CN114958806A - 一种鲤lpl1重组蛋白及其制备方法 - Google Patents
一种鲤lpl1重组蛋白及其制备方法 Download PDFInfo
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- CN114958806A CN114958806A CN202210663779.XA CN202210663779A CN114958806A CN 114958806 A CN114958806 A CN 114958806A CN 202210663779 A CN202210663779 A CN 202210663779A CN 114958806 A CN114958806 A CN 114958806A
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- lpl1
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Abstract
本发明提供一种鲤LPL1重组蛋白，并公开了氨基酸序列和核苷酸序列，氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。本发明提供一种包含鲤LPL1重组蛋白基因的质粒。本发明提供一种鲤LPL1重组蛋白的制备方法。本发明采用SlyD或Skp作为重组蛋白的融合标签，可以促进目标蛋白的正确折叠和复性，可以提高目标蛋白的表达量和可溶性。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种鲤LPL1重组蛋白及其制备方法。
背景技术
原核表达系统是体外获得大量蛋白，用以研究蛋白结构、功能以及应用于治疗、生产等的首要选择，其中大肠杆菌表达系统具有遗传背景清楚、成本低廉、生产率高、操作简单等优点，是常见的原核表达系统。但是大肠杆菌原核表达系统具有以下几个方面的不足：1、由于存在密码子的喜好，很多真核基因在大肠杆菌中不表达；2、大部分外源蛋白以包涵体形式表达；3、表达蛋白纯化难获得。因此，针对以上不足之处，常常采用以下办法：选取合适的表达载体、表达条件优化、密码子优化、融合标签及分子伴侣共表达等方法均能够提高外源蛋白的可溶性。
脂蛋白脂肪酶(lipoprotein lipase，LPL)是一种由脂肪细胞、心肌细胞、骨骼肌细胞以及乳腺细胞等实质细胞合成和分泌的一种糖蛋白，属于酯酶家族。LPL主要催化乳糜微粒和极低密度脂蛋白的甘油三酯水解，产生供组织利用的脂肪酸和单酰甘油。LPL能分解卵磷脂、磷脂酰乙醇胺，并促使脂蛋白之间转移胆固醇、磷脂及载脂蛋白，在机体脂质代谢调控上起关键作用。鲤LPL包含3个同源蛋白，LPL1、LPL2、LPL3，分子量分别为56.51kDa、54.85kDa和59.49kDa。为促进鲤脂肪代谢的机制的理论基础研究，对鲤LPL进行体外重组蛋白原核表达及纯化非常必要。但是实际操作中，面临表达效率低、可溶性差、纯化效率低的问题。
发明内容
针对现有技术存在的问题，本发明提供一种鲤LPL1重组蛋白，氨基酸序列如SEQID NO.1或SEQ ID NO.2所示。
进一步的，SEQ ID NO.1对应的核苷酸序列如SEQ ID NO.3所示；SEQ ID NO.2对应的核苷酸序列如SEQ ID NO.4所示。
采用SlyD或Skp作为重组蛋白的融合标签；其中：LPL1基因序列如SEQ ID NO.5所示，SlyD基因序列如SEQ ID NO.6所示，Skp基因序列如SEQ ID NO.7所示。LPL1和SlyD之间，LPL1和Skp之间通过碱基序列GAATTC连接，而且实验证实不影响LPL1功能。
对裂解敏感蛋白D(sensitive to lysi s D，SlyD)是一种分子量为28kDa的分子伴侣蛋白，能够促进蛋白的正确折叠，SlyD蛋白的C末端含丰富的组氨酸，能够与Ni2+结合。Skp(Seventeen-Ki lodalton protein)分子量为15.674kDa，在SDS-PAGE中显现的分子量大小通常为17kDa，因此，被命名为Skp。Skp也是一种分子伴侣，可以协助外膜蛋白早期折叠中间体的产生及保持其可溶性，蛋白中不包含组氨酸残基。另外，我们通过实验验证，SlyD和Skp没有脂蛋白脂肪酶的生物活性。
因此，使用SlyD和Skp作为鲤LPL1重组蛋白的融合标签，构建含有SlyD和Skp融合标签的重组质粒，能够提高原核表达重组蛋白的可溶性，而且在后续研究中无需对该融合标签进行切除，减少重组LPL1蛋白酶活性的损失。
进一步的，重组蛋白表达的载体为质粒，进一步的，质粒为改造后的pET43.1a(+)。
本发明提供一种重组质粒，将pET43.1a(+)质粒载体上的NusA-tag、S-tag和HSV-tag替换为SlyD或Skp融合标签。
本发明提供一种包含SEQ ID NO.1或SEQ ID NO.2所示所述鲤LPL1重组蛋白基因的质粒，其特征是：将pET43.1a(+)质粒载体上的NusA-tag、S-tag和HSV-tag替换为SEQ IDNO.3或SEQ ID NO.4。
