CN114107351A - 人pak1蛋白的真核表达载体的构建方法、表达和纯化方法 - Google Patents
人pak1蛋白的真核表达载体的构建方法、表达和纯化方法 Download PDFInfo
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Abstract
本发明公开了一种人PAK1蛋白的真核表达载体的构建方法,包括如下步骤:1)以表达PAK1蛋白的基因序列为模板,采用PCR扩增获得PAK1基因片段;2)将得到的PAK1基因片段连接到真核表达载体上得到重组质粒;3)将得到的重组质粒进行转染转化复制,得到人PAK1蛋白的真核表达载体。还公开了利用真核表达系统进行重组人PAK1蛋白表达的方法:还包括步骤4)利用步骤3)获得的人PAK1蛋白的真核表达载体扩增纯化得到的质粒转染进入真核细胞中进行表达。还公开了获得重组人PAK1蛋白的方法:还包括步骤5)将步骤4)得到的阳性克隆进行培养扩大体系,收集培养细胞,离心收集上清,通过色谱柱纯化,即得。
Description
技术领域
本发明涉及分子生物学技术领域,涉及重组蛋白真核表达技术,尤其涉及一种人PAK1蛋白的真核表达载体的构建方法、表达和纯化方法。
背景技术
蛋白激酶(protein kinases,简称PK),是催化蛋白质磷酸化过程的酶。蛋白质的磷酸化过程是神经信息在细胞内传递的最后环节,导致离子通道蛋白及通道门的状态变化,是体内信号通路极其重要的组成部分,在神经细胞内有许多种类。因此,蛋白激酶的活力受到多种方式的调控,其中一种很常见的方式就是自磷酸化激活。PAK(p21-activatedkinase)蛋白家族在多种细胞过程中起着重要的作用,也受到相当精细的调控;PAK1是这个家族的代表性成员,其活力受到其N 端调控结构域及自磷酸化反应的调节,其活化环上一个保守的苏氨酸位点的磷酸化是其激活所必须的。
目前有很多关于人PAK1蛋白重组及表达的方法,但都是利用大肠杆菌系统表达,另外一个事实是目前市售的PAK1蛋白单抗在建立PAK1蛋白检测方法时,绝大多数抗体都存在特异性差、灵敏度低的缺点。我们认为利用原核系统进行蛋白表达过程中,由于缺乏与天然PAK1蛋白相同的表达环境导致重组的PAK1蛋白与天然PAK1蛋白结构有差异之处,导致制备出来的单抗存在识别天然PAK1 蛋白特异性差和灵敏度低的缺点。
发明内容
针对上述技术问题,本发明的目的是提供一种人PAK1蛋白的真核表达载体的构建方法,包括如下步骤:
1)以表达PAK1蛋白的基因序列为模板,采用PCR扩增获得PAK1基因片段;
2)将步骤1)得到的PAK1基因片段连接到真核表达载体上得到重组质粒;
3)将得到的重组质粒进行转染转化复制,得到人PAK1蛋白的真核表达载体。
在上述技术方案中,步骤2)中所述真核表达载体为pcDNA3.1,将将步骤1) 得到的PAK1基因片段和pcDNA3.1分别采用限制性内切酶BamHI、EcoRI消化后,连接消化产物得到重组质粒。
或者,步骤2)中所述真核表达载体为pCMV-blank,先将步骤1)得到的PAK1 基因片段采用T载体克隆,鉴定测序正确的克隆质粒与pCMV-blank分别采用限制性内切酶ApaⅠ、PstⅠ消化后,连接消化产物得到重组质粒。
在上述技术方案中,步骤3)将得到的重组质粒转化大肠杆菌复制得到大量的重组质粒。
在上述技术方案中,所述步骤1)中PCR扩增采用的引物为PAK1-F/PAK1-R,引物序列为:
PAK1-F:5’-AGCTCAACTATGATCTTTTT-3’,
PAK1-R:5’-GAATATTTTCATCTCCTTTT-3’。
本发明的另一目的是提供一种利用真核表达系统进行重组人PAK1蛋白表达的方法,在前述的构建方法基础上,还包括如下步骤:
4)利用步骤3)获得的人PAK1蛋白的真核表达载体的正确重组子扩增纯化得到的质粒转染进入真核细胞中进行表达。
优选地,步骤4)所述真核细胞包括用于外源基因表达的CHO细胞、COS-7 细胞、NSO细胞、酵母、昆虫细胞及人源细胞,所述转染采用脂质体法转染。
本发明的再一目的是提供一种获得重组人PAK1蛋白的方法,在前述的方法步骤基础上,还包括步骤5):
5)将步骤4)得到的阳性克隆进行培养扩大体系,收集培养细胞,破碎细胞,离心后收集上清,通过色谱柱将目的蛋白纯化,即获得高纯度重组人PAK1蛋白。
优选地,所述步骤5)中,收集培养细胞,采用超声破碎法破碎细胞,离心后收集上清,使用Ni柱进行亲和层析。
本发明的最后的目的是提供一种重组人PAK1蛋白的真核表达体系,是采用前述的方法构建得到的人PAK1蛋白的真核表达载体转染真核细胞得到的表达体系,所述真核细胞选自用于外源基因表达的CHO细胞、COS-7细胞、NSO细胞、酵母、昆虫细胞及人源细胞。
