CN109402134A - 一种高效表达生长激素的工程菌的制备方法及其应用 - Google Patents
一种高效表达生长激素的工程菌的制备方法及其应用 Download PDFInfo
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- CN109402134A CN109402134A CN201811442625.8A CN201811442625A CN109402134A CN 109402134 A CN109402134 A CN 109402134A CN 201811442625 A CN201811442625 A CN 201811442625A CN 109402134 A CN109402134 A CN 109402134A
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Abstract
本发明公开了一种高效表达生长激素的工程菌的制备方法,包括以下步骤:1)通过PCR法对人生长激素hGH基因进行扩增,得到扩增后的hGH基因;2)将步骤1)中的扩增后的hGH基因连接到pMal‑p2x载体上,得到重组质粒pMal‑hGH;4)将步骤3)中的重组质粒导入到大肠杆菌BL21(DE3)中,通过IPTG诱导蛋白表达,并确定表达正确后,获得高效表达生长激素的工程菌。本发明分别采用pMal‑p2x作为载体,获得了重组质粒pMal‑p2x‑GH,pMal‑p2x‑GH的重组质粒的氨基端带有一个MBP蛋白标签,因而将重组质粒导入到大肠杆菌的受体中,可以将hGH和MBP标签进行融合表达,从而将hGH带到外周质空间,更有利于hGH蛋白的纯化,而且由于外周质的氧化环境,hGH蛋白能够正确形成二硫键,使蛋白具有较好的活性。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种高效表达生长激素的工程菌的制备方法及其应用。
背景技术
人生长激素(human growth bormone,hGH),其成熟形式是去除了信号肽的,由191个氨基酸组成的亲水性单链球蛋白,其相对分子量约为22kD,等电点为4.9,分子内存在两对二硫键,分别位于Cys53与Cys165之间和Cys182与Cys189之间。hGH是由人脑垂体前叶嗜酸性细胞分泌的一种重要的非糖基化蛋白质激素。脑垂体在下丘脑生长激素释放因子的刺激下,便能释放生长激素,通过血液运输到各个器官组织。hGH的受体遍及人的全身,因此生长激素能够影响几乎所有的组织类型和细胞。其主要作用是刺激骨、软骨细胞的生长和分化;调节蛋白质,糖类及脂肪的代谢等。临床上主要用于治疗侏儒症,近年来的研究发现hGH还能治疗烧伤、创伤、骨折及骨质疏松等多种疾病。
生长激素的种属特异性强,不同物种的生长激素差异很大,不能混用。因此,传统临床应用的生长激素只能是从人脑的垂体中提取的,一个人的垂体最多只能提取出3-5mg的人生长激素。来源受限造成价格昂贵,无法大量医用。随着分子生物学和基因工程的发展,生产重组蛋白的技术突飞猛进,使得大规模生产hGH成为现实。这样能够大大地满足了社会的需求。
由于生长激素不需要糖基化修饰,使得大肠杆菌成为了表达hGH的理想载体。传统的利用大肠杆菌表达重组hGH,有两种不同技术途径,一种是生产胞内型的重组人生长激素,在胞内的尽管表达量较高,但是提纯比较麻烦,另外重组蛋白容易降解,并且不能很好的形成二硫键;另一种是生产分泌型的重组人生长激素,纯化步骤简单,杂蛋白干扰少,氧化型的环境有利于二硫键形成,但是通常其分泌效率不高,大部分的表达蛋白可能滞留在胞内。
发明内容
本发明的目的是提供一种表达量高,且易提取纯化的高效表达生长激素的工程菌的制备方法及其应用。
本发明这种高效表达生长激素的工程菌的制备方法,包括以下步骤:
1)通过PCR法对人生长激素hGH基因进行扩增,得到扩增后的hGH基因;
2)将步骤1)中的扩增后的hGH基因连接到pMal-p2x载体上,得到重组质粒pMal-hGH;
3)将步骤2)中的重组质粒导入到大肠杆菌BL21(DE3)中,通过IPTG 诱导蛋白表达,并确定表达正确后,获得高效表达生长激素的工程菌。
所述的人生长激素hGH基因,其核苷酸序列如SEQ ID NO:1所示。
所述hGH扩增基因的上游引物1如SEQ ID NO:2所示;下游引物2如SEQ ID NO:3所示。
所述的工程菌在细胞外周质表达人生长激素hGH中的应用。
所述的人生长激素hGH的氨基酸序列如SEQ ID NO:10。
