CN114621959B - 一种编码牙鲆igf2可溶性蛋白的基因及蛋白重组表达方法与应用 - Google Patents
一种编码牙鲆igf2可溶性蛋白的基因及蛋白重组表达方法与应用 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域，具体涉及一种编码牙鲆IGF2可溶性蛋白的基因、编码蛋白及在促细胞增殖中的应用。具体公开了编码牙鲆IGF2可溶性蛋白的基因并利用该基因编码牙鲆IGF2可溶性蛋白，本发明将该基因构建真核表达载体和转染至人胚胎肾细胞HEK293T，在体外获得牙鲆IGF2可溶性蛋白，该蛋白具有促进细胞增殖的作用。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及一种编码牙鲆IGF2可溶性蛋白的基因、编码蛋白及在促细胞增殖中的应用。
背景技术
牙鲆(Paralichthys olivaceus)隶属于鲽形目(Pleuronectiformes)、鲽亚目(Pleuronectoi)、牙鲆科(Paralichthyidae)、牙鲆属 (Paralichthys)，为冷温性底栖鱼类，主要分布于日本、朝鲜半岛及中国北方沿海海域。其肉质鲜嫩，蛋白品质和不饱和脂肪酸的含量较高，是营养丰富的名贵鱼类。
与哺乳动物类似，鱼类的生长主要受生长激素(growth hormone,GH) －类胰岛素生长因子(insulin-like growth factors,IGFs)轴的调控， GH促进生长的作用主要是通过IGFs途径实现。人们早已认识到IGF2在鱼类生长中的重要性，体外重组该蛋白可以作为促生长剂或添加剂等用于水产养殖，然而，尚未见有关牙鲆IGF2的相关报道。尽管已有半滑舌鳎 (Cynoglossus semilaevis Günther)IGF2体外重组表达的报道，但是其采用原核表达，获得的IGF2重组蛋白为的包涵体蛋白，而包涵体蛋白复性实验庞杂、繁琐、效率低；此外，原核表达蛋白没有蛋白修饰，即使获得可溶性蛋白或包涵体蛋白复性蛋白，本领域技术人员所知其理化性质可能并不能预期与体内蛋白性质相同。本发明目的在于提供一种通过真核细胞直接表达牙鲆IGF2可溶性蛋白的方法，不存在包涵体蛋白现象，存在蛋白修饰，比原核表达的重组蛋白更接近牙鲆IGF2蛋白的体内形式。
发明内容
本发明要解决的技术问题在于提供一种编码牙鲆IGF2可溶性蛋白的基因、编码蛋白及在促细胞增殖中的应用。
为实现上述目的，本发明采用技术方案为：
一种编码牙鲆IGF2可溶性蛋白的基因，编码牙鲆IGF2可溶性蛋白的基因的核苷酸序列如SEQ ID NO:1所示。
所述基因编码蛋白的氨基酸序列为SEQ ID NO:2。
一种真核表达载体，表达载体含所述的SEQ ID NO:1序列核苷酸序列。
所述真核表达载体pcDNA3.1。
利用PCR扩增获得的SEQ ID NO:1，经纯化后，重组到真核表达载体 pcDNA3.1的EcoRⅠ酶切位点，即构建成所述真核表达载体pcDNA3.1-IGF2。
一种转染细胞，所述转染细胞含所述的真核表达载体。
所述权利要求3所述的真核表达载体pcDNA3.1-IGF2转染至HEK293T 细胞而得pcDNA3.1-IGF2转染细胞。
一种重组牙鲆IGF2可溶性蛋白的获得方法，所述重组蛋白为牙鲆IGF2 可溶性蛋白。
所述pcDNA3.1-IGF2转染细胞在转染后2-3天，转染细胞用DMEM培养基清洗，清洗后将其于DMEM培养基中于37℃、5％的CO2培养箱中孵育 30-60分钟，而后更换DMEM培养基再次培养2-3天，培养后收集DMEM培养基的上清，上清蛋白浓缩，获得重组牙鲆IGF2可溶性蛋白。
一种所述编码牙鲆IGF2可溶性蛋白的基因、所述真核表达载体、所述转染细胞或所述重组蛋白在促细胞增殖中的应用。
进一步的说该蛋白具有促进HEK293T或人宫颈癌细胞Hela增殖的作用。
本发明与现有技术相比具有以下有益效果：
本发明首次扩增编码牙鲆IGF2可溶性蛋白的基因，其核苷酸序列如 SEQ ID NO:1，并将该基因构建真核表达载体和转染HEK293T细胞，利用 pcDNA3.1-IGF2转染细胞在体外获得牙鲆IGF2可溶性蛋白，所述方法能够得到该基因编码的重组牙鲆IGF2可溶性蛋白，重组牙鲆IGF2可溶性蛋白能够促进细胞增殖。
