CN114958915A - 一种人源otop1基因的真核重组质粒和表达人源酸味受体otop1蛋白的真核细胞系 - Google Patents
一种人源otop1基因的真核重组质粒和表达人源酸味受体otop1蛋白的真核细胞系 Download PDFInfo
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Abstract
本发明公开一种人源OTOP1基因的真核重组质粒,其由以下步骤制备得到:(1)将OTOP1基因cDNA序列与FLAG标签序列连接,得到如SEQ ID No.1所示的FLAG‑OTOP1基因序列;(2)将步骤(1)得到的序列插入到真核表达载体的多克隆酶切位点处,即得到所述真核重组质粒。本发明还公开稳定表达重组人源酸味受体OTOP1蛋白的细胞系,其包含上述人源OTOP1基因的真核重组质粒。本发明还公开了所述细胞系的构建过程及酸味受体OTOP1蛋白在细胞中的定位和表达。本发明可在细胞水平检测外源酸味剂的“酸度”,建立了一种可以快速、高效、灵敏检测酸味物质的方法,具有好的应用前景。
Description
技术领域
本发明涉及人源OTOP1基因的真核重组质粒和表达人源酸味受体OTOP1细胞系的构建,具体地说,涉及将含有人源酸味受体OTOP1的基因转入HEK293细胞系,进而得到表达酸味受体OTOP1蛋白的细胞系,将为食品酸度的检测、酸味拮抗剂和激动剂的筛选与研制提供一种技术方法,可以用于食品工业、医学及药学等领域。
背景技术
味觉受体细胞(Taste Receptor Cells,TRCs)是味觉探测的解剖学单位,这些细胞组装成味蕾,分布在舌头和腭部的不同乳头上。味觉首先是在TRCs水平上激活的,通过感觉传入纤维将信息传递到大脑中的味觉皮层,以进行味觉感知。酸味检测是一种重要的感官输入,可以警告人们不要摄入酸性(例如变质或未成熟)食物来源。
人的酸味受体是氢离子(H+)选择性离子通道OTOP1,编码OTOP1亚基的基因位于4号染色体的4p16.3位点,OTOP1亚基的基因组DNA全长38092bp;含有6个外显子,转录mRNA的ORF为1838bp。OTOP1蛋白亚基全长610个氨基酸,分子量为67.4KD,主要分布在细胞质膜上。
酸刺激后,H+通过OTOP1通道进入酸味感受细胞,导致细胞内pH值降低,从而阻断向内整流性钾离子(K+)通道Kir2.1。OTOP1和Kir2.1通道协同工作,将酸刺激转化为膜去极化,触发动作电位,激活电压门控的钙离子(Ca2+)通道,细胞内Ca2+增加使得Ca2+依赖性的味觉神经元突触胞吐释放神经递质,激活传入神经纤维,Ca2+是可供检测的下游信号分子。
关于酸味受体的检测,常规方法包括免疫荧光技术(Immunofluorescencetechnique)、蛋白质免疫印迹技术(Western blotting)等方法,这些方法能够检测到酸味受体蛋白 OTOP1在细胞内的表达定位及其表达水平,但通过检测酸味受体蛋白含量来定量检测酸度的方法过于复杂,人们希望有更简便的检测酸度定量检测方法。
发明内容
本发明第一方面提供了一种人源OTOP1基因(其核酸序列如SEQ ID No.1)的真核重组质粒,其中OTOP1基因与FLAG标签序列连接,得到如SEQ ID No.1所示的 FLAG-OTOP1基因序列,然后该基因序列被插入到真核表达载体pLVX-Puro的多克隆酶切位点处,得到所述人源OTOP1基因的真核重组质粒。
优选地,,步骤(2)中所述的真核表达载体为pLVX-Puro,用此载体得到的人源OTOP1基因的真核重组质粒命名为pLVX-Puro-OTOP1。
优选地,所述的OTOP1基因通过基因合成扩增得到。
本发明第二方面提供了一种表达人源酸味受体OTOP1蛋白(其氨基酸序列如SEQID No.2)的细胞系,其包含根据本发明第一方面所示的人源OTOP1基因的真核重组质粒。
本发明第三方面提供了根据本发明第二方面表达人源OTOP1蛋白的细胞系的制备方法,该方法包括以下步骤:
(1)将人源OTOP1基因cDNA序列与FLAG标签序列连接后,插入到真核表达载体的多克隆酶切位点处,构建含有OTOP1基因的重组质粒;
(2)将步骤(1)得到的重组质粒转染进入真核细胞中,并通过药物抗性筛选,得到具有抗生素抗性的阳性细胞克隆;
(3)将步骤(2)中得到的阳性细胞克隆扩增培养即得到稳定表达融合蛋白FLAG-OTOP1的细胞系。
优选地,所述步骤(1)中的真核表达载体为pLVX-Puro;
优选地,所述步骤(2)中的转染方法为电穿孔转染法,所述真核细胞为人胚胎肾细胞HEK293,所述药物为嘌呤霉素。
本发明第四方面提供了一种在细胞水平上定量检测酸味剂酸度的方法,其特征在于,使用根据本发明第三方面所示的表达人源OTOP1蛋白的细胞系作为检测细胞,向含有该检测细胞的体系中加入待测酸味剂,并检测该检测细胞内的钙离子浓度变化,与标准酸度的酸味剂导致的钙离子浓度变化进行比较,即定量检测到该酸味剂的酸度。