本发明的有益效果:MBP蛋白是由大肠杆菌基因malE表达的一个前体蛋白,带有一个26个氨基酸的信号肽,该信号肽能够帮助它通过大肠杆菌的 SecYEG转运系统分泌到外周质空间,参与大肠杆菌对麦芽糖吸收和有效利用。 MBP蛋白除了帮助融合蛋白分泌表达到外周质空间之外,它还具有增强重组蛋白的可溶性的特点,能够降低蛋白的翻译速率,有利于蛋白的正确折叠,避免重组蛋白形成包涵体或者沉淀;另外它还作为亲和层析的标签在蛋白纯化领域被广泛使用,它和麦芽糖亲和柱特异、高效结合,进一步方便蛋白纯化。本发明分别采用pMal-p2x作为载体,获得了重组质粒pMal-hGH,pMal-hGH的重组质粒的氨基端带有一个MBP蛋白标签,因而将重组质粒导入到大肠杆菌的受体中,可以将hGH和MBP标签进行融合表达,从而将hGH带到外周质空间,更有利于hGH蛋白的纯化,而且由于外周质的氧化环境,hGH蛋白能够正确形成二硫键,使蛋白具有较好的活性。
附图说明
图1实施例3中hGH蛋白的表达情况分析图;其中:泳道1为蛋白marker,泳道2和3为pET22b-hGH诱导后的样品,泳道4和5为未诱导的对照菌液,泳道6和7为pMal-hGH诱导后的样品。
图2实施例3中hGH蛋白表达位置的确定;其中:泳道1为蛋白marker,泳道2-5分别为pET22b-hGH菌体经上清液1和上清液2处理后得到蛋白上清液;泳道6-9分别为pMal-hGH菌体经上清液1和上清液2处理后得到蛋白上清液;泳道10-11代表pET22b-hGH处理之后的细胞裂解液;泳道12-13代表pMal-hGH 处理之后的细胞裂解液。
图3实施例4中切除BMP标签蛋白后的hGH蛋白测试图;其中:泳道1 为蛋白marker;泳道2-6分别为收集的被ppase酶酶切之后的hGH;上面的一条多余的条带为过量的ppase酶。
图4实施例5中hGH蛋白分子筛纯化结果,其中:A.显示hGH的分子筛色谱图;B.取fraction number:15-22进行SDS-PAGE分析图。
图5.实施例6中hGH蛋白分子内活性二硫键的分析图;其中:A.hGH蛋白样品在reduced和no-reduced下SDS-PAGE检测;B.使用hGH抗体对hGH蛋白进行定性检测。
图中★代表释放的hGH蛋白。
具体实施方式
实施例1
1)以HG16122-CF(http://www.sinobiologicalcdn.com/reagent/HG16122-CF.pdf)为模板质粒(SEQ ID NO:1所示),以含HRV3C蛋白酶切位点的上游引物1和含PST1限制性内切酶酶切位点下游引物2,对hGH基因进行PCR扩增,纯化后,得到扩增后hGH基因,其中:
上游引物1为:CTGGAAGTCCTGTTTCAGGGACCCTTCCCAACCATTCCCTTATCCAG(SEQ IDNO:2所示);
下游引物2为:CGGCCAGTGCCAAGCTTGCCTGCAGCTAGAAGCCACAGCTGCCCTCC(SEQ IDNO:3所示);
KOD FX PCR反应体系为:94℃2min,98℃10sec,62℃30Sec,68℃50sec,重复35cycles;68℃延伸5min;4℃hold。
2)使用PST1酶切位点同源臂的反向互补序列上游引物1和含HRV3C蛋白酶切位点反向互补序列下游引物2,通过PCR扩增获得pMal-p2X载体骨架片段,切胶回收之后和hGH基因片段进行同源重组反应。将hGH基因插入到 pMal-p2x(HRV 3C)的HRV3C蛋白酶切位点和PST1限制性内切酶酶切位点之间尽量避免引入非天然的氨基酸,将同源重组产物转化大肠杆菌DH5α,然后挑取克隆,测序正确后,得到重组质粒命名为pMal-hGH。
上游引物3为:CCAGTGCCAAGCTTGCCTGCAG(SEQ ID NO:4所示);
下游引物4为:GGGTCCCTGAAACAGGACTTCCAGC(SEQ ID NO:5所示);
Primestar PCR反应体系为:95℃2min,98℃10sec,55℃15sec,72℃1min,重复30cycles;72℃延伸3min;4℃hold。
3)将测序正确的重组质粒与大肠杆菌感受态细胞(E.coli DH5α)混匀,放置在冰浴中30min,42℃水浴下90s,取出后再次冰浴5min;接着将其加入到 LB(Luria-Bertani培养基)液体培养基,并在37℃,150rpm条件下,振荡培养1h;取菌液于含50mg/L卡那霉素的LB固体培养基,涂布均匀,在37℃过夜培养,获得单克隆菌落,将获得的单克隆菌落进行测序,测序正确的单菌落进行活化培养,即得重组基因工程菌1。
对比例1
1)以HG16122-CF为模板质粒cDNA,利用上游引物5和下游引物6进行 PCR(聚合酶链式反应)扩增,纯化后,得到扩增hGH基因,其中:
上游引物5为:CCTCGCTGCCCAGCCGGCGATGGCCTTCCCAACCATTCCCTTATCCAG(SEQ IDNO:6所示);
下游引物6为:CAGTGGTGGTGGTGGTGGTGCTCGAGGAAGCCACAGCTGCCCTCC(SEQ ID NO:7所示);
KOD FX PCR反应程序为:94℃2min,98℃10sec,62℃30Sec68℃50sec,重复35cycles;68℃延伸5min;4℃hold。
2)使用上游引物7和下游引物8分别以载体pET-22b为模板进行PCR扩增得到,PCR产物纯化后,得到pET-22b载体骨架片段;将扩增hGH基因片段和 pET-22b载体骨架片段通过同源重组反应,将hGH基因插入到pET-22b的pelB 信号肽和Xho1酶切位点之间,将同源重组产物其转化大肠杆菌DH5α,然后挑取克隆,测序正确后,得到重组质粒命名为pET-hGH。
上游引物7为:CTCGAGCACCACCACCACCACCACTG(SEQ ID NO:8 所示);
下游引物8为:GGCCATCGCCGGCTGGGCAGCGAGG(SEQ ID NO:9 所示);
PrimestarPCR反应体系为:95℃2min,98℃10sec,55℃15sec,72℃1min,重复30cycles;72℃延伸3min;4℃hold。
3)将测序正确的重组质粒与大肠杆菌感受态细胞(E.coli DH5α)混匀,放置在冰浴中30min,42℃水浴下90s,取出后再次冰浴5min;接着将其加入到 LB(Luria-Bertani培养基)液体培养基,并在37℃,150rpm条件下,振荡培养1h;取菌液于含50mg/L卡那霉素的LB固体培养基,涂布均匀,在37℃过夜培养,获得单克隆菌落,将获得的单克隆菌落进行测序,测序正确的单菌落进行活化培养,即得重组基因工程菌2。
实施例3
实施例1与对比例1中的重组基因工程菌的小量表达:
分别将重组基因工程菌1和2进行以下处理:
1)挑选的单克隆菌株接到5ml氨苄抗性的TB液体培养基中,37℃培养24h。
2)第二天取1ml菌液转接到100ml的TB液体培养基中,细菌在37℃培养到OD600=0.7-0.9时,加入IPTG至终浓度为0.2mM,18℃诱导表达16h。
3)取100μl菌液直接加入30μl SDS上样缓冲液,95℃加热20min。然后取出10μl进行SDS-page电泳检测hGH的表达情况;
4)将剩余的菌液6000rpm离心20min收集菌体沉淀。
5)hGH蛋白的收集:将收集的菌体沉淀使用10ml的高渗溶液(bufferⅠ:30mM Tris,20%W/V sucrose,1mM EDTA)重悬,并且在4℃搅拌30min。8000g 离心20min,收集上清液1。然后将菌体沉淀使用10ml低渗溶液(buffer Ⅱ:5mM MgSO4)重悬菌体,4℃搅拌10-30min;再次8000g离心20min,收集低渗上清液2。
将菌体沉淀使用10ml的细菌裂解液进行重悬,然后取出100μl菌液直接加入30μlSDS上样缓冲液,95℃加热20min,得到细菌裂解液。
所述步骤3)中hGH的表达情况如图1所示,从图1中可以看出,无论载体采用pMal-p2x还是pET-22b,都能是hGH基因进行表达。
将步骤5)中两种工程菌的上清液1、上清液2和细菌裂解液的也分别进行 SDS-page电泳检测分析,其结果如图2所示:工程菌2中上清液1和上清液2 中hGH蛋白很少,大部分存在于细菌裂解液中;工程菌1中上清液2含有大量的hGH蛋白,说明采用pMal-p2x载体可以使hGH蛋白表达至细胞的外周质。(因为pET22b-hGH是在hGH的C末端带有一个histag(约0.8kD),而pMal-hGH 是在N端带有一个MBPtag(约40kDa)所以两种工程菌表达的hGH蛋白存在差异)
实施例4:hGH蛋白的大量表达
1)采用实施例1中的工程菌1接种到10ml的TB液体培养基中,37℃培养24h。
2)第二天将10ml菌液转接到1L的TB液体培养基中,继续在37℃培养细菌到OD600=0.7-0.9,加入IPTG至其终浓度0.2mM,18℃诱导表达16h,收集菌体。
3)将收集的菌体使用100ml的高渗溶液(bufferⅠ:30mM Tris,20%W/V sucrose,1mM EDTA)重悬,并且在4℃搅拌30min后,8000g离心20min,弃上清液,然后使用100ml低渗溶液(buffer Ⅱ:5mM MgSO4)重悬菌体,4℃搅拌10-30min后,再次8000g离心20min,收集低渗上清液。
4)取2ml糊精配体亲和层析介质装到重力柱中,使用至少5倍介质体积的结合缓冲液(20ml tris pH8.0,100mM NaCl)进行洗涤,去除乙醇。接着将步骤 3)中得到低渗上清液使用0.45μm的滤器过滤之后,与洗涤好的糊精配体亲和层析介质在4℃共孵育3h。