本发明提供一种鲤LPL1重组蛋白的制备方法，其特征是，包含如下步骤：
(4)PCR扩增及扩增片段回收；
(5)重组质粒的构建及测序；
(6)转化，蛋白诱导表达。
进一步的，鲤LPL1重组蛋白的制备方法，其特征是，诱导温度分为20℃和25℃。
本发明提供一种重组质粒的制备方法，其特征是，包含如下步骤：
(5)载体、融合标签基因片段双酶切，切除原质粒载体上的NusA-tag和S-tag片段；
(6)重组质粒pET43.1a-Skp或pET43.1a-SlyD的构建；
(7)重组质粒pET43.1a-Skp或pET43.1a-SlyD及鲤LPL1双酶切，切除原质粒载体上的HSV-tag片段；
(8)pET43.1a-Skp-LPL1或pET43.1a-SlyD-LPL1构建。
本发明的有益效果：
(1)SlyD和Skp没有脂蛋白脂肪酶的生物活性，添加融合标签Skp和SlyD的蛋白表达载体可以提高目标蛋白的表达量和可溶性。
(2)添加融合标签SlyD的蛋白表达载体能起到促进蛋白分离纯化的作用。
(3)切除原质粒载体上的Nus-tag、S-tag和HSV-tag等，使融合蛋白无冗余氨基酸序列，利于后续研究。
(4)获得大量具有活性的鲤LPL1重组蛋白，为其功能研究提供了基础。
(5)对包涵体蛋白的复性发现含有融合标签Skp和SlyD可以促进目标蛋白的正确折叠和复性。
附图说明
图1：pET43.1a-Skp-LPL1或pET43.1a-SlyD-LPL1重组质粒构建流程图。
其中：Amp为氨苄抗性基因；Ori为质粒复制起始位点；LacI为启动子，来自大肠杆菌的乳糖操纵子；NusA-tag能增加融合蛋白的溶解度和表达；S-tag可使用S-tag抗体进行免疫检测，也可用于蛋白质纯化；HSV-tag可用于免疫检测；Nde I、EcoR I、Xho I为酶切位点；6×his Tag为组氨酸标签，用于蛋白纯化。
图2：Skp-LPL1诱导表达。
其中：M：蛋白Marker；1：不加IPTG；2：20℃IPTG总菌；3：20℃IPTG上清；4：25℃IPTG总菌；5：25℃IPTG上清；6：28℃IPTG总菌；7：28℃IPTG上清；8：37℃IPTG总菌；9：37℃IPTG上清。
图3：Skp-LPL1在20℃、25℃、28℃、37℃诱导温度下上清中可溶性重组蛋白占总菌中该蛋白的比率。
图4：SlyD-LPL1表达图。
其中：M：蛋白Marker；1:不加IPTG；2：16℃IPTG总菌；3：16℃IPTG上清；4：16℃IPTG沉淀；5：20℃IPTG总菌；6：20℃IPTG上清；7：20℃IPTG沉淀；8：25℃IPTG总菌；9：25℃IPTG上清；10：25℃IPTG沉淀；11：37℃IPTG总菌；12：37℃IPTG上清；13：37℃IPTG沉淀。
图5：SlyD-LPL1在16℃、20℃、25℃、37℃诱导温度下上清中可溶性重组蛋白占总菌中该蛋白的比率。
图6：可溶性蛋白纯化得及浓度测定
其中：A.纯化后的Skp-LPL1。M：蛋白marker；1～6分别为浓度为5.0μg、10.0μg、15.0μg、20.0μg、25.0μg、30.0μg的BSA蛋白。B.纯化后的SlyD-LPL1。M：蛋白marker；1～6分别为浓度为3.75μg、5.0μg、10.0μg、15.0μg、20.0μg、25.0μg的BSA蛋白。
具体实施方式
为使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施方式，进一步阐述本发明。
实施例1：
1.实验试剂：限制性核酸内切酶NcoⅠ，EcoRⅠ和Nde I购自美国NEB公司；2×Phanta-MaxMaster Mix、质粒提取试剂盒、胶回收试剂盒购自中国Vazyme公司；预染蛋白Mark、5×蛋白质加样缓冲液购自中国生工公司。氨苄青霉素，异丙基-β-D-1-硫代半乳糖吡喃糖苷(IPTG)。
2.PCR扩增及扩增片段回收。
slyD基因片段和skp基因片段的扩增：使用primer6.0设计slyD和skp特异性引物，并在slyD正向引物(
SEQ IDNO.8所示)、反向引物(
SEQ IDNO.9所示)的5'端分别加入限制性内切酶Nde I和EcoR I的酶切位点及保护碱基，在skp正向引物(
SEQ ID NO.10所示)、反向引物(
SEQ IDNO.11所示)的5'端分别加入限制性内切酶Nde I和EcoR I的酶切位点及保护碱基。以大肠杆菌基因组为模板，应用PCR分别扩增slyD基因片段和skp基因片段，反应体系为20μL，其中2×Taq Plus Master MixⅡ(Vazyme，中国)10μL，去离子水8μL，大肠杆菌基因组模板1μL扩增，正反向引物各0.5μL。PCR扩增程序为：94℃变性5min；94℃30s，56℃10s，72℃10s，共30个循环；最后在72℃延伸1min。
LPL1基因片段的扩增：采用正向引物为(
所示)、反向引物为(