本发明具有以下有益效果:
(1)本发明利用电穿孔转染法建立细胞系,OTOP1表达稳定,在多次传代后仍保持很好的蛋白表达水平;
(2)本发明构建的重组质粒pLVX-Puro-OTOP1中,除了包含插入编码OTOP1蛋白的基因序列外,还含有FLAG标签序列,便于后期的蛋白质检测;
(3)本发明用检测HEK293-FLAG-OTOP1细胞系胞内钙离子的浓度来评估细胞系表达的OTOP1酸味受体的活化水平,所述方法易标准化、重复性好,且本发明的细胞系制备方法简单、成本低廉;
(4)本发明中提供的表达OTOP1蛋白的HEK293-FLAG-OTOP1细胞系可用于通过检测添加外源酸味剂后胞内钙离子的浓度变化来评估外源酸味剂的酸度,建立了一种可以快速、高效、稳定、可靠地筛选酸味剂酸度的方法,具有好的应用前景。
附图说明
图1是稳定表达FLAG-OTOP1的细胞系克隆RT-PCR检测结果。
图2是稳定表达FLAG-OTOP1的细胞系克隆激光共聚焦检测结果。
图3是稳定表达FLAG-OTOP1的细胞系克隆Western blot检测结果。
图4是稳定表达FLAG-OTOP1的细胞系在加酸前后的钙离子荧光图。
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。此外,下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
实施例1
本实施例涉及pLVX-Puro-FLAG-OTOP1重组质粒的构建及检测。
1.编码FLAG-OTOP1蛋白的基因序列设计及制备
1.1OTOP1蛋白的基因序列设计
以人源OTOP1基因cDNA序列(SEQ ID No.1)为模板,设计用于编码表达重组 OTOP1蛋白(SEQ ID No.2)的特异性正向引物OTOP1-F(SEQ ID No.3)和特异性反向引物OTOP1-R(SEQ ID No.4)。OTOP1-F在起始密码子前加入EcoRI酶切位点和保护性碱基。OTOP1-R在OTOP1编码末端加有终止子、BamH I酶切位点与保护性碱基,并在起始密码子后加入编码FLAG的碱基序列,以在真核表达的OTOP1蛋白N端加入 FLAG标签。
1.2编码OTOP1重组蛋白的核酸片段制备
以人源OTOP1的cDNA为模板,加入正向引物OTOP1-F和反向引物OTOP1-R进行基因扩增。扩增体系见表1:
表1
2X Taq Mix | 17μL |
Primer Otop1-F(10μM) | 1μL |
Primer Otop1-R(10μM) | 1μL |
模板(0.1-1μg) | cDNA |
扩增条件:为95℃预变性8min,再进行35个循环,循环条件为95℃变性30s, 65℃退火30s,72℃延伸30s;最后72℃延伸5min。
2.重组质粒的构建
将真核表达载体pLVX-Puro用EcoR I和BamH I酶切后回收,然后与经EcoR I和BamH I酶切后回收的FLAG-OTOP1基因扩增产物在T4连接酶作用下连接,转化DH5α感受态细胞后涂布在含氨苄的LB平板,培养16h后,挑取单菌落扩增培养并提取质粒,送至测序公司测序鉴定,鉴定的阳性质粒即为本实施例所需重组质粒,提取质粒保存。将经过测序验证后的重组质粒命名为pLVX-Puro-FLAG-OTOP1。
实施例2
本实施例涉及表达FLAG-OTOP1蛋白的重组细胞系的构建及检测。
1.重组FLAG-OTOP1质粒转染细胞
为构建稳定表达FLAG-OTOP1的真核细胞系,利用电穿孔方法将重组质粒 pLVX-Puro-FLAG-OTOP1转染入HEK293细胞,具体步骤如下所示:
取传代的HEK 293细胞,移除培养基,用3mL PBS溶液洗涤两次,用1mL 0.25%的胰蛋白酶(Trypsin)消化细胞5min,加入1mL培养基洗涤以终止反应,并多次吹吸,1000rpm离心5min;再用1mL PBS溶液重悬细胞,1000rpm离心5min,移除PBS。加入适量电转缓冲液悬浮细胞。配制电转溶液,体系如下表2所示,
表2
溶液 | 体积 |
pLVX-Puro-FLAG-Otop1(1λ) | 10μL |
1X电转缓冲液 | 1mL |
MgSO4 | 25μL |
用上述电转缓冲液重悬HEK 293细胞,轻轻混匀,转移至电转杯中。将电转杯放入电转槽,1000μF,260V电转。转移细胞至相应10mL培养基中,混匀细胞后轻轻加入培养皿中,使其均匀分布,转入CO2培养箱中培养。
2.阳性克隆的筛选
