CN106749615A - 一种大菱鲆生长催乳素sl基因、重组蛋白及其制备方法 - Google Patents
一种大菱鲆生长催乳素sl基因、重组蛋白及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种大菱鲆生长催乳素SL基因、重组蛋白及其制备方法。本发明克隆得到了生长催乳素的编码基因，其核苷酸序列为SEQ ID NO:2，催乳素SL多肽的氨基酸序列为SEQ ID NO:1。将生长泌乳素SL基因与PET‑24a表达载体相连接构建表达载体pET‑24a‑SL，转化入表达菌株进行原核表达，后经纯化复性获得大菱鲆生长催乳素重组蛋白，本发明将为研究大菱鲆的生长发育调控机理和开发可应用于生产的调控大菱鲆生长发育的新产品打下良好基础。
Description
技术领域
本发明属于生物工程技术领域，涉及一种大菱鲆生长催乳素SL基因、重组蛋白及其制备方法。
背景技术
生长催乳素(Somatolactin，SL)是生长激素 (growth hormone，GH)／催乳激素(prolactin，PRL)家族的一种新的脑垂体蛋白质激素, SL是由类脑垂体中部呈过碘酸雪夫(PAS)阳性细胞分泌产生的，具有促进性腺成熟和排卵、参与发育调控、低渗调节、色素细胞的发生、应激反应和脂肪代谢等重要生理作用，随着垂体促性腺激素释放激素（GnRH）、生长激素（GH）等神经多肽在养殖生产中成熟地应用于调控鱼类的生长和繁殖以来，如何借助生长催乳素来人为调控养殖鱼类的生长、性腺发育、营养代谢、能量平衡及其他生理活动也逐渐成为关注的重点。当前中国的海水鱼类养殖业发展迅速，为了促进产业朝着规模化、集约化、环境友好型的新型养殖模式发展，对鱼类发育机理、生长代谢等方面的基础研究显得尤为重要，为此急需开展鱼类SL等神经内分泌因子相关研究，为研制可应用于生产安全的鱼类生长调控产品打下基础，以推动海水鱼类养殖大产业向集约化、可持续、环境友好型的方向发展。
发明内容
本发明的目的是提供了一种大菱鲆生长催乳素SL基因、重组蛋白及其制备方法。本发明构建了含有大菱鲆生长催乳素SL基因的重组表达载体，并将其转入表达菌株进行表达，经纯化获得大菱鲆生长催乳素重组蛋白。
为实现上述发明目的，本发明采用以下技术方案予以实现：
本发明提供了一种大菱鲆生长催乳素SL，所述大菱鲆生长催乳素SL的氨基酸序列如SEQ ID NO:1所示。
本发明提供了编码所述的大菱鲆生长催乳素SL的SL基因，所述SL基因的核苷酸序列如SEQ ID NO:2所示。
进一步的：克隆所述SL基因所需的引物Fs1和Rs1序列如下：
Fs1:5′- ACGAGCACGGCCTGTTCCAGTACGACG -3′
Rs1:5′- CGTCGTACTGGAACAGGCCGTGCTCGT -3′。
本发明提供了含有所述的SL基因的重组表达载体。
进一步的：所述重组表达载体为pET-24a-SL，其制备过程为：使用正向引物F和反向引物R，通过PCR扩增获得所述SL基因，并在SL基因的两端分别添加XhoI和EcoRI酶切位点，将SL基因经过限制性内切酶XhoI和EcoRI酶切后，插入pET-24a(+)表达载体XhoI和Eco4RI酶切位点之间，获得重组表达载体pET-24a-SL。
进一步的：所述正向引物F和反向引物R序列如下：
F：5′- CCGGAATTCATGAACTCGATGACAGGCAC -3′；
R：5′- CCGCTCGAGTGCACAGTTGTAGTTGTCG-3′；
PCR扩增的反应条件为： 95℃5min；95℃35s，55℃退火35s，72℃延伸1min，35个循环。
本发明还提供了所述的大菱鲆生长催乳素SL重组蛋白的制备方法，所述制备方法包括以下步骤：
（1）将重组表达载体pET-24a-SL转化大肠杆菌，将挑取的阳性克隆菌株接种于含有卡那霉素的LB液体培养基中，37℃振荡培养过夜，次日转接到含有LB 培养基的发酵罐中，37℃培养到OD600=0.5~1.0时，加入IPTG诱导重组蛋白表达，离心收集细菌培养物；
（2）将诱导表达后的细菌培养物重悬于裂解液中，超声破碎，将超声破碎的细胞裂解液离心，收集沉淀即为包涵体，使用包涵体洗涤液洗涤包涵体；用溶解缓冲液溶解包涵体，4℃放置过夜；室温下离心；将上述包涵体溶液稀释后装入透析袋中，4℃透析过夜；
（3）利用低压层析系统，将透析后的包涵体溶液上样至Ni柱；收集洗脱后的蛋白流出液；装入透析袋中，透析过夜；获得纯化后的催乳素SL重组蛋白。
进一步的：所述步骤（1）中IPTG的浓度为0.5 ~ 0.7mmol/L，诱导表达的时间为3 ~5h。
进一步的：所述步骤（3）中透析后的包涵体溶液以0.5 ml/min流速上样至Ni-IDA-Sepharose CL-6B亲和层析柱。
本发明的优点和技术效果是：本发明利用分子生物学手段获得大菱鲆生长催乳素SL的成熟肽编码序列，通过构建重组大菱鲆生长催乳素SL的原核表达载体，转化表达宿主菌，诱导菌体表达，收集菌体及纯化获得重组表达的大菱鲆生长催乳素SL重组蛋白。本发明可用于制备ELISA试剂盒和饲料添加剂等产品，将为研究大菱鲆的食欲调控机理和开发可应用于生产的促进鱼类摄食和生长的新产品打下良好基础。