所示)，在LPL1的正向引物5'端加入了EcoR I的酶切位点及保护碱基，在反向引物5'端加入了Xho I的酶切位点及保护碱基，同时在反向引物5'端加入了6个组氨酸标签蛋白。以鲤基因组为模板进行PCR扩增，反应体系为20μL，其中2×Taq Plus Master MixⅡ(Vazyme，中国)10μL，去离子水8μL，鲤基因组DNA 1μL，正反向引物各0.5μL。反应条件为PCR扩增程序为：94℃变性5min；94℃30s，60℃30s，72℃10s，共30个循环；2℃延伸7min。
PCR扩增产物均使用浓度为1％左右的琼脂糖凝胶检测，产物纯化回收使用FastPure Gel DNA Extraction Mini Kit(Vazyme，中国)。
3.重组质粒的构建及测序
(1)载体、Skp和SlyD基因片段双酶切：选取质粒pET43.1a(+)作为重组蛋白表达的载体。用限制性内切酶Nde I和EcoR I对pET43.1a(+)、Skp和SlyD基因片段进行双酶切，体系为30μL，其中基因片段或者载体为12μL，Nde I和EcoR I各1μL，10×H buffer为3μL，13μLddH2O。置于37℃金属浴12h，酶切后使用浓度为1％的琼脂糖凝胶检测，产物回收使用DNA胶回收试剂盒(FastPure Gel DNA Extraction Mini Kit，Vazyme，中国)。pET43.1a(+)双酶切后，将原质粒上的Nus-tag片段(Nus-tag是一种反转录终止因子，能够增加融合蛋白的溶解度和表达)和S-tag(S-tag可用于蛋白质纯化，可以使用商业化的S-tag抗体进行免疫检测)进行了切除。Skp和SlyD基因片段双酶切后分别获得片段如SEQ ID NO.7和SEQ IDNO.6。
(2)重组质粒pET43.1a-Skp和pET43.1a-SlyD的构建。连接：将上述双酶切回收后的pET43.1a(+)载体分别和Skp、SlyD基因片段进行连接，连接体系为10μL，其中pET43.1a2μL，Skp或SlyD片段3μL，Soultion I 5μL(TaKaRa，日本)，混匀后，于恒温循环槽箱(上海比朗科挤有限公司，中国)中，16℃连接10h左右。转化：将连接产物与30μL大肠杆菌Dh5α感受态细胞混合，冰浴30min后，42℃热击45s，冰浴2min，加入500μL LB液体培养基，37℃摇床200r/min培养60min。使用离心机5000r/min离心2min，吸走300μL上清，剩余液体吸打均匀，取100μL涂布在含氨苄抗性的LB固体培养基上，37℃过夜培养，选取单菌落，加入含氨苄抗性的1mL LB液体培养基，摇床过夜培养。验证：以菌液为模板，分别用slyD引物对和skp引物对进行PCR检测，并筛选阳性单克隆送测序，测序公司为上海生工。质粒提取：对经测序验证的菌株进行质粒提取，质粒提取试剂盒为FastPure Plasmid Mini Kit(Vazyme，中国)。
(3)重组质粒pET43.1a-Skp和pET43.1a-SlyD及鲤LPL1双酶切：双酶切体系为30μL，其中重组质粒或LPL1基因片段为12μL，限制性内切酶EcoR I和Xho I分别为1μL，10×Hbuffer为3μL，13μL ddH2O。于37℃恒温酶切12h，酶切后进行1％的琼脂糖凝胶电泳，产物纯化回收使用FastPure Gel DNA Extraction Mini Kit(Vazyme，中国)。重组质粒双酶切后，上面的HSV-tag被切除。LPL1基因片段双酶切后获得片段如SEQ ID NO.5。
(4)pET43.1a-Skp-LPL1和pET43.1a-SlyD-LPL1构建：将酶切纯化后的pET43.1a-Skp、pET43.1a-SlyD分别与酶切纯化后的鲤LPL1进行连接。连接、转化、验证及质粒提取步骤同(2)，构建的重组质粒分别命名为pET43.1a-Skp-LPL1和pET43.1a-SlyD-LPL1。连接后，载体上的添加的片段分别如SEQ ID NO.3和SEQ ID NO.4。
pET43.1a-Skp-LPL1或pET43.1a-SlyD-LPL1重组质粒构建流程图，如图1所示。
4.蛋白诱导表达及纯化
(1)转化：重组质粒pET43.1a-Skp-LPL1和pET43.1a-SlyD-LPL1转化大肠杆菌Transetta(DE3)感受态细胞，1μL质粒和30μL感受态混合，冰浴30min后，42℃水浴热击45s，冰浴2min，加入500μL LB液体培养基，37℃摇床200r/min培养60min，取100μL涂在含氨苄抗性的LB固体培养基上，37℃培养12h，挑取单克隆菌落接种于10mL含氨苄的LB液体培养基中培养12h。