转染细胞培养约48h后,在细胞培养基中加入嘌呤霉素(puromycin)使其终浓度为0.6μg/mL,48h后换入新的、嘌呤霉素终浓度为0.6μg/mL的新鲜培养基培养细胞。每隔2天换含0.6μg/mL的嘌呤霉素的新鲜培养基培养2天,重复6次。消化、收集细胞,用无限稀释法将细胞分在96孔板中,加入含0.6μg/mL的嘌呤霉素培养基培养,培养、等待10天左右。挑取96孔板中由单个细胞增殖来的一团细胞到1个24孔板中,待其长到合适的数量后,消化、收集细胞,传代到12孔板中。重复此过程,将细胞依次培养在6孔板、60mm培养皿、100mm培养皿,并保种。
3.阳性克隆的鉴定
将克隆细胞在培养皿中培养2天,收集细胞提取RNA,用RT-PCR进行鉴定,以获得克隆细胞中FLAG-OTOP1的表达情况。利用FLAG特异性抗体,我们发现挑选有FLAG-OTOP1的表达的单克隆细胞,继续扩大培养后保存,将该细胞系命名为HEK293- FLAG-OTOP1,进行后续实验。RT-PCR结果如图1所示,显影结果如图3所示。
实施例3
本实施例涉及共聚焦显微镜检测FLAG-OTOP1酸味受体蛋白在细胞中的表达。
1样品的制备
通过使用共聚焦显微技术,我们检测了上述细胞系中酸味受体蛋白OTOP1亚基的表达和定位。使用DMEM培养基(Gibco)将5-10x105个瞬时或稳定表达FLAG-OTOP1 酸味受体蛋白的HEK293细胞种植在3.5cm的培养皿中,培养皿中放置13mm的圆形盖玻片。在细胞密度达到50%-80%时,将盖玻片取出,经过4%的多聚甲醛固定、3%BSA 封闭后,用FLAG特异性抗体(鼠源)孵育,去除未结合抗体,最后用抗鼠的二抗TRITC 抗体孵育,去除未结合抗体,制成可用于共聚焦显微镜检测免疫荧光的样品,所述TRITC 为荧光标签罗丹明。
2荧光检测
将制备好的玻片倒置在共聚焦显微镜下,在明场下,用60x物镜找到形态较好的细胞,粗、微调调节焦距,清晰地观测细胞样品。转换为荧光观察,选用TRITC染料,图像满足要求时,尽量使用低激光强度。并通过PMTHV(增强图像信号),Gain(增强信号)和Offset(扣除背景)调节图像。然后进行荧光扫描。
结合FLAG-OTOP1蛋白抗体上的TRITC,在541nm激光法激发下,呈红色荧光信号,拍摄结果如图2;可以观察到OTOP1蛋白分布在细胞的质膜上。
实施例4
本实施例涉及使用表达人源FLAG-OTOP1蛋白的细胞系来定量检测酸味剂酸度。
200μM乙酸溶液下,HEK293细胞和HEK293-OTOP1细胞胞内钙离子浓度变化:
(1)共聚焦培养皿中种植细胞:将细胞按比例传代到共聚焦培养皿中。待培养皿中细胞贴壁生长24h-48h,在光学显微镜下观察到细胞覆盖培养皿底部的面积达到 40%-60%。
(2)荧光探针标记细胞:将HBSS溶液放在37℃水浴锅中温浴30min。HBSS溶液即Hanks的平衡盐溶液,为商购Thermo公司产品,具体成分见下表:
将10μl浓度为1mM的Fluo-4AM稀释到10ml Flex缓冲液,得到Fluo-4AM(10μM) 稀释液,避光。除去共聚焦培养皿中的培养液,每皿加入1ml的Fluo-4AM(10μM)稀释液,37℃避光培养45-60min。培养结束后,除去Fluo-4AM稀释液(10μM),加入1ml HBSS 缓冲液清洗一遍。每皿加入1ml HBSS缓冲液,以备后续FV3000激光扫描共聚焦显微镜检测。
(3)激光共聚焦扫描显微镜获取TIME轴荧光图片
按顺序打开激光扫描共聚焦显微,打开488nm激发光激光器;
打开软件,调节扫描速度为8μs/Pixel,像素为640x 640,打开时间轴扫描模式,激发光为488nm,接受光通道为500nm-600nm;将物镜调节为60倍镜,在镜头上滴加一滴香柏油,将培养皿固定在在载物台上方;将镜头上升,使香柏油与培养皿下表面接触,再调节细准焦螺旋寻找细胞,直至在目镜下可清楚观察到细胞形态;寻找细胞形态好,分散均匀的视野(方便后续荧光定量);快速扫描荧光图像,通过调节激发光强度、 HV(增强图像信号)、Gain(增强信号)、Offset(扣除背景)以及焦平面,得到能清晰地扫描出细胞中钙离子荧光图像;用移液枪将培养皿中的HBSS缓冲液移除,再次调节焦平面;开始扫描荧光图像,扫描时长为3min,总帧数为70;开始扫描后的第16s时,在检测目的细胞周围悬空滴加200μl的200μM乙酸(切勿触碰培养皿,防止视野移动);等待扫描时间结束,保存图像数据,后续进行数据收集和处理。拍摄结果如图4。
(4)定量单细胞胞内钙离子浓度变化
将图像文件在Image J软件中打开,在(4)步骤中拍摄的荧光图片中随机选取三个视野,每个视野中有2-3个细胞,分别提取细胞在各帧图像的荧光数值。以前16s的背景的最低荧光值为Fo,以前2min的细胞的最大荧光值为Fm,依以下公式获得单个细胞的荧光变化比值:△F/Fo=(Fm-Fo)/Fo;对图像文件中多个细胞的荧光变化比值平均,将HEK293-OTOP1细胞荧光强度变化比率△F/Fo值减去HEK293细胞荧光强度变化比率△F/Fo的值即得到荧光变化比值△Rs,其值为3.399553,可见本发明的细胞系能响应酸味剂的存在而产生增强的钙离子荧光,通过检测钙离子荧光强度,能够定量检测酸味剂的酸度。
