gctttgatac cttcagttgg gcttttttgt ccttgtttcg actaatgact 1140
caggacttct gggaaaatct ttatcaactg acattacgtg ctgctgggaa aacgtacatg 1200
atattttttg tattggtcat tttcttgggc tcattctacc taataaattt gatcctggct 1260
gtggtggcca tggcctacga ggaacagaat caggccacct tggaagaagc agaacagaaa 1320
gaggccgaat ttcagcagat gattgaacag cttaaaaagc aacaggaggc agctcagcag 1380
gcagcaacgg caactgcctc agaacattcc agagagccca gtgcagcagg caggctctca 1440
gacagctcat ctgaagcctc taagttgagt tccaagagtg ctaaggaaag aagaaatcgg 1500
aggaagaaaa gaaaacagaa agagcagtct ggtggggaag agaaagatga ggatgaattc 1560
caaaaatctg aatctgagga cagcatcagg aggaaaggtt ttcgcttctc cattgaaggg 1620
aaccgattga catatgaaaa gaggtactcc tccccacacc agtctttgtt gagcatccgt 1680
ggctccctat tttcaccaag gcgaaatagc agaacaagcc ttttcagctt tagagggcga 1740
gcaaaggatg tgggatctga gaacgacttc gcagatgatg agcacagcac ctttgaggat 1800
aacgagagcc gtagagattc cttgtttgtg ccccgacgac acggagagag acgcaacagc 1860
aacctgagtc agaccagtag gtcatcccgg atgctggcag tgtttccagc gaatgggaag 1920
atgcacagca ctgtggattg caatggtgtg gtttccttgg ttggtggacc ttcagttcct 1980
acatcgcctg ttggacagct tctgccagag gtgataatag ataagccagc tactgatgac 2040
aatggaacaa ccactgaaac tgaaatgaga aagagaaggt caagttcttt ccacgtttcc 2100
atggactttc tagaagatcc ttcccaaagg caacgagcaa tgagtatagc cagcattcta 2160
acaaatacag tagaagaact tgaagaatcc aggcagaaat gcccaccctg ttggtataaa 2220
ttttccaaca tattcttaat ctgggactgt tctccatatt ggttaaaagt gaaacatgtt 2280
gtcaacctgg ttgtgatgga cccatttgtt gacctggcca tcaccatctg tattgtctta 2340
aatactcttt tcatggccat ggagcactat ccaatgacgg accatttcaa taatgtgctt 2400
acagtaggaa acttggtttt cactgggatc tttacagcag aaatgtttct gaaaattatt 2460
gccatggatc cttactatta tttccaagaa ggctggaata tctttgacgg ttttattgtg 2520
acgcttagcc tggtagaact tggactcgcc aatgtggaag gattatctgt tctccgttca 2580
tttcgattgc tgcgagtttt caagttggca aaatcttggc caacgttaaa tatgctaata 2640
aagatcatcg gcaattccgt gggggctctg ggaaatttaa ccctcgtctt ggccatcatc 2700
gtcttcattt ttgccgtggt cggcatgcag ctctttggta aaagctacaa agattgtgtc 2760
tgcaagatcg ccagtgattg tcaactccca cgctggcaca tgaatgactt cttccactcc 2820
ttcctgattg tgttccgcgt gctgtgtggg gagtggatag agaccatgtg ggactgtatg 2880
gaggttgctg gtcaagccat gtgccttact gtcttcatga tggtcatggt gattggaaac 2940
ctagtggtcc tgaatctctt tctggccttg cttctgagct catttagtgc agacaacctt 3000
gcagccactg atgatgataa tgaaatgaat aatctccaaa ttgctgtgga taggatgcac 3060
aaaggagtag cttatgtgaa aagaaaaata tatgaattta ttcaacagtc cttcattagg 3120
aaacaaaaga ttttagatga aattaaacca cttgatgatc taaacaacaa gaaagacagt 3180
tgtatgtcca atcatacagc agaaattggg aaagatcttg actatcttaa agatgtaaat 3240
ggaactacaa gtggtatagg aactggcagc agtgttgaaa aatacattat tgatgaaagt 3300
gattacatgt cattcataaa caaccccagt cttactgtga ctgtaccaat tgctgtagga 3360
gaatctgact ttgaaaattt aaacacggaa gactttagta gtgaatcgga tctggaagaa 3420
agcaaagaga aactgaatga aagcagtagc tcatcagaag gtagcactgt ggacatcggc 3480
gcacctgtag aagaacagcc cgtagtggaa cctgaagaaa ctcttgaacc agaagcttgt 3540
ttcactgaag gctgtgtaca aagattcaag tgttgtcaaa tcaatgtgga agaaggcaga 3600
ggaaaacaat ggtggaacct gagaaggacg tgtttccgaa tagttgaaca taactggttt 3660
gagaccttca ttgttttcat gattctcctt agtagtggtg ctctggcatt tgaagatata 3720
tatattgatc agcgaaagac gattaagacg atgttggaat atgctgacaa ggttttcact 3780
tacattttca ttctggaaat gcttctaaaa tgggtggcat atggctatca aacatatttc 3840