发明人认为现有技术利用原核系统进行蛋白表达过程中,由于缺乏与天然 PAK1蛋白相同的表达环境导致重组的PAK1蛋白与天然PAK1蛋白结构有差异之处,导致制备出来的单抗存在识别天然PAK1蛋白特异性差和灵敏度低的缺点。为此我们选择真核表达系统模拟天然PAK1蛋白的表达环境可选用CHO等真核表达系统进行人PAK1蛋白表达、纯化。CHO表达系统具有以下的优点:
(1)具有准确的转录后修饰功能,表达的蛋白在分子结构、理化特性和生物学功能方面最接近于天然蛋白分子;
(2)既可贴壁生长,又可以悬浮培养,且有较高的耐受剪切力和渗透压能力;
(3)具有重组基因的高效扩增和表达能力,外源蛋白的整合稳定;
(4)具有产物胞外分泌功能,并且很少分泌自身的内源蛋白,便于下游产物分离纯化;
(5)能以悬浮培养方式或在无血清培养基中达到高密度培养。且培养体积能达到1000L以上,可以大规模生产。
本发明中所指真核表达载体是具有可以携带基因片段并可以通过调控元件在真核表达系统调控表达携带基因片段的分子生物学载体工具,更进一步的说是可以通过信号肽进行分泌或不带有信号肽进行胞内表达的载体或者可以通过包装病毒进行感染宿主细胞将携带基因整合到宿主基因组而进行该基因表达的载体;本发明不限制选择使用真核表达载体。本发明中指述的真核表达体系是可以适合外源基因表达的所有真核细胞,本发明不限制选择使用宿主细胞。
本发明的有益效果是:利用本发明的真核表达系统克服了原核表达的缺点,获得了高活性的重组人PAK1蛋白,且经过本发明方法纯化后的蛋白纯度高,纯度达到95%以上,本发明方法获得的重组人PAK1蛋白与天然PAK1蛋白结构相同,采用本发明方法获得的重组人PAK1蛋白制备出来的单抗识别天然PAK1蛋白特异性高、且灵敏度高,为获得高质量抗体提供了有力的基础。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下述实施例中的实验方法,如无特别说明,均为常规方法;各实施例中所用生物、化学试剂,如无特殊说明,均为常规试剂,均可通过商购获得。
主要试剂来源:
pcDNA3.1(+)载体:从Invitrogen公司采购
限制酶BamHI、EcoRI、ApaⅠ、PstⅠ:使用NEB公司的产品
PCR扩增用试剂PCR反应MIX:使用北京全式金公司的产品
lipofectAMINE试剂:品牌Invitrogen
pGEM-T载体:从PROMEGA公司采购
含真核表达载体pCMV-blank的菌株:使用北京全式金公司的产品
实施例1PAK1蛋白真核表达载体的构建及其表达、纯化
1、PAK1蛋白真核表达载体的构建
按照PAK1蛋白的序列设计扩增引物:
上游引物PAK1-F(SEQ ID NO.1):5’-AGCTCAACTATGATCTTTTT-3’,
下游引物PAK1-R(SEQ ID NO.2):5’-GAATATTTTCATCTCCTTTT-3’。
其中在上游引物引入BamHI酶切位点,在下游引物引入EcoRI酶切位点及6×组氨酸标签。
采用HELA细胞为模板,用引物对PAK1-F/PAK1-R进行PCR扩增,反应体系是:PCR反应MIX 25μl、浓度为10μM的正反向引物各1μl、DNA模板2μl、加ddH2O至50μl。PCR扩增程序是:95℃180秒;94℃30秒,60℃30秒, 72℃30秒,35个循环;最后72℃300秒。
PCR扩增完毕进行琼脂糖凝胶电泳检测,采用胶回收试剂盒对凝胶回收后,用限制酶BamHI、EcoRI进行酶切,然后与同样用BamHI、EcoRI消化的 pcDNA3.1(+)载体采用连接酶连接并转化大肠杆菌DH5a感受态细胞,经过PCR 鉴定,选择阳性重组子进行测序鉴定,阳性重组子测得的序列如SEQ ID NO.3所示。
2、PAK1蛋白在CHO细胞中的表达
经过测序鉴定正确的克隆,大量制备质粒。
使用lipofectAMINE试剂,依据产品说明书进行CHO细胞转染,加入筛选药物G418Sulfate(0.8mg/ml),进行阳性克隆的筛选,挑选出含有质粒的CHO细胞。
将培养体系增大到1L,经过72小时的培养收集细胞,采用超声破碎法破碎细胞,离心后收集上清,使用Ni柱进行亲和层析。经过SDS-PAGE分析,蛋白大小为21kD左右,与理论值相同,产量为3mg/L。根据SDS-PAGE的条带灰度估算蛋白浓度后,将蛋白浓缩到3mg/ml。
本实施例用真核表达得到的纯化蛋白是使用ABCOM公司兔单克隆抗体[EPR20045]测定的,操作步骤如下:
(1)将ABCOM公司兔单克隆抗体[EPR20045]以0.1mg/ml的浓度稀释到碳酸缓冲液中,再ELISA反应孔中,每个孔加0.1ml,再2~8℃反应过夜。第二天洗去抗体,并用BSA封闭。
(2)将纯化后的蛋白用蛋白保护液稀释到约1.0μg/ml,2.0μg/ml,3.0μg/ml 用上述ELISA孔进行检测,用ABCOM公司人PAK1蛋白(ab67946)同样稀释到1.0μg/ml,2.0μg/ml,3.0μg/ml进行检测。