(2)诱导表达及重组蛋白获取：扩大培养，取1mL前述菌液接种于100mL含氨苄的LB液体培养基，37℃培养2～3h。菌液吸光度OD 600值达到0.6时，加入IPTG(终浓度0.1mM)进行蛋白表达诱导，使用不同的诱导培养温度，时间为12h。将诱导后的菌液4℃，5000rpm离心10min，去上清。加入10mL非变性裂解液(NaH2PO4 50mmol/L，NaCl 300mmol/L，pH8.0)和终浓度为0.1mg/mL蛋白酶抑制剂(PMSF)，使用超声波细胞破碎仪(南京舜玛)对菌液进行破碎，4℃，12000rpm离心5min，留上清液。取10μL上清，加入2.5μL 5×蛋白上样缓冲液(250mmol/L Tris-HCl，pH6.8，10％SDS，0.5％溴酚蓝，50％甘油，7.5％DTT)，吹打混匀后，100℃水浴锅20min。取10μL用12％SDS-PAGE凝胶进行电泳分析，考马斯亮蓝R-250染色1h显示电泳结果。通过Image LabTM软件(Bio-Rad)进行灰度分析。
(3)可溶性重组蛋白的纯化：选择20℃和25℃的诱导温度分别诱导Skp-LPL1、SlyD-LPL1重组蛋白，180r/min摇床12h，离心收集菌，加入非变性裂解液、蛋白酶抑制剂超声，离心后取上清，用0.22μm滤膜过滤，使用碧云天镍柱纯化柱纯化目的蛋白，用BSA标准蛋白确定浓度。
5.结果分析
(1)诱导表达获得的重组蛋白氨基酸序列如SEQ ID NO.1和SEQ ID NO.2。在上清中均获得Skp-LPL1和SlyD-LPL1重组蛋白，说明Skp-和SlyD-融合标签有效促进重组蛋白的可溶性。所获得Skp-LPL1和SlyD-LPL1重组蛋白的分子量分别是72.46kDa和77.62kDa(图2和图4)。不同温度诱导蛋白表达结果灰度分析显示，Skp-LPL1在20℃、25℃、28℃、37℃诱导温度下上清中可溶性重组蛋白占总菌中该蛋白的比率分别是62％、54％、43％和26％(图3)。SlyD-LPL1在16℃、20℃、25℃、37℃诱导温度下上清中可溶性重组蛋白占总菌中该蛋白的比率分别是74％、51％、89％和22％(图5)。因此，Skp-LPL1和SlyD-LPL1可溶性重组蛋白得量最高的最适诱导温度分别为20℃和25℃。最适温度下，上清中Skp-LPL1和SlyD-LPL1在总融合蛋白中占的比例分别为62％和89％，可见融合标签Skp-和SlyD-均能促进可溶性蛋白的表达，而且含SlyD-的重组蛋白较含有Skp-的具有更好的可溶性效果。
(2)结果显示：可溶性Skp-LL1和SlyD-LPL1重组蛋白均可经纯化获得纯化蛋白，每获得1g总菌获得可溶性Skp-LL1和SlyD-LPL1重组蛋白的质量为分别为0.24mg和0.31mg(图6)。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。
序列表
<110> 中国水产科学研究院淡水渔业研究中心
<120> 一种鲤LPL1重组蛋白及其制备方法
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gccgttgctt acaacagcag cgatgtaaaa gacatcactg ccgacgtact gaaacaggtt 420
aaagaattcg caacttcgat tgaaccagcg agtgaatttc ccacttttaa taacatcatg 480
gacaatggca cagaatggat gacggacttc agtgatatag tgtccaagtt ttccttcagg 540
accagcgaag aacccgaaga tgatctatgc tacatagttc caggtcaacc cgaaaccatc 600
aaagaatgta acttcaatcc agataacaag actttcatag ttattcatgg atggacggtt 660
acgggtatgt ttgaaagctg ggttcccaaa ctggtaacag ccctgtatga acgagagcca 720
acagccaatg tgattgtggt ggactggctg tcccgtgcgc aacaacacta ccttacatca 780
gccggctaca ccaaactagt ggggagggat gtggccaagt ttgtcaactg gttacaggct 840
gagattgact atccttggga gaggctgcat ctgttgggct acagtcttgg tgctcatgta 900
gcaggaatcg ctggtcttct caccaaacat aaagttaaca gaatcacagg catggatcct 960
gctggcccta gttttgagta tgcagatgcc caaagcactc tttctcccga tgatgccctt 1020
ttcgtggacg ttcttcacac caacactcgc ggttctccag atcgcagcat tgggattcag 1080
aggccagtgg gccacattga catctacccc aatggaggaa ccttccaacc cggctgtgac 1140