accaatgcct ggtgttggct ggacttctta attgttgatg tttcattggt cagtttaaca 3900
gcaaatgcct tgggttactc agaacttgga gccatcaaat ctctcaggac actaagagct 3960
ctgagacctc taagagcctt atctcgattt gaagggatga gggtggttgt gaatgccctt 4020
ttaggagcaa ttccatccat catgaatgtg cttctggttt gtcttatatt ctggctaatt 4080
ttcagcatca tgggcgtaaa tttgtttgct ggcaaattct accactgtat taacaccaca 4140
actggtgaca ggtttgacat cgaagacgtg aataatcata ctgattgcct aaaactaata 4200
gaaagaaatg agactgctcg atggaaaaat gtgaaagtaa actttgataa tgtaggattt 4260
gggtatctct ctttgcttca agttgccaca ttcaaaggat ggatggatat aatgtatgca 4320
gcagttgatt ccagaaatgt ggaactccag cctaagtatg aagaaagtct gtacatgtat 4380
ctttactttg ttattttcat catctttggg tccttcttca ccttgaacct gtttattggt 4440
gtcatcatag ataatttcaa ccagcagaaa aagaagtttg gaggtcaaga catctttatg 4500
acagaagaac agaagaaata ctataatgca atgaaaaaat taggatcgaa aaaaccgcaa 4560
aagcctatac ctcgaccagg aaacaaattt caaggaatgg tctttgactt cgtaaccaga 4620
caagtttttg acataagcat catgattctc atctgtctta acatggtcac aatgatggtg 4680
gaaacagatg accagagtga atatgtgact accattttgt cacgcatcaa tctggtgttc 4740
attgtgctat ttactggaga gtgtgtactg aaactcatct ctctacgcca ttattatttt 4800
accattggat ggaatatttt tgattttgtg gttgtcattc tctccattgt aggtatgttt 4860
cttgccgagc tgatagaaaa gtatttcgtg tcccctaccc tgttccgagt gatccgtctt 4920
gctaggattg gccgaatcct acgtctgatc aaaggagcaa aggggatccg cacgctgctc 4980
tttgctttga tgatgtccct tcctgcgttg tttaacatcg gcctcctact cttcctagtc 5040
atgttcatct acgccatctt tgggatgtcc aactttgcct atgttaagag ggaagttggg 5100
atcgatgaca tgttcaactt tgagaccttt ggcaacagca tgatctgcct attccaaatt 5160
acaacctctg ctggctggga tggattgcta gcacccattc tcaacagtaa gccacccgac 5220
tgtgacccta ataaagttaa ccctggaagc tcagttaagg gagactgtgg gaacccatct 5280
gttggaattt tcttttttgt cagttacatc atcatatcct tcctggttgt ggtgaacatg 5340
tacatcgcgg tcatcctgga gaacttcagt gttgctactg aagaaagtgc agagcctctg 5400
agtgaggatg actttgagat gttctatgag gtttgggaga agtttgatcc cgatgcaact 5460
cagttcatgg aatttgaaaa attatctcag tttgcagctg cgcttgaacc gcctctcaat 5520
ctgccacaac caaacaaact ccagctcatt gccatggatt tgcccatggt gagtggtgac 5580
cggatccact gtcttgatat cttatttgct tttacaaagc gggttctagg agagagtgga 5640
gagatggatg ctctacgaat acagatggaa gagcgattca tggcttccaa tccttccaag 5700
gtctcctatc agccaatcac tactacttta aaacgaaaac aagaggaagt atctgctgtc 5760
attattcagc gtgcttacag acgccacctt ttaaagcgaa ctgtaaaaca agcttccttt 5820
acgtacaata aaaacaaaat caaaggtggg gctaatcttc ttataaaaga agacatgata 5880
attgacagaa taaatgaaaa ctctattaca gaaaaaactg atctgaccat gtccactgca 5940
gcttgtccac cttcctatga ccgggtgaca aagccaattg tggaaaaaca tgagcaagaa 6000
ggcaaagatg aaaaagccaa agggaaacat catcatcatc atcattaa 6048

Claims (5)

1.一种6His-Nav1.1融合蛋白稳定表达细胞系的构建方法，其特征在于，所述的构建方法包括如下步骤：
(1)构建携带SEQ ID No.3所示序列的重组载体；
(2)用所述重组载体转化EPI400感受态细胞，挑选转化菌落、培养，扩增所述重组载体；
(3)用所述扩增得到的重组载体转染HEK293细胞，传代培养，筛选，获得6His-Nav1.1融合蛋白稳定表达细胞系;
其中，步骤(1)中构建所述重组载体采用pcDNA3.1/Hygro(+)。
2.根据权利要求1所述的6His-Nav1.1融合蛋白稳定表达细胞系的构建方法，其特征在于，步骤(2)中，所述培养的过程中，培养液中添加有illumina公司的CopyCutterTM诱导剂。
3.根据权利要求2所述的6His-Nav1.1融合蛋白稳定表达细胞系的构建方法，其特征在于，所述培养液与诱导剂的体积比为(1200～800)：1。
4.根据权利要求1至3任一项所述的6His-Nav1.1融合蛋白稳定表达细胞系的构建方法，其特征在于，步骤(3)中，所述转染采用的转染试剂为Lipofectamine 3000转染试剂。
5.根据权利要求1至3任一项所述的6His-Nav1.1融合蛋白稳定表达细胞系的构建方法，其特征在于，步骤(3)中，所述转染采用的转染试剂为X-tremeGENE HP DNATransf.Reag。
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