本领域的技术人员容易理解,以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
序列表
<110> 云南中烟工业有限责任公司
<120> 一种人源OTOP1基因的真核重组质粒和表达人源酸味受体OTOP1蛋白的真核细胞系
<130> RIB220348
<160> 6
<170> SIPOSequenceListing 1.0
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<211> 1863
<212> DNA
<213> 人工序列(Homo sapiens)
<400> 1
atggattaca aggacgacga tgacaagctc gagggcctgg ggtcgcccgc ctcgccccgg 60
gcagctgcaa gcgcctcggt cgcagggtcg tcggggccag cggcctgctc gcctccctcg 120
tcctcggccc cgaggtcccc ggaatccccg gccccccggc ggggcggtgt gcgcgccagc 180
gtcccacaga aactggccga gatgctgagc agccagtatg ggctgatcgt gttcgtggcg 240
gggctgctgc tgctgctggc ctgggccgtg cacgccgcgg gcgtgagcaa gagcgacctg 300
ctgtgcttcc tgacggcgct catgctgctg cagctgctgt ggatgctgtg gtacgtgggc 360
cgcagctccg cgcaccgccg cctcttccgc ctcaaggaca cgcacgcggg tgccggctgg 420
ctgcgcggta gtatcacatt gtttgcagtc attaccgtca tcctgggatg ccttaaaatt 480
ggatacttca ttggattttc agaatgttta tcagccactg aaggagtttt cccagtcacc 540
cattcagtgc atactttgtt gcaggtatat tttctttggg ggcatgcaaa ggatattatc 600
cagtctttca aaacactgga aaggtttgga gtgatccact cggtgttcac caacctgctt 660
ctgtgggcca atggcgtcct caatgagtca aagcaccaac tcaatgagca caaggaacgg 720
ctcatcactc tgggttttgg gaacataaca acagttttag atgaccacac accgcagtgt 780
aactgcacgc ccccaactct gtgcactgcc atctcccacg ggatctacta cctctacccc 840
ttcaacatag agtatcagat cctggcctcc acaatgctct acgtcctgtg gaagaacatc 900
gggcgcaaag ttgacagcca tcagcaccag aagatgcagt tcaagtctga tggggtcatg 960
gtgggcgcag tcctgggcct gaccgtgctg gccgccacca ttgctgtggt ggtggtatac 1020
ctgattcata ttgggcgctc caagaccaag agcgagtcgg cactcatcat gttctacctg 1080
tatgccatca ccctgctgat gcttatgggg gctgcggggc tggctggaat ccggatttac 1140
aggatagacg agaagtcact ggatgagtcc aaaaatccgg cccgcaaact ggactcggac 1200
ctcttggtgg gcactgcctc gggctcctgg cttatctcct ggggctcaat cttggccatc 1260
ctctgtgctg agggccaccc ccgctacacc tggtacaacc tgccctactc catcctggcg 1320
atcgtggaga agtacatcca gaacctcttc atctttgaat ccattcaccg agagcctgaa 1380
aaactctctg aggacatcca aacccttcgg gtggtcacag tctgcaatgg caacaccatg 1440