附图说明
图1：表达载体pET-24a-SL示意图。
图2：重组表达载体pET-24a-SL的双酶切核酸电泳图；其中：Lane M：DL2 ,000DNAMarker；Lane 1：重组表达载体双酶切产物。
图3：SDS-PAGE分析重组大菱鲆SL蛋白表达情况，其中：Lane M：蛋白分子质量标准。
图4：SDS-PAGE分析纯化的大菱鲆SL重组蛋白，其中：Lane M：蛋白分子质量标准；Lane 1：经Ni柱纯化后的大菱鲆SL重组蛋白。
具体实施方式
下面结合附图和具体实施例对本发明的技术方案做进一步详细的说明。
1、大菱鲆下丘脑总RNA提取
大菱鲆下丘脑总RNA提取采用的EasyPureTMRNA Kit动物组织提取试剂盒(北京全式金生物技术有限公司提供)。具体操作方法如下：
1）取20mg左右下丘脑组织，快速用液氮速冻组织，用研磨杵将组织研磨成粉未，将粉未转移到1.5ml离心管中加入0.6mlBinding Buffer和15ml Proteinase K，混匀后56℃处理20分钟。
2）室温12000 r/min离心5分钟，吸上清于RNase-free 的离心管中，向上清中加入等倍体积70%乙醇。
3）彻底混匀，将得到的沉淀和溶液一起加入离心柱中，12000 r/min离心30秒，弃掉流出液。
4）向离心柱中加500ml CB4（Clean Buffer 4），室温12000 r/min离心30秒，弃掉流出液。然后向离心柱中加入80ml的DNase I 工作液，室温放置15分钟。
5）重复步骤2）一次。
6）加500mlWB4（Wash Buffer 4）室温12000 r/min离心30秒，弃掉流出液。
7）重复步骤4）一次。
8）室温12000 r/min离心2分钟，彻底去除残留的乙醇，在室温中静置数分钟彻底晾干离心柱。
9）将离心柱放入一个新的1.5ml RNase-free 的管中，并向离心柱中央加入50mlRNase-free Water，室温静置1分钟。
10）室温12000 r/min离心2分钟，用45ml洗脱液洗脱RNA。
11）取5 ml RNA样品在2.0%的琼脂糖凝胶中电泳，EB染色观察结果。再取2ml RNA样品在NanoDrop 2000c 分光光度计检测RNA的浓度和纯度。
2、大菱鲆生长催乳素SL基因全长序列RACE克隆
1）3’RACE 扩增大菱鲆生长催乳素SL基因下游序列
使用SMARTRACE试剂盒以及特异引物Fs1（SEQ ID NO:3）和试剂盒中的3’CDS通用引物，进行 3’RACE扩增。反应条件如下：95°C预变性5min：以下重复30个循环：95°C变性35s，54°C退火35s，72°C延伸50S；最后72°C延伸10min。
3’RACE 扩增PCR体系：
| 反应体系 | 反应体积（20μl） |
| 2×PCRMix | 10μl |
| ddH2O | 7μl |
| Fs1 | 1μl |
| 3’ CDS | 1μl |
| cDNA | 1μl |
2） 5’RACE扩增大菱鲆生长催乳素SL基因cDNA上游序列
使用SMARTRACE试剂盒以及特异引物Rs1（SEQ ID NO:4）和试剂盒中的5’Oliogo通用引物，进行 5’RACE扩增。反应条件如下：95°C预变性5min：以下重复35个循环：95°C变性35s，52°C退火35s，72°C延伸40min；最后72°C延伸10min。
Fs1:5′- ACGAGCACGGCCTGTTCCAGTACGACG -3′（SEQ ID NO:3）
Rs1:5′- CGTCGTACTGGAACAGGCCGTGCTCGT -3′（SEQ ID NO:4）。
5’RACE扩增PCR体系：
| 反应体系 | 反应体积（20μl） |
| 2×PCRMix | 10μl |
| ddH2O | 7μl |
| Rs1 | 1μl |
| 5’Oliogo | 1μl |
| cDNA | 1μl |
3）用2.0%琼脂糖凝分离PCR 扩增得到DNA片段，并利用胶回收试剂盒（北京天根生化科技有限公司）纯化分离得到目的DNA片段，然后亚克隆到pMD18-载体上，挑选阳性克隆委托上海生工进行测序，获得核苷酸序列如SEQ ID NO:2所示的SL基因，编码产生的大菱鲆生长催乳素SL的氨基酸序列如SEQ ID NO:1所示。
3、大菱鲆生长催乳素SL的蛋白重组表达质粒构建
1）以大菱鲆下丘脑总RNA反转录cDNA为模板，以带有酶切位点和保护碱基的引物：正向引物F和反向引物R为引物进行PCR扩增。反应条件为： 95℃5min；95℃35s，55℃退火35s，72℃延伸1min，35个循环。
正向引物(F):Forward 5’-CCG GAATTCATGAACTCGATGACAGGCAC -3’ （SEQ ID NO:5）；
反向引物(R)：Forward 5’-CCG CTCGAGTGCACAGTTGTAGTTGTCG-3’
（斜体CCG为保护碱基；下划线GAATTC为Xhol位点；下划线CTCGAG为EcoR1位点）（SEQ IDNO:6）。