附图说明
图1为本发明实施例提供构建的pcDNA3.1-IGF2真核表达载体的质粒图谱。
图2为本发明实施例提供的pcDNA3.1-IGF2转染细胞表达IGF2可溶性蛋白的Western blot鉴定图，Western blot用His标签抗体检测。其中： 1：pcDNA3.1转染细胞对照上清蛋白；2：pcDNA3.1-IGF2转染细胞上清蛋白
图3为本发明实施例提供重组牙鲆IGF2可溶性蛋白对HEK293T细胞和 Hela细胞增殖的影响。其中：A是HEK293T细胞；B是Hela细胞，*表示有显著性差异(P<0.05)。
具体实施方式
下面结合附图和实施例对本发明作进一步说明，但不以任何形式限制本发明。
本发明突破传统方法，将编码牙鲆IGF2成熟肽的核苷酸序列克隆到真核表达载体，成功构建重组真核表达载体pcDNA3.1-IGF2，并在HEK293T细胞中表达，直接获得牙鲆IGF2可溶性蛋白。该发明可以直接获得牙鲆IGF2 可溶性蛋白，不存在包涵体现象；该发明获得牙鲆IGF2可溶性蛋白为真核细胞表达，存在蛋白修饰，比原核表达的重组蛋白更接近牙鲆IGF2蛋白的体内形式。
实施例1：含编码牙鲆IGF2可溶性蛋白的真核表达载体pcDNA3.1-IGF2的构建
以心跳期和孵化期的牙鲆的混合cDNA为模板，利用IGF2-689F和 IGF2-689R为引物对，PCR扩增获得牙鲆IGF2的开放阅读框，用全式金的 pEASY-T3克隆试剂盒将其克隆到pEASY-T3载体，获得含有牙鲆IGF2开放阅读框的IGF2-T3质粒，以IGF2-T3质粒为模板，利用IGF2-F和IGF2-R作为引物对，PCR扩增获得编码牙鲆IGF2可溶性蛋白的基因，碱基序列为SEQ ID NO:1所示；其中：
IGF2-689F:5’-CTACTGCCATCTGACATG-3’
IGF2-689R:5’-GTCTGTGCAAAGGGCTG-3’
IGF2-F：5’-TGTGCTGGATATCTGCAGgccaccATGTCTTCGTCCAGTCGTGC-3’
IGF2-R：5’-ACTAGTCCAGTGTGGTGGCCTTTCGGACTTGGCGGGTTTGGCAC-3’
将上述获得的编码牙鲆IGF2可溶性蛋白的基因以及用限制性内切酶 EcoRⅠ线性化的真核表达载体pcDNA3.1，用康为世纪的快速琼脂糖凝胶 DNA回收试剂盒回收，进行琼脂糖凝胶电泳，分离纯化编码牙鲆IGF2可溶性蛋白的基因片段和pcDNA3.1线性化载体；用CloneSmarter的无缝克隆试剂盒进行重组后转化入大肠杆菌中，用LB平板筛选转化子，以标准方法提取质粒，用引物IGF2-F和IGF2-R进行PCR扩增，得到与编码牙鲆IGF2可溶性蛋白的基因大小相同片段，将含该片段的质粒用T7引物(5’- TAATACGACTCACTATAGGG-3’)进行测序，序列分析证明编码牙鲆IGF2可溶性蛋白的基因已克隆至pcDNA3.1真核表达载体中，重组质粒命名为pcDNA3.1-IGF2(参见图1)。
由图1可见编码牙鲆IGF2可溶性蛋白的基因已经重组到pcDNA3.1真核表达载体，同时破坏掉EcoRⅠ重组位点；该IGF2基因的羧基端带有myc 和His标签，受CMV启动子驱动表达。
实施例2：表达牙鲆IGF2可溶性蛋白的HEK293T细胞的构建
将上述获得制备好的重组质粒pcDNA3.1-IGF2用标准氯化钙转化法转入大肠杆菌中，在LB平板上37℃进行培养。过夜后长出单克隆，挑取单克隆接种在5ml LB液体培养基中，37℃，220转/分培养过夜，然后用康为世纪的无内毒素质粒小提试剂盒提取质粒，获得pcDNA3.1-IGF2无内毒素质粒；在6孔板上接种HEK293T细胞，按照英潍捷基Lipofectamine3000:pcDNA3.1-IGF2:P3000＝5μl:2.5μg:5μl进行转染，获得表达牙鲆IGF2 可溶性蛋白的HEK293T细胞。
实施例3：利用pcDNA3.1-IGF2瞬时转染细胞表达牙鲆IGF2可溶性蛋白
在上述获得转染HEK293T细胞2天左右后，用DMEM培养基洗2次细胞，而后将细胞于1ml DMEM培养基中在37℃5％的CO2培养箱孵育30分钟，之后换成1ml DMEM培养基，在37℃5％的CO2培养箱继续培养；2天左右后，收集细胞培养基，用Millipore 3K超滤管按照标准方法进行浓缩，得到牙鲆 IGF2的可溶性蛋白，氨基酸序列如SEQ ID NO:2所示，Westernblot分析结果如图2所示。