ccccttgctt cttcctgccc caagagtgga ggtgtggcca gagatgtggc tccccagggc 1500
aaggacatgc caccagcagc caatggaaat gtgtgcatga gagaaagcca tgacaaggag 1560
gaggagaagc aggaggagag cagctgggga gggagcccaa gcccagtccg ccttccccgt 1620
ttcttacagg gcaacgccaa gagaaaagtc ctgaggaata ttgcagcctt cttgttcctc 1680
tgcaatattt cgctttggat acctcccgcc tttggctgtc gacctgagta tgacaatgga 1740
ttggaggaga ttgtctttgg ctttgaaccc tggataattg tggtcaacct ggccatgcct 1800
ttttctattt tctatcgaat gcacgcagct gcctccctct ttgaggtcta ttgtaagata 1860
tag 1863
<210> 2
<211> 620
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 2
Met Asp Tyr Lys Asp Asp Asp Asp Lys Leu Glu Gly Leu Gly Ser Pro
1 5 10 15
Ala Ser Pro Arg Ala Ala Ala Ser Ala Ser Val Ala Gly Ser Ser Gly
20 25 30
Pro Ala Ala Cys Ser Pro Pro Ser Ser Ser Ala Pro Arg Ser Pro Glu
35 40 45
Ser Pro Ala Pro Arg Arg Gly Gly Val Arg Ala Ser Val Pro Gln Lys
50 55 60
Leu Ala Glu Met Leu Ser Ser Gln Tyr Gly Leu Ile Val Phe Val Ala
65 70 75 80
Gly Leu Leu Leu Leu Leu Ala Trp Ala Val His Ala Ala Gly Val Ser
85 90 95
Lys Ser Asp Leu Leu Cys Phe Leu Thr Ala Leu Met Leu Leu Gln Leu
100 105 110
Leu Trp Met Leu Trp Tyr Val Gly Arg Ser Ser Ala His Arg Arg Leu
115 120 125
Phe Arg Leu Lys Asp Thr His Ala Gly Ala Gly Trp Leu Arg Gly Ser
130 135 140
Ile Thr Leu Phe Ala Val Ile Thr Val Ile Leu Gly Cys Leu Lys Ile
145 150 155 160
Gly Tyr Phe Ile Gly Phe Ser Glu Cys Leu Ser Ala Thr Glu Gly Val
165 170 175
Phe Pro Val Thr His Ser Val His Thr Leu Leu Gln Val Tyr Phe Leu
180 185 190
Trp Gly His Ala Lys Asp Ile Ile Gln Ser Phe Lys Thr Leu Glu Arg
195 200 205
Phe Gly Val Ile His Ser Val Phe Thr Asn Leu Leu Leu Trp Ala Asn
210 215 220
Gly Val Leu Asn Glu Ser Lys His Gln Leu Asn Glu His Lys Glu Arg
225 230 235 240
Leu Ile Thr Leu Gly Phe Gly Asn Ile Thr Thr Val Leu Asp Asp His
245 250 255
Thr Pro Gln Cys Asn Cys Thr Pro Pro Thr Leu Cys Thr Ala Ile Ser
260 265 270
His Gly Ile Tyr Tyr Leu Tyr Pro Phe Asn Ile Glu Tyr Gln Ile Leu
275 280 285
Ala Ser Thr Met Leu Tyr Val Leu Trp Lys Asn Ile Gly Arg Lys Val
290 295 300
Asp Ser His Gln His Gln Lys Met Gln Phe Lys Ser Asp Gly Val Met
305 310 315 320