(3)每个浓度的检测重复5次。
(4)以上各浓度值测定取平均值,相关系数R>0.99。
采用原核表达的PAK1蛋白用作对照,使用ABCOM公司兔单克隆抗体 [EPR20045]对在大肠杆菌中表达的纯化蛋白进行测定:
(1)将ABCOM公司兔单克隆抗体[EPR20045]以0.1mg/ml的浓度稀释到碳酸缓冲液中,再ELISA反应孔中,每个孔加0.1ml,再2~8℃反应过夜。第二天洗去抗体,并用BSA封闭。
(2)将纯化后的蛋白用蛋白保护液稀释到约1.0μg/ml,2.0μg/ml,3.0μg/ml 用上述ELISA孔进行检测,用ABCOM公司人PAK1蛋白(ab67946)同样稀释到 1.0μg/ml,2.0μg/ml,3.0μg/ml进行检测。
(3)每个浓度的检测重复5次。
(4)以上各浓度值测定取平均值,相关系数R<0.95。
实施例2
一、PAK1基因的分子克隆
采用HELA细胞为模板,用实施例1中的引物对PAK1-F/PAK1-R进行PCR 扩增,反应体系是:PCR反应MIX 25μl、浓度为10μM的正反向引物各1μl、DNA 模板2μl、加ddH2O至50μl。PCR扩增程序是:95℃180秒;94℃30秒,60℃ 30秒,72℃30秒,35个循环;最后72℃300秒。
PCR扩增完毕进行琼脂糖凝胶电泳检测,采用胶回收试剂盒对凝胶回收纯化 PCR扩增产物,根据pGEM-TEasy载体系统产品说明书建立如下连接反应:10×T4 DNA连接反应缓冲液1μl,pGEM-T载体1μl(50ng),纯化的PCR扩增片段3μl, T4 DNA连接酶1μl(3U),加ddH2O至总体积为10μl,于16℃连接2小时,得到连接反应物。
二、制备大肠杆菌感受态细胞
用接种环从平板上挑取大肠杆菌DH5α单个菌落接种于3ml不含抗生素的 LB液体培养基中,37℃振摇培养过夜,取对数生长期的菌液0.5ml转种于50ml 不含抗生素的LB液体培养基中,继续于37℃振摇4小时,然后将培养液转入50ml 离心管中,冰浴10分钟,于4℃以4000转/分离心5分钟,弃培养液,用预冷 CaCl2(0.1mol/L)溶液重悬菌体,冰浴20分钟,于4℃以4000转/分离心20分钟,去上清,加1ml冰浴的CaCl2溶液(0.1mol/L)轻轻混匀,分装到1.5ml离心管中,冰浴2小时后,放4℃贮存备用。
取200μl DH5α感受态细胞菌液于1.5ml离心管中,加入2μl连接反应物,轻轻混匀,冰浴20分钟,放入42℃水浴中45秒,取出离心管迅速放入冰浴2分钟,加不含抗生素的SOC液体培养基800μl,37℃振摇(150转/分)1小时,取转化后的菌液100μl铺于LB平板上(含氨苄青霉素100μg/ml,表层铺有40μl 20mg/ml X-gal及4μl 200mg/ml IPTG)37℃培养过夜。将转化后37℃过夜培养的平板置于4℃ 4小时,使蓝白色菌落充分显现。从转化平板随机挑取白色菌落,接种于3ml含氨苄青霉素的LB液体培养基中,37℃振摇14小时,根据质粒提取试剂盒操作指南提取质粒DNA。测序确定所含目标序列准确,所得质粒为PGEM-TEasy-PAK1。三、真核表达载体pCMV-PAK1的构建及鉴定
将冻存的含真核表达载体pCMV-blank的菌株由冰箱取出,室温溶解;取 200ul冻存菌液,加入Kana的LB溶液中;37℃振荡培养箱过夜培养16h,提取质粒,测定质粒浓度后,将质粒浓度标记在EP管上,-20℃保存备用。
将测序正确的克隆质粒pGEM-TEasy-PAK1双酶切,反应体系为:质粒12μl, ApaⅠ、PstⅠ各1μl,10×反应缓冲液2μl,补足ddH2O至20μl,37℃酶切2h,存于 -20℃备用,取3μl酶切产物于1.5%琼脂糖凝胶电泳,观察结果。在紫外灯下用干净的手术刀切下含目的片段的琼脂块,放入1.5ml离心管中,用胶回收试剂盒回收目的基因PAK1基因。取3μl电泳观察提取效果,剩余样品-20℃保存备用。
pCMV-blank双酶切:取pCMV-blank 10μl,加入离心管中,加10×反应缓冲液2μl,ApaⅠ、PstⅠ各1μl,加纯水至20μl,37℃水浴3小时,取3μl酶切后的反应混合物于1.5%琼脂糖凝胶中电泳检测酶切效果。