2）克隆得到编码大菱鲆生长催乳素（SL）蛋白的cDNA 序列，并且两端分别带有XhoI和EcoRI酶切位点，将该序列经过限制性内切酶XhoI和EcoRI酶切后，插入pET-24a(+)表达载体XhoI和EcoRI酶切位点之间，获得重组表达载体pET-24a-SL。表达载体示意图如图1所示，大菱鲆生长催乳素SL基因的cDNA 序列经XhoI和EcoRI双酶切后的电泳结果如图2所示。
4、大菱鲆生长催乳素（SL）重组蛋白在大肠杆菌中的表达
将重组表达载体pET-24a-SL转化大肠杆菌BG21，挑选阳性克隆，测序正确后，将挑取的BG21菌株接种于含有卡那霉素的LB液体培养瓶中，37℃振荡培养过夜，次日按1:100 ~ 1:500(V/V) 转接到含有10L LB 培养基的15L 的发酵罐中， 37℃培养到OD600=0.5~1.0时，按浓度0.5 ~ 0.7mmol/L 加入IPTG 诱导3 ~5h，6000rpm 离心10min，收集细菌培养物放到置于-20℃保存。采用SDS-PAGE分析重组大菱鲆SL蛋白表达情况，结果如图3所示。
5、大菱鲆生长催乳素（SL）重组包涵体蛋白的变复性
将诱导表达后的菌体沉淀重悬于裂解液Lysis buffer (20 mM Tris-HCl containing1 mM PMSF and bacteria protease inhibitor cocktail, pH 8.0)中，超声破碎，将超声破碎的细胞裂解液4℃ 10000g离心20 min，收集沉淀即为包涵体，使用包涵体洗涤液(20mMTris，1mM EDTA，2M尿素，1M NaCl，1％Triton X-100，pH8.0)洗涤包涵体3-5次；用溶解缓冲液(20mM Tris，5mM DTT，8M尿素 pH8.0)，按一定比例溶解包涵体，4度放置过夜；室温，15000rpm离心15min；将上述溶液滴加到20 mM Tris-HCL 5mM EDTA Buffer PH7.8缓冲液中，逐步成倍梯度稀释缓慢搅拌，剪取处理好适量长度的透析袋，在纯水中冲洗，将蛋白溶液装入透析袋中，放入1*PBS（PH7.4）缓冲液中，4℃透析过夜。
6、大菱鲆生长催乳素（SL）重组蛋白的Ni柱亲和纯化
利用低压层析系统，将透析后的包涵体溶液以0.5 ml/min流速上样至Ni-IDABinding-Buffer预平衡的Ni-IDA -Sepharose CL-6B亲和层析柱；用Ni-IDA Binding-Buffer以0.5 ml/min流速冲洗，至流出液OD280值到达基线；用Ni-IDA Washing-Buffer(20mM Tris-HCl，20 mM咪唑，0.15 M NaCl，pH8.0)以1 ml/min流速冲洗，至流出液OD280值到达基线；用Ni-IDA Elution-Buffer(20 mM Tris-HCl，250 mM咪唑，0.15 M NaCl，pH8.0)以1 ml/min流速洗脱目的蛋白，收集蛋白流出液；剪取处理好适量长度的透析袋，在纯水中冲洗，将上述收集的蛋白溶液装入透析袋中，放入1*PBS（PH7.4）缓冲液中，4℃透析过夜；分步收集洗脱液，检测纯度和含量。分别取3μL未纯化包涵体蛋白、3μL变复性的蛋白和1μL洗脱的纯化产物与蛋白质分子量标准进行12％的聚丙烯酰胺凝胶电泳，经考马斯蓝染色后，于凝胶成像系统观察重组蛋白纯化效果，结果见图4所示。经亲和层析法纯化后获得纯化的重组蛋白，收集后作为样品备用。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
SEQUENCE LISTING
<110> 中国水产科学研究院黄海水产研究所
<120> 一种大菱鲆生长催乳素SL基因、重组蛋白及其制备方法
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 233
<212> PRT
<213> 人工序列
<400> 1
Met Asn Ser Met Thr Gly Thr Val Ala Trp Arg Ala Val Trp Ala Ala
1 5 10 15
Leu Leu Trp Pro Cys Leu Leu Thr Ala Ala Met Pro Leu Asp Cys Thr
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Glu Glu Gln Gly Ser Leu Tyr Arg Cys Pro Ser Ile Ser Gln Glu Lys
35 40 45
Leu Leu Asp Arg Val Ile Gln His Ala Glu Leu Ile Tyr Arg Val Ser
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Glu Glu Ser Cys Ala Met Phe Glu Glu Met Phe Val Pro Phe Pro Leu
