CN117384275B - 胰岛素样生长因子突变体igf1m及其应用 - Google Patents
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Abstract
本发明属于基因工程领域,具体涉及胰岛素样生长因子突变体IGF1M及其应用。本发明提供了一种来源于家燕(Hirundo rustica)的胰岛素样生长因子突变体IGF1M,其氨基酸序列如SEQ ID NO.1所示,且本发明提供了编码上述胰岛素样生长因子突变体IGF1M的基因。该突变增强了与其受体的亲和力,能够促进家禽的生长发育,提高家禽个体重量。
Description
技术领域
本发明属于基因工程领域,具体涉及胰岛素样生长因子突变体IGF1M及其应用。
背景技术
胰岛素样生长因子(insulin like growth factor,IGF)是一组具有促生长作用的多肽类物质,其分泌细胞广泛分布在人体肝、肾、肺、心、脑和肠等组织中。IGF族有IGF-Ⅰ和IGF-Ⅱ两种。胰岛素样生长因子是功能比较多样的调控因子,主要在肝脏内部合成,会广泛存在于血液循环中。胰岛素样生长因子介质会刺激和调节身体各大组织的生长发育,可调节肌肉的力量,维持身体的成分,调节营养代谢等。胰岛素样生长因子能够通过内分泌、自分泌等方式,发挥生物学效应,同时受到结合蛋白的调节,促进个体细胞增殖(刘芝亮等人通过原核表达体系获得了半滑舌鳎胰岛素样生长因子I,并发现该多肽能够促进乳腺细胞MDA231细胞的增殖;张建峰等人也发现鸡胰岛素生长因子I能够促进鸡成骨细胞的增殖;张飞燕等通过真核表达系统克隆并表达了藏猪胰岛素样生长因子I,并发现其能够促进猪外周淋巴细胞的增殖)。除此之外,很多肝外组织也能产生胰岛素样生长因子,而且不用受到生长激素的调控。
在临床上,胰岛素样生长因子在中枢神经系统发育时期是非常重要的信号分子,可传递重要的生长信息。IGF-Ⅰ通常认为是胶质祖细胞的存活因子,可以以此判断胶质细胞的存活情况。IGF-Ⅰ和IGF-Ⅱ都可以作为肌源性神经营养因子,它们有刺激神经突起生长的重要作用。
胰岛素样生长因子还与产肉性能和肌肉的生长密切相关。很多证据表明 IGF和哺乳动物肌肉的生长、分化调控有关。血清 IGF-I和 IGF-Ⅱ浓度与肋条肉的瘦肉率和相关组织的比率呈负相关[1]。低血清 IGF-I浓度的牛,其肉具有较高的大理石花纹面积和质量等级,故血清 IGF-I浓度可成为提高肉牛大理石花纹面积和质量等级的一个有用的选育标准[2]。葛盛芳等对60日龄大蛋系和高产系绍兴鸭的研究发现大蛋系鸭的血清 IGF-I含量高于高产系,这可能与其体重及产蛋量高于高产系有关,表明 IGF-I对鸭的生长和产蛋有一定的影响[3]。Kang 等[ 4]证明 IGF-I 水平与产蛋量有关。以 IGF-Ⅰ基因作为研究产蛋性状的候选标记基因,Nagaraja 等[ 5]报道在白来航鸡中 IGF-Ⅰ基因5'端调控区发现了多态性和蛋重及蛋壳重相关。在此基础上,欧阳建华等[ 6]对万载康乐黄鸡和泰和乌骨鸡 ,以及Kim 等[ 7]对韩国土种Ogo l鸡 ,分别比较分析了IGF-Ⅰ基因型与各产蛋性状,发现存在一定的相关性。
鉴于胰岛素样生长因子的广泛用途,有必要开发促细胞增殖能力强的新胰岛素样生长因子,进而提高动物生产性能,为胰岛素样生长因子在动物产能中的应用提供技术支持。
[1] Nikolic I A, et al. Bio t in Anim Hus, 2000, 16( 3/4): 3-10.
[2] Davis M E, et al. Anim Sci, 2000, 78( 9): 2305-2313.
[3] 葛盛芳 ,等.南京农业大学学报 , 2000, 23( 2): 76-79.