胶回收pCMV-blank酶切后大片段,将前面胶回收的目的基因PAK1基因和 pCMV-blank大片段进行连接。反应体系如下:目的基因12μl,pCMV-blank大片段3μl,SolutionⅠ(TaKaRa DNA Ligation Kit Ver.2)15μl,在16℃连接2h,得到连接反应物。取200μl DH5α感受态细胞菌液于1.5ml离心管中,加入2μl连接反应物,轻轻混匀,冰浴20分钟,放入42℃水浴中45秒,取出离心管迅速放入冰浴 2分钟,加不含抗生素的SOC液体培养基800μl,37℃振摇(150转/分)1小时,取转化后的菌液100μl铺于含Kana的LB平板上,37℃培养过液。从转化平板挑取单菌落,接种于3ml含Kana的LB液体培养基中,37℃振摇过夜,根据质粒提取试剂盒操作指南提取质粒DNA,得到50μl质粒DNA,取3μl于1.5%琼脂糖凝胶中电泳检测质粒DNA。将所得重组质粒DNA 10μl,加入1.5ml离心管中,加10×反应缓冲液2μl,ApaⅠ、PstⅠ各1μl,加纯水至20μl,37℃水浴3小时,取 10μl酶切后的反应混合物于1.5%琼脂糖凝胶中电泳检测酶切效果。将经过限制性核酸内切酶双酶消化增鉴定为阳性的重组质粒测序,测得的序列如SEQ ID NO.4所示。结果显示重组的目的基因序列与Genbank中PAK1蛋白基因序列完全一致,并且方向正确。
结果表明成功构建了PAK1的真核表达载体pCMV-PAK1。
四、获得稳定表达PAK1的细胞株
COS-7细胞用含10%胎牛血清的DMEM高糖完全培养液,37℃、5%CO2 培养箱中恒温恒湿培养。转染前一天将成长汇合度达到85%~95%的细胞铺于 12孔板中,培养24h后,更换为无血清无抗生素的DMEM高糖培养液,参照说明书,将Lipofectin阳离子脂质体和重组质粒pCMV-PAK1混匀静置20min,之后均匀滴入12孔板的各孔中,培养6h后,吸弃各孔内的培养液,更换为无抗生素的新鲜DMEM高糖培养液继续培养。
以转染空白质粒pCMV-blank的COS-7细胞作为对照。细胞转染24h后,更换为含有筛选药物G418的细胞培养液,初始浓度为400μg/ml,压力筛选7天后细胞开始大量死亡,PBS清洗2遍后,调整G418浓度为200μg/ml,继续培养三天后,将残存细胞按照1个细胞/孔点种于96孔板内,维持G418浓度为200μg/ml 扩增培养8周,获得单克隆细胞株。PCR筛选PAK1的阳性单克隆细胞株,之后将阳性单克隆细胞株继续在G418浓度200μg/ml的环境中继续培养,连续传代十次后,再次以PCR鉴定,挑取仍然表达目的基因PAK1的阳性单克隆细胞株,以此细胞株作为PAK1稳定表达细胞株进行后续试验。收集PAK1蛋白稳定表达细胞株细胞培养上清液2ml,冷冻干燥浓缩后,作为蛋白样品进行Westernblotting 实验。蛋白检测方法与实施例1相同,检测出蛋白大小为21kD左右,与理论值相同。
序列表
<110> 绍兴守仁医疗健康科技有限公司
<120> 人PAK1蛋白的真核表达载体的构建方法、表达和纯化方法
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> 人工序列
<400> 1
agctcaacta tgatcttttt 20
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<211> 20
<212> DNA
<213> 人工序列
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gaatattttc atctcctttt 20
<210> 3
<211> 3266
<212> DNA
<213> 人工序列
<400> 3
agctcaacta tgatcttttt tgagaaacct cggatggtct gagttctacc agttctgcta 60
ctcattagtg aaagaaaaca taatacatgt ctggctgtga gatcacacga catcggatag 120
ttttcaaatt cagcccttga caacgagctc atagatgcgg tggtgctttg ttaacctgaa 180
aaggaccctc agcgccggaa gttagccgtc tttctccatc ctgactacta cagtagccct 240
cccagaaatc gtcccaaaat tgtctgatag aaaggctgca cggaaacaac ccgccacgga 300
cagcggaccg agaacagtga cgcgacgcga tgacgtcacg cagcaagcgg tagctgctgc 360
tggtggtgac aatgtcaaat aacggcctag acattcaaga caaaccccca gcccctccga 420
tgagaaatac cagcactatg attggagccg gcagcaaaga tgctggaacc ctaaaccatg 480
gttctaaacc tctgcctcca aacccagagg agaagaaaaa gaaggaccga ttttaccgat 540
ccattttacc tggagataaa acaaataaaa agaaagagaa agagcggcca gagatttctc 600