ctccagaaca ctgtgttgat ggtggccacc tctggtttaa gaaacatgga tcagatcgtg 1200
aagtgctccc atgagcgctc cattcacctg ttcatcgact cgctggtgaa ccaggaccaa 1260
gaaagcatgg cttaccgctg cagctccaaa gacagcttca ataagggcat gtgtctcagc 1320
tgccgcaaga accgttgcaa caaggtgggc tacggagtca acaaaattcg cacacgcaga 1380
agcagcaaga tgtacatgaa gaccagaaat gtgatgccat ataaagtttt ccattatcaa 1440
gttaaggtcc acttcttcgg caagacaaaa ttaagctaca ctgatcagcc cattaagatc 1500
tcactgtacg gaatccatgg cgagaaggaa aatatcccct acattttgcc tgctttgaac 1560
acaaacacca ccgtgtcttt cctgctgacc acggacacag acatcggaga gctgctgatg 1620
gtaaaacttc tctgggagaa agacaccctc atcagctggc catggtggaa ccccgatacc 1680
ttccacatcc gcaaactgcg catcaaatca ggagaaagac agtccaagat catcttcagt 1740
gccaaagaag gtgaattttc ctacctttcc cgtggagggg aggccgccgt ctttgtgaaa 1800
gaaaaagaag cccagtcaag ccgcaaaaac cagagattgc acaagttgaa gatgcatgga 1860
agttcattca aacagagcac cgag 1884
<210> 4
<211> 2046
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aaagtagcaa aagacctggt ggtcagcctg gcctatcagg tacgtacaga agacggtgtg 60
ttggttgatg agtctccggt gagtgcgccg ctggactacc tgcatggtca cggttccctg 120
atctctggcc tggaaacggc gctggaaggt catgaagttg gcgacaaatt tgatgtcgct 180
gttggcgcga acgacgctta cggtcagtac gacgaaaacc tggtgcaacg tgttcctaaa 240
gacgtattta tgggcgttga tgaactgcag gtaggtatgc gtttcctggc tgaaaccgac 300
cagggtccgg taccggttga aatcactgcg gttgaagacg atcacgtcgt ggttgatggt 360
aaccacatgc tggccggtca gaacctgaaa ttcaacgttg aagttgtggc gattcgcgaa 420
gcgactgaag aagaactggc tcatggtcac gttcacggcg cgcacgatca ccaccacgat 480
cacgaccacg acggttgctg cggcggtcat ggccacgatc acggtcatga acacggtggt 540
gaaggctgct gtggcggtaa aggcaacggc ggttgcggtt gccacgaatt cgcaacttcg 600
attgaaccag cgagtgaatt tcccactttt aataacatca tggacaatgg cacagaatgg 660
atgacggact tcagtgatat agtgtccaag ttttccttca ggaccagcga agaacccgaa 720
gatgatctat gctacatagt tccaggtcaa cccgaaacca tcaaagaatg taacttcaat 780
ccagataaca agactttcat agttattcat ggatggacgg ttacgggtat gtttgaaagc 840
tgggttccca aactggtaac agccctgtat gaacgagagc caacagccaa tgtgattgtg 900
gtggactggc tgtcccgtgc gcaacaacac taccttacat cagccggcta caccaaacta 960
gtggggaggg atgtggccaa gtttgtcaac tggttacagg ctgagattga ctatccttgg 1020