5)让低渗上清液从重力柱流穿,然后使用结合缓冲液(20ml tris pH8.0, 100mMNaCl)洗涤亲和层析介质至少5次,每次10ml,得到纯化后的上清液。
6)向纯化处理过上清液中加入1ml的用结合缓冲液稀释的ppase酶,4℃下过夜酶切,切除MBP标签,然后使用2ml的结合缓冲液收集hGH蛋白,直至监测A280<0.05时停止收集,得到hGH蛋白溶液。将hGH蛋白溶液进行 SDS-PAGE电泳分析,其结果如图3所示,从图3可知:切除MBP标签蛋白后,泳道2~6有两条蛋白带,上面的那条蛋白带主要是过量的ppase酶。
实施例5
将实施例4中,分离出的hGH蛋白溶液通过分子筛Superdex75(10/300GL) 进行分离纯化,(纯化的缓冲液为PBS,设置流速为0.5ml/min),然后检测280nm 处的吸光值。其结果如图4A所示,洗脱峰显示hGH蛋白的分子量为22kD。
收集含有hGH的(Fractionnumber:15-20)进一步浓缩。蛋白浓度按照消光系数=17670(fromwww.expasy.org)进行估算。最后浓缩至终浓度为3mg/ml 的hGH蛋白2ml。SDS-PAGE电泳检测,GH的纯度能够达到95%以上(如图 4B所示)。
实施例6
纯化的hGH蛋白在reducing and non-reducing(还原与非还原)SDS-PAGE 迁移率
由于hGH蛋白含有两个cys,成熟的hGH会形成内部二硫键,这对于其活性非常重要。因此为了检测纯化之后的GH蛋白是否是成熟形式的蛋白。对纯化的GH蛋白分别制备还原性和非还原性蛋白样品,然后进行电泳检测。如图 5A所示,在非还原条件下hGH蛋白由于能够形成天然的二硫键,从而会形成一种更加紧密的空间结构,因此它会具有更快迁移率。
Western blot定性检测纯化的hGH蛋白
为了定性检验得到的hGH蛋白,使用hGH特异抗体进行了westernblot分析。取纯化的hGH蛋白分别进行还原和非还原处理之后进行SDS-PAGE,电泳结束后转膜,室温牛奶封闭1h,使用hGH抗体(proteintech)孵育过夜,然后用TBST洗涤三次,每次15min。二抗1:5000稀释后室温孵育1h。再用TBST 洗涤3次。使用化学发光液ECL曝光(图5B)。Western blot得到的条带和图 5A中考染的结果一致,表明纯化得到的蛋白为hGH蛋白,其氨基酸序列如SEQID NO:10所示。
序列表
<110> 湖南百尔泰克生物科技有限公司
<120> 一种高效表达生长激素的工程菌的制备方法及其应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 576
<212> DNA
<213> hGH基因(人工序列)
<400> 1
ttcccaacca ttcccttatc caggcttttt gacaacgcta tgctccgcgc ccatcgtctg 60
caccagctgg cctttgacac ctaccaggag tttgaagaag cctatatccc aaaggaacag 120
aagtattcat tcctgcagaa cccccagacc tccctctgtt tctcagagtc tattccgaca 180
ccctccaaca gggaggaaac acaacagaaa tccaacctag agctgctccg catctccctg 240
ctgctcatcc agtcgtggct ggagcccgtg cagttcctca ggagtgtctt cgccaacagc 300
ctggtgtacg gcgcctctga cagcaacgtc tatgacctcc taaaggacct agaggaaggc 360
atccaaacgc tgatggggag gctggaagat ggcagccccc ggactgggca gatcttcaag 420
cagacctaca gcaagttcga cacaaactca cacaacgatg acgcactact caagaactac 480
gggctgctct actgcttcag gaaggacatg gacaaggtcg agacattcct gcgcatcgtg 540
cagtgccgct ctgtggaggg cagctgtggc ttctag 576
<210> 2
<211> 47
<212> DNA
<213> 上游引物1(人工序列)
<400> 2
ctggaagtcc tgtttcaggg acccttccca accattccct tatccag 47