tcccttcaga ttttgaacac acaattcatg tcggttttga tgctgtcaca ggggagttta 660
cgggaatgcc agagcagtgg gcccgcttgc ttcagacatc aaatatcact aagtcggagc 720
agaagaaaaa cccgcaggct gttctggatg tgttggagtt ttacaactcg aagaagacat 780
ccaacagcca gaaatacatg agctttacag ataagtcagc tgaggattac aattcttcta 840
atgccttgaa tgtgaaggct gtgtctgaga ctcctgcagt gccaccagtt tcagaagatg 900
aggatgatga tgatgatgat gctaccccac caccagtgat tgctccacgc ccagagcaca 960
caaaatctgt atacacacgg tctgtgattg aaccacttcc tgtcactcca actcgggacg 1020
tggctacatc tcccatttca cctactgaaa ataacaccac tccaccagat gctttgaccc 1080
ggaatactga gaagcagaag aagaagccta aaatgtctga tgaggagatc ttggagaaat 1140
tacgaagcat agtgagtgtg ggcgatccta agaagaaata tacacggttt gagaagattg 1200
gacaaggtgc ttcaggcacc gtgtacacag caatggatgt ggccacagga caggaggtgg 1260
ccattaagca gatgaatctt cagcagcagc ccaagaaaga gctgattatt aatgagatcc 1320
tggtcatgag ggaaaacaag aacccaaaca ttgtgaatta cttggacagt tacctcgtgg 1380
gagatgagct gtgggttgtt atggaatact tggctggagg ctccttgaca gatgtggtga 1440
cagaaacttg catggatgaa ggccaaattg cagctgtgtg ccgtgagtgt ctgcaggctc 1500
tggagttctt gcattcgaac caggtcattc acagagacat caagagtgac aatattctgt 1560
tgggaatgga tggctctgtc aagctaactg actttggatt ctgtgcacag ataaccccag 1620
agcagagcaa acggagcacc atggtaggaa ccccatactg gatggcacca gaggttgtga 1680
cacgaaaggc ctatgggccc aaggttgaca tctggtccct gggcatcatg gccatcgaaa 1740
tgattgaagg ggagcctcca tacctcaatg aaaaccctct gagagccttg tacctcattg 1800
ccaccaatgg gaccccagaa cttcagaacc cagagaagct gtcagctatc ttccgggact 1860
ttctgaaccg ctgtctcgag atggatgtgg agaagagagg ttcagctaaa gagctgctac 1920
agcatcaatt cctgaagatt gccaagcccc tctccagcct cactccactg attgctgcag 1980
ctaaggaggc aacaaagaac aatcactaaa accacactca ccccagcctc attgtgccaa 2040
gccttctgtg agataaatgc acatttcaga aattccaact cctgatgccc tcttctcctt 2100
gccttgcttc tcccatttcc tgatctagca ctcctcaaga ctttgatcct tggaaaccgt 2160
gtgtccagca ttgaagagaa ctgcaactga atgactaatc agatgatggc catttctaaa 2220
taaggaattt cctcccaatt catggatatg agggtggttt atgattaagg gtttatataa 2280
ataaatgttt ctagtcttcc gtgtgtcaaa atcctcacct ccttcataac catctcccac 2340