[4] Kang W J, J S Yun , D S S eo .Study on the associ ation of earlyegg productivit y w ith serum insulin-like grow th fact or-Ⅰ in Korean NativeOgol C hicken[ J].J Anim S ci Technol, 2000 , 42(6):767-776.
[5] Nagaraja S C , S E Aggrey , J Yao , et al .T rait association ofa geneti c marker near the IGF-Ⅰ gene in egg-laying chicken s[ J].J H ered ,2000 , 91(2):150-156.
[6] 欧阳建华, 黄建华, 孙 汉, 等.鸡 IGF-Ⅰ 基因的遗传多态性与繁殖性状的相关研究[ J].江西畜牧兽医杂志, 2003(6):6-8.
[7] Kim M H , D S Seo , Y Ko .Relati onship Between E gg Productivity and Insulin-Lik e Grow th Fact or-I Genoty pes in Korean Native Ogol Chickens[ J].Poult Sci, 2004 , 83(7):1203-1208.
发明内容
为了解决上述问题,本发明提供了一种胰岛素样生长因子的突变体,将野生型胰岛素样生长因子进行突变后,增强了该多肽胰岛素样生长因子受体I的结合能力。与野生型的胰岛素样生长因子相比对细胞增殖的促进能力得到了显著提升。
一方面,本发明提供了一种胰岛素样生长因子突变体IGF1M,所述的胰岛素样生长因子突变体IGF1M的氨基酸序列如SEQ ID NO. 1所示。
具体地,所述的突变为3个位点的突变,突变位点为E9G,F16I,C18L。
进一步具体地,所述的E9G是第9位的谷氨酸突变为甘氨酸;所述的F16I是第16位的苯丙氨酸突变为异亮氨酸;所述的C18L是第18位的半胱氨酸突变为亮氨酸。
具体地,所述的序列SEQ ID NO. 1的N端连接信号肽序列SEQ ID NO.2。
又一方面,本发明提供了编码前述的胰岛素样生长因子突变体IGF1M的核酸。
具体地,所述核酸的序列为SEQ ID NO.3或与SEQ ID NO.3具有90%以上序列同源性的序列。
优选地,所述核酸的序列为SEQ ID NO.3或与SEQ ID NO.3具有95%以上序列同源性的序列。
进一步优选地,所述核酸的序列为SEQ ID NO.3或与SEQ ID NO.3具有98%以上序列同源性的序列。
又一方面,本发明提供了一种包括前述的核酸的重组载体。
具体地,所述的载体可以是质粒、噬菌体和病毒中的一种。
优选地,所述的载体是质粒。
进一步优选地,所述的载体是pPIC9K质粒。
具体地,将核酸插入到质粒pPIC9K上的XhoI和EcoR I限制性酶切位点之间。
又一方面,本发明提供了一种包括前述的重组载体的细胞。
具体地,所述的细胞可以是用于表达蛋白的基因工程细胞,包括但不限于:植物细胞、动物细胞、细菌或酵母。
进一步具体地,所述的细胞可以是工程菌。
优选地,所述的细胞可以是大肠杆菌、酵母菌、芽孢杆菌或乳酸杆菌。
进一步优选地,所述的细胞可以是酿酒酵母菌、红法夫酵母菌或毕赤酵母菌。
更进一步优选地,所述的细胞是毕赤酵母菌GS115。
又一方面,本发明提供了包含前述的胰岛素样生长因子突变体IGF1M的细胞培养物。
又一方面,本发明提供了一种提取物,所述的提取物是从前述的细胞培养物中提取到的;所述的提取物中含有IGF1M。
具体地,所述的提取物为细胞培养上清、细胞裂解液和/或细胞外泌体。
又一方面,本发明提供了一种制备IGF1M的方法,包括以下步骤:
(1)用前述的重组载体转化宿主细胞,得重组菌株;
(2)培养重组菌株,诱导类胰岛素因子突变体表达;
(3)回收并纯化所表达的IGF1M。
具体地,所述的细胞可以是用于表达蛋白的基因工程细胞,包括但不限于:植物细胞、动物细胞、细菌、酵母。