<210> 3
<211> 47
<212> DNA
<213> 下游引物2(人工序列)
<400> 3
cggccagtgc caagcttgcc tgcagctaga agccacagct gccctcc 47
<210> 4
<211> 22
<212> DNA
<213> 上游引物3(人工序列)
<400> 4
ccagtgccaa gcttgcctgc ag 22
<210> 5
<211> 25
<212> DNA
<213> 下游引物4(人工序列)
<400> 5
gggtccctga aacaggactt ccagc 25
<210> 6
<211> 48
<212> DNA
<213> 上游引物5(人工序列)
<400> 6
cctcgctgcc cagccggcga tggccttccc aaccattccc ttatccag 48
<210> 7
<211> 45
<212> DNA
<213> 下游引物6(人工序列)
<400> 7
cagtggtggt ggtggtggtg ctcgaggaag ccacagctgc cctcc 45
<210> 8
<211> 26
<212> DNA
<213> 上游引物7(人工序列)
<400> 8
ctcgagcacc accaccacca ccactg 26
<210> 9
<211> 25
<212> DNA
<213> 下游引物8(人工序列)
<400> 9
ggccatcgcc ggctgggcag cgagg 25
<210> 10
<211> 191
<212> PRT
<213> hGH蛋白(人工序列)
<400> 10
Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg
1 5 10 15
Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu
20 25 30
Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro
35 40 45
Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg
50 55 60
Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu
65 70 75 80
Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val
85 90 95
Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp
100 105 110
Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu
115 120 125
Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser
130 135 140
Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr
145 150 155 160
Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe
165 170 175
Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe
180 185 190
Claims (5)
1.一种高效表达生长激素的工程菌的制备方法,包括以下步骤:
1)通过PCR法对人生长激素hGH基因进行扩增,得到扩增后的hGH基因;
2)将步骤1)中的扩增后的hGH基因连接到pMal-p2x载体上,得到重组质粒pMal-hGH;
3)将步骤2)中的重组质粒导入到大肠杆菌BL21(DE3)中,通过IPTG诱导蛋白表达,并确定表达正确后,获得高效表达生长激素的工程菌。
2.根据权利要求1所述的工程菌的制备方法,其特征在于,所述的人生长激素hGH基因,其核苷酸序列如SEQ ID NO:1所示。
3.根据权利要求1所述的工程菌的制备方法,其特征在于,所述步骤1)中,所述hGH扩增基因的上游引物1如SEQ ID NO:2所示;下游引物2如SEQ ID NO:3所示。
4.根据权利要求1所述的工程菌在细胞外周质表达人生长激素hGH中的应用。
5.根据权利要求所述的人生长激素hGH的氨基酸序列如SEQ ID NO:10。
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