aattaattct tgactatata aatttatggt ttgataatat tatcaatttg taatcaattg 2400
agatttcttt agtgcttgct tttctgtgac tcaactgccc agacacctca ttgtacttga 2460
aaactggaac agcttgggaa tgccatgggg tttgataatc tgccagggac atgaagaggc 2520
tcagcttcct ggaccatgac tttggctcag ctgatcctga catgggagaa caaccacatt 2580
tttctttgtg tgtgcttcta gcagctgttc gggaggacct tgacccaata gtgttcccat 2640
gctgtttctt gtgaaatgct ctcggctatg tagcagcttt tgattccctg cataccctag 2700
gctgctgccc ctatcctgtc ccttgtttat aacattgaga ggttttctag ggcacatact 2760
gagtgagagc agtgttgaga agtcggggaa aatggtgact acttttagag caaggctggg 2820
catcagcacc tgtccagctc tacttgtgtg atgtttcagg aactcagccc ctttttctgc 2880
ctaggataag gagctgaaag attaacttgg atcttctaat ggtccaaatc ttttggtcac 2940
aataaagagt ctccaaatta gagactgcat gttagttctg gatggatttg gtggcctgac 3000
atgataccct gccagctgtg aggggacccc gtttttaaga tgcatggcca agctctctgc 3060
aaatggaaat gcttacactg ggtgttgggg atgtttgcta cctcctgcta tttttgtggt 3120
tttggttctc ccactatggt aggacccctg gccagcattg tggcttgtca tgtcagcccc 3180
attgactacc ttctcatgct ctgaggtact actgcctctg cagcacaaat ttctatttct 3240
gtcaataaaa ggagatgaaa atattc 3266
<210> 4
<211> 3266
<212> DNA
<213> 人工序列
<400> 4
agctcaacta tgatcttttt tgagaaacct cggatggtct gagttctacc agttctgcta 60
ctcattagtg aaagaaaaca taatacatgt ctggctgtga gatcacacga catcggatag 120
ttttcaaatt cagcccttga caacgagctc atagatgcgg tggtgctttg ttaacctgaa 180
aaggaccctc agcgccggaa gttagccgtc tttctccatc ctgactacta cagtagccct 240
cccagaaatc gtcccaaaat tgtctgatag aaaggctgca cggaaacaac ccgccacgga 300
cagcggaccg agaacagtga cgcgacgcga tgacgtcacg cagcaagcgg tagctgctgc 360
tggtggtgac aatgtcaaat aacggcctag acattcaaga caaaccccca gcccctccga 420
tgagaaatac cagcactatg attggagccg gcagcaaaga tgctggaacc ctaaaccatg 480
gttctaaacc tctgcctcca aacccagagg agaagaaaaa gaaggaccga ttttaccgat 540
ccattttacc tggagataaa acaaataaaa agaaagagaa agagcggcca gagatttctc 600
tcccttcaga ttttgaacac acaattcatg tcggttttga tgctgtcaca ggggagttta 660
cgggaatgcc agagcagtgg gcccgcttgc ttcagacatc aaatatcact aagtcggagc 720
agaagaaaaa cccgcaggct gttctggatg tgttggagtt ttacaactcg aagaagacat 780