Val Gly Ala Val Leu Gly Leu Thr Val Leu Ala Ala Thr Ile Ala Val
325 330 335
Val Val Val Tyr Leu Ile His Ile Gly Arg Ser Lys Thr Lys Ser Glu
340 345 350
Ser Ala Leu Ile Met Phe Tyr Leu Tyr Ala Ile Thr Leu Leu Met Leu
355 360 365
Met Gly Ala Ala Gly Leu Ala Gly Ile Arg Ile Tyr Arg Ile Asp Glu
370 375 380
Lys Ser Leu Asp Glu Ser Lys Asn Pro Ala Arg Lys Leu Asp Ser Asp
385 390 395 400
Leu Leu Val Gly Thr Ala Ser Gly Ser Trp Leu Ile Ser Trp Gly Ser
405 410 415
Ile Leu Ala Ile Leu Cys Ala Glu Gly His Pro Arg Tyr Thr Trp Tyr
420 425 430
Asn Leu Pro Tyr Ser Ile Leu Ala Ile Val Glu Lys Tyr Ile Gln Asn
435 440 445
Leu Phe Ile Phe Glu Ser Ile His Arg Glu Pro Glu Lys Leu Ser Glu
450 455 460
Asp Ile Gln Thr Leu Arg Val Val Thr Val Cys Asn Gly Asn Thr Met
465 470 475 480
Pro Leu Ala Ser Ser Cys Pro Lys Ser Gly Gly Val Ala Arg Asp Val
485 490 495
Ala Pro Gln Gly Lys Asp Met Pro Pro Ala Ala Asn Gly Asn Val Cys
500 505 510
Met Arg Glu Ser His Asp Lys Glu Glu Glu Lys Gln Glu Glu Ser Ser
515 520 525
Trp Gly Gly Ser Pro Ser Pro Val Arg Leu Pro Arg Phe Leu Gln Gly
530 535 540
Asn Ala Lys Arg Lys Val Leu Arg Asn Ile Ala Ala Phe Leu Phe Leu
545 550 555 560
Cys Asn Ile Ser Leu Trp Ile Pro Pro Ala Phe Gly Cys Arg Pro Glu
565 570 575
Tyr Asp Asn Gly Leu Glu Glu Ile Val Phe Gly Phe Glu Pro Trp Ile
580 585 590
Ile Val Val Asn Leu Ala Met Pro Phe Ser Ile Phe Tyr Arg Met His
595 600 605
Ala Ala Ala Ser Leu Phe Glu Val Tyr Cys Lys Ile
610 615 620
<210> 3
<211> 62
<212> DNA
<213> 人工序列(Homo sapiens)
<400> 3
caagcttcga attcatggat tacaaggacg acgatgacaa gctcgagggc ctggggtcgc 60
cc 62
<210> 4
<211> 38
<212> DNA
<213> 人工序列(Homo sapiens)
<400> 4
cgcgggatcc ctatatctta caatagacct caaagagg 38
<210> 5
<211> 27
<212> DNA
<213> 人工序列(Homo sapiens)
<400> 5
atggattaca aggacgacga tgacaag 27
<210> 6
<211> 9
<212> PRT
<213> 人工序列(Homo sapiens)
<400> 6
Met Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
Claims (6)
1.一种人源OTOP1基因的真核重组质粒,其特征在于,由以下步骤制备得到:
(1)将OTOP1基因cDNA序列与FLAG标签序列连接,得到如SEQ ID No.1所示的FLAG-OTOP1基因序列;
(2)将步骤(1)得到的序列插入到真核表达载体的多克隆酶切位点处,即得到所述真核重组质粒。
2.根据权利要求1所述的人源OTOP1基因的真核重组质粒,其特征在于,步骤(2)中所述的真核表达载体为pLVX-Puro。