gagaggctgc atctgttggg ctacagtctt ggtgctcatg tagcaggaat cgctggtctt 1080
ctcaccaaac ataaagttaa cagaatcaca ggcatggatc ctgctggccc tagttttgag 1140
tatgcagatg cccaaagcac tctttctccc gatgatgccc ttttcgtgga cgttcttcac 1200
accaacactc gcggttctcc agatcgcagc attgggattc agaggccagt gggccacatt 1260
gacatctacc ccaatggagg aaccttccaa cccggctgtg acctccagaa cactgtgttg 1320
atggtggcca cctctggttt aagaaacatg gatcagatcg tgaagtgctc ccatgagcgc 1380
tccattcacc tgttcatcga ctcgctggtg aaccaggacc aagaaagcat ggcttaccgc 1440
tgcagctcca aagacagctt caataagggc atgtgtctca gctgccgcaa gaaccgttgc 1500
aacaaggtgg gctacggagt caacaaaatt cgcacacgca gaagcagcaa gatgtacatg 1560
aagaccagaa atgtgatgcc atataaagtt ttccattatc aagttaaggt ccacttcttc 1620
ggcaagacaa aattaagcta cactgatcag cccattaaga tctcactgta cggaatccat 1680
ggcgagaagg aaaatatccc ctacattttg cctgctttga acacaaacac caccgtgtct 1740
ttcctgctga ccacggacac agacatcgga gagctgctga tggtaaaact tctctgggag 1800
aaagacaccc tcatcagctg gccatggtgg aaccccgata ccttccacat ccgcaaactg 1860
cgcatcaaat caggagaaag acagtccaag atcatcttca gtgccaaaga aggtgaattt 1920
tcctaccttt cccgtggagg ggaggccgcc gtctttgtga aagaaaaaga agcccagtca 1980
agccgcaaaa accagagatt gcacaagttg aagatgcatg gaagttcatt caaacagagc 2040
accgag 2046
<210> 5
<211> 1455
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gcaacttcga ttgaaccagc gagtgaattt cccactttta ataacatcat ggacaatggc 60
acagaatgga tgacggactt cagtgatata gtgtccaagt tttccttcag gaccagcgaa 120
gaacccgaag atgatctatg ctacatagtt ccaggtcaac ccgaaaccat caaagaatgt 180
aacttcaatc cagataacaa gactttcata gttattcatg gatggacggt tacgggtatg 240
tttgaaagct gggttcccaa actggtaaca gccctgtatg aacgagagcc aacagccaat 300
gtgattgtgg tggactggct gtcccgtgcg caacaacact accttacatc agccggctac 360
accaaactag tggggaggga tgtggccaag tttgtcaact ggttacaggc tgagattgac 420
tatccttggg agaggctgca tctgttgggc tacagtcttg gtgctcatgt agcaggaatc 480
gctggtcttc tcaccaaaca taaagttaac agaatcacag gcatggatcc tgctggccct 540
agttttgagt atgcagatgc ccaaagcact ctttctcccg atgatgccct tttcgtggac 600
gttcttcaca ccaacactcg cggttctcca gatcgcagca ttgggattca gaggccagtg 660
ggccacattg acatctaccc caatggagga accttccaac ccggctgtga cctccagaac 720
actgtgttga tggtggccac ctctggttta agaaacatgg atcagatcgt gaagtgctcc 780