ccaacagcca gaaatacatg agctttacag ataagtcagc tgaggattac aattcttcta 840
atgccttgaa tgtgaaggct gtgtctgaga ctcctgcagt gccaccagtt tcagaagatg 900
aggatgatga tgatgatgat gctaccccac caccagtgat tgctccacgc ccagagcaca 960
caaaatctgt atacacacgg tctgtgattg aaccacttcc tgtcactcca actcgggacg 1020
tggctacatc tcccatttca cctactgaaa ataacaccac tccaccagat gctttgaccc 1080
ggaatactga gaagcagaag aagaagccta aaatgtctga tgaggagatc ttggagaaat 1140
tacgaagcat agtgagtgtg ggcgatccta agaagaaata tacacggttt gagaagattg 1200
gacaaggtgc ttcaggcacc gtgtacacag caatggatgt ggccacagga caggaggtgg 1260
ccattaagca gatgaatctt cagcagcagc ccaagaaaga gctgattatt aatgagatcc 1320
tggtcatgag ggaaaacaag aacccaaaca ttgtgaatta cttggacagt tacctcgtgg 1380
gagatgagct gtgggttgtt atggaatact tggctggagg ctccttgaca gatgtggtga 1440
cagaaacttg catggatgaa ggccaaattg cagctgtgtg ccgtgagtgt ctgcaggctc 1500
tggagttctt gcattcgaac caggtcattc acagagacat caagagtgac aatattctgt 1560
tgggaatgga tggctctgtc aagctaactg actttggatt ctgtgcacag ataaccccag 1620
agcagagcaa acggagcacc atggtaggaa ccccatactg gatggcacca gaggttgtga 1680
cacgaaaggc ctatgggccc aaggttgaca tctggtccct gggcatcatg gccatcgaaa 1740
tgattgaagg ggagcctcca tacctcaatg aaaaccctct gagagccttg tacctcattg 1800
ccaccaatgg gaccccagaa cttcagaacc cagagaagct gtcagctatc ttccgggact 1860
ttctgaaccg ctgtctcgag atggatgtgg agaagagagg ttcagctaaa gagctgctac 1920
agcatcaatt cctgaagatt gccaagcccc tctccagcct cactccactg attgctgcag 1980
ctaaggaggc aacaaagaac aatcactaaa accacactca ccccagcctc attgtgccaa 2040
gccttctgtg agataaatgc acatttcaga aattccaact cctgatgccc tcttctcctt 2100
gccttgcttc tcccatttcc tgatctagca ctcctcaaga ctttgatcct tggaaaccgt 2160
gtgtccagca ttgaagagaa ctgcaactga atgactaatc agatgatggc catttctaaa 2220
taaggaattt cctcccaatt catggatatg agggtggttt atgattaagg gtttatataa 2280
ataaatgttt ctagtcttcc gtgtgtcaaa atcctcacct ccttcataac catctcccac 2340
aattaattct tgactatata aatttatggt ttgataatat tatcaatttg taatcaattg 2400
agatttcttt agtgcttgct tttctgtgac tcaactgccc agacacctca ttgtacttga 2460
aaactggaac agcttgggaa tgccatgggg tttgataatc tgccagggac atgaagaggc 2520
tcagcttcct ggaccatgac tttggctcag ctgatcctga catgggagaa caaccacatt 2580