3.一种表达人源酸味受体OTOP1蛋白的细胞系,其特征在于,其包含根据权利要求1所示的人源OTOP1基因的真核重组质粒,其中所述人源酸味受体OTOP1蛋白的氨基酸序列如SEQ ID No.2所示。
4.根据权利要求3所述的表达人源OTOP1蛋白的细胞系的制备方法,其特征在于,该方法包括以下步骤:
(1)将人源OTOP1基因cDNA序列与FLAG标签序列连接后,插入到真核表达载体的多克隆酶切位点处,构建含有OTOP1基因的重组质粒;
(2)将步骤(1)得到的重组质粒转染进入真核细胞中,并通过药物抗性筛选,得到具有抗生素抗性的阳性细胞克隆;
(3)将步骤(2)中得到的阳性细胞克隆扩增培养即得到稳定表达融合蛋白FLAG-OTOP1的细胞系。
5.根据权利要求4所述的表达人源FLAG-OTOP1蛋白的细胞系的制备方法,其特征在于:
所述步骤(1)中的真核表达载体为pLVX-Puro;
所述步骤(2)中的转染方法为电穿孔转染法,所述真核细胞为人胚胎肾细胞HEK293,所述药物为嘌呤霉素。
6.一种在细胞水平上定量检测酸味剂酸度的方法,其特征在于,使用根据权利要求3所示的表达人源OTOP1蛋白的细胞系作为检测细胞,向含有该检测细胞的体系中加入待测酸味剂,并检测该检测细胞内的钙离子浓度变化,与标准酸度的酸味剂导致的钙离子浓度变化进行比较,即定量检测到该酸味剂的酸度。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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US20070009912A1 (en) * | 2005-07-06 | 2007-01-11 | The Regents Of The University Of California | Novel ion channel preferentially expressed in mammalian taste receptor cells |
US7629134B2 (en) * | 2006-07-10 | 2009-12-08 | Duke University | Sour taste receptor compositions and methods |
CN108611324A (zh) * | 2018-05-07 | 2018-10-02 | 云南中烟工业有限责任公司 | 一种稳定共表达重组人源甜味受体蛋白的真核细胞系及其制备方法 |
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Patent Citations (3)
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US20070009912A1 (en) * | 2005-07-06 | 2007-01-11 | The Regents Of The University Of California | Novel ion channel preferentially expressed in mammalian taste receptor cells |
US7629134B2 (en) * | 2006-07-10 | 2009-12-08 | Duke University | Sour taste receptor compositions and methods |
CN108611324A (zh) * | 2018-05-07 | 2018-10-02 | 云南中烟工业有限责任公司 | 一种稳定共表达重组人源甜味受体蛋白的真核细胞系及其制备方法 |
Non-Patent Citations (3)
Title |
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BOCHUAN TENG等: "Cellular and Neural Responses to Sour Stimuli Require the Proton Channel Otop1", CURR BIOL., vol. 29, no. 21, pages 2 * |
INNA HUGHES等: "Otopetrin 1 activation by purinergic nucleotides regulates intracellular calcium", PROC NATL ACAD SCI USA, vol. 104, no. 29, pages 12028 * |
NCBI: "GenBank:NM_177998.3", NCBI * |
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