catgagcgct ccattcacct gttcatcgac tcgctggtga accaggacca agaaagcatg 840
gcttaccgct gcagctccaa agacagcttc aataagggca tgtgtctcag ctgccgcaag 900
aaccgttgca acaaggtggg ctacggagtc aacaaaattc gcacacgcag aagcagcaag 960
atgtacatga agaccagaaa tgtgatgcca tataaagttt tccattatca agttaaggtc 1020
cacttcttcg gcaagacaaa attaagctac actgatcagc ccattaagat ctcactgtac 1080
ggaatccatg gcgagaagga aaatatcccc tacattttgc ctgctttgaa cacaaacacc 1140
accgtgtctt tcctgctgac cacggacaca gacatcggag agctgctgat ggtaaaactt 1200
ctctgggaga aagacaccct catcagctgg ccatggtgga accccgatac cttccacatc 1260
cgcaaactgc gcatcaaatc aggagaaaga cagtccaaga tcatcttcag tgccaaagaa 1320
ggtgaatttt cctacctttc ccgtggaggg gaggccgccg tctttgtgaa agaaaaagaa 1380
gcccagtcaa gccgcaaaaa ccagagattg cacaagttga agatgcatgg aagttcattc 1440
aaacagagca ccgag 1455
<210> 6
<211> 585
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aaagtagcaa aagacctggt ggtcagcctg gcctatcagg tacgtacaga agacggtgtg 60
ttggttgatg agtctccggt gagtgcgccg ctggactacc tgcatggtca cggttccctg 120
atctctggcc tggaaacggc gctggaaggt catgaagttg gcgacaaatt tgatgtcgct 180
gttggcgcga acgacgctta cggtcagtac gacgaaaacc tggtgcaacg tgttcctaaa 240
gacgtattta tgggcgttga tgaactgcag gtaggtatgc gtttcctggc tgaaaccgac 300
cagggtccgg taccggttga aatcactgcg gttgaagacg atcacgtcgt ggttgatggt 360
aaccacatgc tggccggtca gaacctgaaa ttcaacgttg aagttgtggc gattcgcgaa 420
gcgactgaag aagaactggc tcatggtcac gttcacggcg cgcacgatca ccaccacgat 480
cacgaccacg acggttgctg cggcggtcat ggccacgatc acggtcatga acacggtggt 540
gaaggctgct gtggcggtaa aggcaacggc ggttgcggtt gccac 585
<210> 7
<211> 423
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gctgacaaaa ttgcaatcgt caacatgggc agcctgttcc agcaggtagc gcagaaaacc 60
ggtgtttcta acacgctgga aaatgagttc aaaggccgtg ccagcgaact gcagcgtatg 120
gaaaccgatc tgcaggctaa aatgaaaaag ctgcagtcca tgaaagcggg cagcgatcgc 180
actaagctgg aaaaagacgt gatggctcag cgccagactt ttgctcagaa agcgcaggct 240
tttgagcagg atcgcgcacg tcgttccaat gaagaacgcg gcaaactggt tactcgtatc 300
cagactgctg tgaaatccgt tgccaacagc caggatatcg atctggttgt tgatgcaaac 360
gccgttgctt acaacagcag cgatgtaaaa gacatcactg ccgacgtact gaaacaggtt 420
aaa 423
<210> 8