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Arg Leu Gln Arg Asn Gln Ala Gly Tyr Ser Cys Ile Thr Lys Ala Leu
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Pro Ile Pro Ser Ser Lys Ser Glu Ile Gln His Ile Ser Asp Lys Trp
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Leu Leu His Ser Val Leu Met Leu Val Gln Ser Trp Ile Glu Pro Leu
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Val Tyr Leu Gln Thr Thr Leu Asp Arg Tyr Glu Asn Ala Pro Asp Met
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Leu Leu Asn Lys Thr Lys Trp Val Ser Asp Lys Leu Ile Ser Leu Glu
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Gln Gly Val Val Val Leu Ile Arg Lys Met Leu Asp Glu Gly Met Leu
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Thr Thr Thr Tyr Asn Glu His Gly Leu Phe Gln Tyr Asp Ala Leu Pro
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Asp Met Leu Glu Ser Val Met Arg Asp Tyr Thr Leu Ile Ser Cys Phe
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Lys Lys Asp Ala His Lys Met Glu Val Phe Leu Lys Leu Leu Lys Cys
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<210> 2
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<213> 人工序列
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atgaactcga tgacaggcac agtcgcgtgg agggccgtgt gggccgcgct gctctggccc 60
tgcctgctca ccgcggccat gccgctggac tgcaccgagg agcagggcag cctctaccgc 120
tgcccgtcca tctcccagga gaagctcctg gaccgcgtca tccagcacgc cgagctcatc 180
taccgcgtct ccgaggagtc gtgtgccatg tttgaggaga tgttcgtgcc cttcccgctg 240
cggctccaga ggaaccaggc cggctactcg tgcatcacca aggcgttgcc catccccagc 300
tccaagagtg aaatccaaca catatctgat aaatggctgc tgcactcggt gctgatgctg 360
gtccagtcgt ggatcgagcc cctggtctac ctgcagacca cgctcgatcg ctacgagaac 420
gcgccggaca tgctgctcaa caagaccaag tgggtgtctg acaaactcat cagcctggag 480
caaggggtgg tggtccttat caggaagatg ttggacgagg ggatgttgac cacgacctac 540
aacgagcacg gcctgttcca gtacgacgcg ctgcccgaca tgttggaatc ggtcatgcgg 600
gactacaccc tgatcagctg cttcaagaag gacgcccaca agatggaggt cttcctcaag 660
ctcctcaagt gtcggcagtc cgacaactac aactgtgcat ag 702
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acgagcacgg cctgttccag tacgacg 27
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cgtcgtactg gaacaggccg tgctcgt 27