tttctttgtg tgtgcttcta gcagctgttc gggaggacct tgacccaata gtgttcccat 2640
gctgtttctt gtgaaatgct ctcggctatg tagcagcttt tgattccctg cataccctag 2700
gctgctgccc ctatcctgtc ccttgtttat aacattgaga ggttttctag ggcacatact 2760
gagtgagagc agtgttgaga agtcggggaa aatggtgact acttttagag caaggctggg 2820
catcagcacc tgtccagctc tacttgtgtg atgtttcagg aactcagccc ctttttctgc 2880
ctaggataag gagctgaaag attaacttgg atcttctaat ggtccaaatc ttttggtcac 2940
aataaagagt ctccaaatta gagactgcat gttagttctg gatggatttg gtggcctgac 3000
atgataccct gccagctgtg aggggacccc gtttttaaga tgcatggcca agctctctgc 3060
aaatggaaat gcttacactg ggtgttgggg atgtttgcta cctcctgcta tttttgtggt 3120
tttggttctc ccactatggt aggacccctg gccagcattg tggcttgtca tgtcagcccc 3180
attgactacc ttctcatgct ctgaggtact actgcctctg cagcacaaat ttctatttct 3240
gtcaataaaa ggagatgaaa atattc 3266
Claims (10)
1.一种人PAK1蛋白的真核表达载体的构建方法,其特征在于,包括如下步骤:
1)以表达PAK1蛋白的基因序列为模板,采用PCR扩增获得PAK1基因片段;
2)将步骤1)得到的PAK1基因片段连接到真核表达载体上得到重组质粒;
3)将得到的重组质粒进行转染转化复制,得到人PAK1蛋白的真核表达载体。
2.如权利要求1所述的人PAK1蛋白的真核表达载体的构建方法,其特征在于:步骤2)中所述真核表达载体为pcDNA3.1,将将步骤1)得到的PAK1基因片段和pcDNA3.1分别采用限制性内切酶BamHI、EcoRI消化后,连接消化产物得到重组质粒。
3.如权利要求1所述的人PAK1蛋白的真核表达载体的构建方法,其特征在于:步骤2)中所述真核表达载体为pCMV-blank,先将步骤1)得到的PAK1基因片段采用T载体克隆,鉴定测序正确的克隆质粒与pCMV-blank分别采用限制性内切酶ApaⅠ、PstⅠ消化后,连接消化产物得到重组质粒。
4.如权利要求1所述的人PAK1蛋白的真核表达载体的构建方法,其特征在于:步骤3)将得到的重组质粒转化大肠杆菌复制得到大量的重组质粒。
5.如权利要求1-4任一项所述的人PAK1蛋白的真核表达载体的构建方法,其特征在于:所述步骤1)中PCR扩增采用的引物为PAK1-F/PAK1-R,引物序列为:
PAK1-F:5’-AGCTCAACTATGATCTTTTT-3’,
PAK1-R:5’-GAATATTTTCATCTCCTTTT-3’。
6.一种利用真核表达系统进行重组人PAK1蛋白表达的方法,其特征在于:在权利要求1-5任一项所述的构建方法基础上,还包括如下步骤:
4)利用步骤3)获得的人PAK1蛋白的真核表达载体的正确重组子扩增纯化得到的质粒转染进入真核细胞中进行表达。
7.如权利要求6所述的方法,其特征在于:步骤4)所述真核细胞包括用于外源基因表达的CHO细胞、COS-7细胞、NSO细胞、酵母、昆虫细胞及人源细胞,所述转染采用脂质体法转染。
8.一种获得重组人PAK1蛋白的方法,其特征在于:在权利要求6或7所述的方法步骤基础上,还包括步骤5):
5)将步骤4)得到的阳性克隆进行培养扩大体系,收集培养细胞,破碎细胞,离心后收集上清,通过色谱柱将目的蛋白纯化,即获得高纯度重组人PAK1蛋白。
9.如权利要求8所述的方法,其特征在于:所述步骤5)中,收集培养细胞,采用超声破碎法破碎细胞,离心后收集上清,使用Ni柱进行亲和层析。
10.一种重组人PAK1蛋白的真核表达体系,其特征在于:是采用权利要求1至5任一项所述的方法构建得到的人PAK1蛋白的真核表达载体转染真核细胞得到的表达体系,所述真核细胞选自用于外源基因表达的CHO细胞、COS-7细胞、NSO细胞、酵母、昆虫细胞及人源细胞。
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