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggaattccat atgaaagtag caaaagacc 29
<210> 9
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccggaattcg tggcaaccgc aaccg 25
<210> 10
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggaattccat atggctgaca aaattgcaat c 31
<210> 11
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ccggaattct ttaacctgtt tcagtacgtc ggcag 35
<210> 12
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cggaattcgc aacttcgatt gaaccag 27
<210> 13
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ccgctcgagt tagtggtgat gatgatggtg atagaagcgc agcgcgtt 48
Claims (8)
1.一种鲤LPL1重组蛋白，其特征是，氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
2.如权利要求1所述的重组蛋白，其特征是，SEQ ID NO.1对应的核苷酸序列如SEQ IDNO.3所示；SEQ ID NO.2对应的核苷酸序列如SEQ ID NO.4所示。
3.如权利要求1所述的鲤LPL1重组蛋白，其特征是，重组蛋白表达的载体为质粒，进一步的，质粒为改造后的pET43.1a(+)。
4.一种重组质粒，其特征是：将pET43.1a(+)质粒载体上的NusA-tag、S-tag和HSV-tag替换为SlyD或Skp融合标签。
5.一种包含如权利要求1所述鲤LPL1重组蛋白基因的质粒，其特征是：将pET43.1a(+)质粒载体上的NusA-tag、S-tag和HSV-tag替换为SEQ ID NO.3或SEQ ID NO.4。
6.如权利要求1所述的鲤LPL1重组蛋白的制备方法，其特征是，包含如下步骤：
(1)PCR扩增及扩增片段回收；
(2)重组质粒的构建及测序；
(3)转化，蛋白诱导表达。
7.如权利要求6所述的鲤LPL1重组蛋白的制备方法，其特征是，诱导温度分为20℃和25℃。
8.如权利要求4或6所述的重组质粒的制备方法，其特征是，包含如下步骤：
(1)载体、融合标签基因片段双酶切，切除原质粒载体上的NusA-tag和S-tag片段；
(2)重组质粒pET43.1a-Skp或pET43.1a-SlyD的构建；
(3)重组质粒pET43.1a-Skp或pET43.1a-SlyD及鲤LPL1双酶切，切除原质粒载体上的HSV-tag片段；
(4)pET43.1a-Skp-LPL1或pET43.1a-SlyD-LPL1构建。
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| US20140170701A1 (en) * | 2012-12-19 | 2014-06-19 | Boehringer Ingelheim Rcv Gmbh & Co Kg | Method for producing a recombinant protein of interest |
| CN105189743A (zh) * | 2012-12-19 | 2015-12-23 | 桑多斯股份公司 | 用于生产重组兴趣蛋白的方法 |
| CN108178787A (zh) * | 2017-12-28 | 2018-06-19 | 广东省农业科学院动物卫生研究所 | pORF131重组蛋白及其制备方法和应用 |
| CN112979781A (zh) * | 2021-03-18 | 2021-06-18 | 中国海洋大学 | 一种许氏平鲉IL-1β重组蛋白及其制备方法和应用 |
| CN114181321A (zh) * | 2021-12-10 | 2022-03-15 | 中国海洋大学 | 花鲈fgf6a、fgf6b以及fgf18重组蛋白及其制备方法和应用 |
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| US20140170701A1 (en) * | 2012-12-19 | 2014-06-19 | Boehringer Ingelheim Rcv Gmbh & Co Kg | Method for producing a recombinant protein of interest |
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