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ccggaattca tgaactcgat gacaggcac 29
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<212> DNA
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ccgctcgagt gcacagttgt agttgtcg 28
Claims (9)
1.一种大菱鲆生长催乳素SL，其特征在于：所述大菱鲆生长催乳素SL的氨基酸序列如SEQ ID NO:1所示。
2.编码权利要求1所述的大菱鲆生长催乳素SL的SL基因，其特征在于：所述SL基因的核苷酸序列如SEQ ID NO:2所示。
3.根据权利要求2所述的SL基因，其特征在于：克隆所述SL基因所需的引物Fs1和Rs1序列如下：
Fs1:5′- ACGAGCACGGCCTGTTCCAGTACGACG -3′
Rs1:5′- CGTCGTACTGGAACAGGCCGTGCTCGT -3′。
4.含有权利要求2所述的SL基因的重组表达载体。
5.根据权利要求4所述的重组表达载体，其特征在于：所述重组表达载体为pET-24a-SL，其制备过程为：使用正向引物F和反向引物R，通过PCR扩增获得所述SL基因，并在SL基因的两端分别添加XhoI和EcoRI酶切位点，将SL基因经过限制性内切酶XhoI和EcoRI酶切后，插入pET-24a(+)表达载体XhoI和Eco4RI酶切位点之间，获得重组表达载体pET-24a-SL。
6.根据权利要求5所述的重组表达载体，其特征在于：所述正向引物F和反向引物R序列如下：
F：5′- CCGGAATTCATGAACTCGATGACAGGCAC -3′；
R：5′- CCGCTCGAGTGCACAGTTGTAGTTGTCG-3′；
PCR扩增的反应条件为： 95℃5min；95℃35s，55℃退火35s，72℃延伸1min，35个循环。
7.权利要求1所述的大菱鲆生长催乳素SL重组蛋白的制备方法，其特征在于：所述制备方法包括以下步骤：
（1）将重组表达载体pET-24a-SL转化大肠杆菌，将挑取的阳性克隆菌株接种于含有卡那霉素的LB液体培养基中，37℃振荡培养过夜，次日转接到含有LB 培养基的发酵罐中，37℃培养到OD600=0.5~1.0时，加入IPTG诱导重组蛋白表达，离心收集细菌培养物；
（2）将诱导表达后的细菌培养物重悬于裂解液中，超声破碎，将超声破碎的细胞裂解液离心，收集沉淀即为包涵体，使用包涵体洗涤液洗涤包涵体；用溶解缓冲液溶解包涵体，4℃放置过夜；室温下离心；将上述包涵体溶液稀释后装入透析袋中，4℃透析过夜；
（3）利用低压层析系统，将透析后的包涵体溶液上样至Ni柱；收集洗脱后的蛋白流出液；装入透析袋中，透析过夜；获得纯化后的催乳素SL重组蛋白。
8.根据权利要求7所述的大菱鲆生长催乳素SL重组蛋白的制备方法，其特征在于：所述步骤（1）中IPTG的浓度为0.5 ~ 0.7mmol/L，诱导表达的时间为3 ~5h。
9.根据权利要求7所述的大菱鲆生长催乳素SL重组蛋白的制备方法，其特征在于：所述步骤（3）中透析后的包涵体溶液以0.5 ml/min流速上样至Ni-IDA -Sepharose CL-6B亲和层析柱。
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| CN108586589A (zh) * | 2018-01-16 | 2018-09-28 | 南京农业大学 | 一种来自贵州木霉的Swollenin蛋白的制备方法及其应用 |
| CN110387373A (zh) * | 2019-07-24 | 2019-10-29 | 上海交通大学 | 环颈雉催乳素蛋白及其编码基因与应用 |
| CN114106142A (zh) * | 2021-11-03 | 2022-03-01 | 中山大学 | 一种黄鳝生长催乳素抗血清及其制备方法与应用 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108586589A (zh) * | 2018-01-16 | 2018-09-28 | 南京农业大学 | 一种来自贵州木霉的Swollenin蛋白的制备方法及其应用 |
| CN110387373A (zh) * | 2019-07-24 | 2019-10-29 | 上海交通大学 | 环颈雉催乳素蛋白及其编码基因与应用 |
| CN114106142A (zh) * | 2021-11-03 | 2022-03-01 | 中山大学 | 一种黄鳝生长催乳素抗血清及其制备方法与应用 |
| CN114106142B (zh) * | 2021-11-03 | 2024-05-14 | 中山大学 | 一种黄鳝生长催乳素抗血清及其制备方法与应用 |
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