由图2可见在转染空载体pcDNA3.1的HEK293T细胞的上清中没有检测到牙鲆IGF2可溶性蛋白的表达，但在转染pcDNA3.1-IGF2的HEK293T细胞的上清中检测到其表达，说明pcDNA3.1-IGF2转染细胞成功表达牙鲆IGF2 可溶蛋白。
实施例4：重组牙鲆IGF2的功能研究——对培养HEK293T和Hela细胞增殖的影响
在96孔板上分别接种合适数量的HEK293T和Hela细胞，接种后第2 天，用DMEM培养基洗2次，而后将细胞于100μl DMEM培养基于37℃5％的CO2培养箱孵育30分钟，之后换成100μl DMEM培养基，每孔加入3μl 浓缩后的牙鲆IGF2可溶性蛋白，浓缩后蛋白浓度为15.3μg/μl，IGF2处理2天左右后，用碧云天的MTT细胞增殖检测试剂盒按照标准方法分析重组牙鲆IGF2可溶性蛋白对培养HEK293T和Hela细胞增殖的影响(参见图 3)。
由图3可见，a图重组牙鲆IGF2可溶性蛋白促进了HEK293T细胞的增殖，b图重组牙鲆IGF2可溶性蛋白促进了Hela细胞的增殖，说明IGF2具有促进细胞增殖的作用。
SEQ ID NO:1
TCGGCGGAGACGCTGTGTGGGGGAGAGCTGGTGGATGCGCTGCAGTT TGTCTGTGAAGACAGAGGCTTCTATTTCAGTAGGCCAACCAGCAGGGGTAGCAACCGGCGCCCCCAGAACCGTGGGATCGTAGAGGAATGTTGT TTCCGTAGCTGTGACCTCAACCTGCTGGAGCAGTACTGTGCCAAACC CGCCAAGTCCGAAAGG
SEQ ID NO:2
SAETLCGGELVDALQFVCEDRGFYFSRPTSRGSNRRPQNRGIVEECCFRSCDLNLLEQYCAKPAKSER。
序列表
<110> 中国科学院海洋研究所
<120> 一种编码牙鲆IGF2可溶性蛋白的基因及蛋白重组表达方法与应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 204
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tcggcggaga cgctgtgtgg gggagagctg gtggatgcgc tgcagtttgt ctgtgaagac 60
agaggcttct atttcagtag gccaaccagc aggggtagca accggcgccc ccagaaccgt 120
gggatcgtag aggaatgttg tttccgtagc tgtgacctca acctgctgga gcagtactgt 180
gccaaacccg ccaagtccga aagg 204
<210> 2
<211> 68
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Ser Ala Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Ala Leu Gln Phe
1 5 10 15
Val Cys Glu Asp Arg Gly Phe Tyr Phe Ser Arg Pro Thr Ser Arg Gly
20 25 30
Ser Asn Arg Arg Pro Gln Asn Arg Gly Ile Val Glu Glu Cys Cys Phe
35 40 45
Arg Ser Cys Asp Leu Asn Leu Leu Glu Gln Tyr Cys Ala Lys Pro Ala
50 55 60
Lys Ser Glu Arg
65
Claims (1)
1.一种重组牙鲆IGF2 可溶性蛋白在促进HEK293T细胞和Hela细胞增殖中的应用；
所述重组牙鲆IGF2 可溶性蛋白的基因的核苷酸序列如SEQ ID NO: 1所示。
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