进一步具体地,所述的细胞可以是工程菌。
优选地,为大肠杆菌、酵母菌、芽孢杆菌或乳酸杆菌。
进一步优选地,毕赤酵母菌GS115。
又一方面,本发明提供了包含前述的胰岛素样生长因子突变体IGF1M或细胞培养物或提取物的饲料。
又一方面,本发明提供了前述的饲料在提高动物生产性能中的应用。
具体地,所述的动物生产性能包括但不限于:产肉和/或产蛋。
本发明所取得的技术效果:
(1)重组胰岛素样生长因子突变体IGF1M产量高,产量达到了130 mg/L左右。
(2)本发明的重组胰岛素样生长因子突变IGF1M与胰岛素样生长因子受体I的结合能力强。
(3)本发明的重组胰岛素样生长因子突变体IGF1M相比于野生型的胰岛素样生长因子细胞增殖的促进能力得到了显著提升。
附图说明
图1为重组胰岛素样生长因子突变体IGF1M蛋白含量测定。
图2为重组胰岛素样生长因子突变体IGF1M酶联免疫分析图。
图3为重组野生型胰岛素样生长因子及其突变体的促细胞增殖实验。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
试验材料和试剂
1、菌株及载体:毕赤酵母表达载体pPIC9K及菌株GS115购自Invitrogen公司。
2、酶类及其它生化试剂:内切酶购自TaKaRa公司,连接酶购自Invitrogen公司。胰岛素样生长因子检测ELISA试剂盒购自Dogesce公司(货号:DG60062C-96T),其它都为国产试剂(均可从普通生化试剂公司购买得到)。
3、培养基:
(1)大肠杆菌培养基LB(100mL) :1g蛋白胨、0.5g酵母提取物、1g NaCl,pH7.0。
(2)BMGY培养基(100mL):1g酵母提取物,2g蛋白胨,0.3g磷酸氢二钾,1.18g磷酸二氢钾,1.34gYNB,0.00004g 生物素,1g甘油。
(3)BMMY培养基:除以1%甲醇代替甘油,其余成分均与BMGY相同,pH4.0。
说明:以下实施例中未作具体说明的分子生物学实验方法,均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行,或者按照试剂盒和产品说明书进行。
实施例1 家燕(Hirundo rustica)胰岛素样生长因子突变体的获得
本发明的类胰岛素因子突变体IGF1M来源于家燕(Hirundo rustica)的胰岛素样生长因子,相对于家燕(Hirundo rustica)的野生型胰岛素样生长因子IGF1的序列(SEQ IDNO.5)发生了3个位点的突变,突变部位为E9G,F16I,C18L。胰岛素样生长因子突变体IGF1M的氨基酸序列如SEQ ID NO. 1所示,对应的核苷酸序列如SEQ ID NO. 3所示;序列SEQ IDNO. 1的N段连接信号肽,信号肽的序列如SEQ ID NO. 2所示,所对应的核苷酸序列如SEQID NO. 4所示。
SEQ ID NO. 1序列如下所示:
GPETLCGAGLVDALQIVLGDRGFYFSKPTGYGSSSRRLHHKGIVDECCFQSCDLRRLEMYCAPIKPPKSA
SEQ ID NO. 2序列如下所示:
MAIPRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLEEAEAEAEPKFINTTIASIAAKEEGVSLEKREAEA
SEQ ID NO.3序列如下所示:
ggcccagaaacactgtgtggtgctgagctggttgatgctcttcagttcgtatgtggagacagaggcttctacttcagtaagcctacagggtatggatccagcagtagacgcttacaccacaagggaatagtggatgagtgctgcttccagagttgtgacctgaggaggctggagatgtactgtgctccaataaagccacctaaatctgca
SEQ ID NO.4序列如下所示:
Atggctattccaagattcccatctatcttcactgctgttttgttcgctgcttcctccgctttggctgctccagtcaacactactaccgaggacgaaactgctcaaattccagctgaggctgtcatcggttactctgacctggagggtgacttcgacgttgctgtcttgccattctccaactccaccaacaacggtttgttggaggaggctgaagctgaagctgaacctaaattcatcaacactactatcgcttctatcgctgctaaggaggagggtgtttccctcgagaaaagagaggctgaagct
SEQ ID NO.5序列如下所示:
GPETLCGAELVDALQFVCGDRGFYFSKPTGYGSSSRRLHHKGIVDECCFQSCDLRRLEMYCAPIKPPKSA
实施例2家燕(Hirundo rustica)胰岛素样生长因子突变体IGF1M基因的克隆
胰岛素样生长因子突变体IGF1M的基因由华大基因进行合成。
以合成的IGF1M的DNA(SEQ ID NO.3)为模板进行PCR扩增,所用突变引物如表1所示,PCR反应参数为:94℃变性5 min;然后94℃变性30 sec,45℃退火30 sec,72℃延伸1min,30个循环后72℃保温10 min。得到一段223 bp片段,将该片段回收后与PM19-T载体进行连接后转化至大肠杆菌DH5α中将获得的阳性克隆进行培养后提取质粒送北京睿博兴科生物技术有限公司测序。
表1. IGF1突变体特异性引物
实施例3 重组胰岛素样生长因子突变体IGF1M重组菌的制备
将表达载体pPIC9K进行双酶切(EcoR I+Xho I),同时将编码类胰岛素因子的基因igf1m双酶切(EcoR I+Xho I),切出编码成熟胰岛素样生长因子的基因片段与表达载体pPIC9K连接,优选为将本发明的胰岛素样生长因子突变体的基因igf1m插入到质粒pPIC9K上的XhoI和EcoRI限制性酶切位点之间,使该核苷酸序列位于AOX1启动子的下游并受其调控,获得含有胰岛素样生长因子突变体的基因igf1m的重组质粒pPIC9K-igf1m并转化毕赤酵母GS115,获得重组毕赤酵母菌株GS115/igf1m。
实施例4 重组胰岛素样生长因子突变体IGF1M重组菌的筛选
取含有重组质粒的GS115菌株单克隆于48孔板接种于900μL BMGY培养液中,30℃、750 rpm振荡培养48h后,离心收集菌体。然后于600μL BMMY培养基重悬,30℃、750 rpm振荡培养。诱导72h后,离心收集上清,对上清中蛋白含量进行初步测定后筛选出产量最高的菌株进行后续培养。
实施例5重组胰岛素样生长因子变体IGF1M的制备
将实施例4获得的高表达菌株单克隆接种于300 mL BMGY培养液中,30℃ 200rpm振荡培养48h后,离心收集菌体。然后于200 mL BMMY培养基重悬,30℃ 200 rpm振荡培养。诱导72h后,离心收集上清。胰岛素样生长因子突变体IGF1M位于发酵液上清中。
实施例6 重组胰岛素样生长因子变体IGF1M蛋白含量测定
蛋白含量测定使用考马斯亮蓝G-250进行测定,首先使用牛血清白蛋白(康为世纪厂家,货号CW0014S)为标准品制备标准曲线,后将样品进行适当稀释后取1 mL稀释后样品加入5 mL考马斯亮蓝染色液,混匀后室温放置5min测定595nm处吸光值,再将吸光值代入标准曲线计算得到蛋白含量如图1所示。经测定,重组胰岛素样生长因子突变体IGF1M在发酵液上清中得到了表达,产量达到了130 mg/L左右,与野生型胰岛素样生长因子IGF1产量接近。
实施例7重组胰岛素样生长因子突变体IGF1M的ELISA分析
依据Dogesce试剂公司购买的胰岛素样生长因子 ELISA试剂盒说明书进行类胰岛素突变体的酶联免疫分析,将于发酵液中纯化得到的IGF1M使用样品稀释液稀释适当倍数后进行酶联免疫分析。挑取转化后的三株菌株IGF1M-1,IGF1M-2,IGF1M-3分别进行发酵,结果表明(图2),在酵母发酵液上清中成功表达了胰岛素因子的突变体IGF1M,表达量为80mg左右,野生型胰岛素样生长因子IGF1表达量为75mg左右。
实施例8重组胰岛素样生长因子突变体IGF1M的细胞增殖实验
将重组类胰岛素因子突变体IGF1M与野生型胰岛素样生长因子IGF1分别进行细胞增殖效果比较。
取第3代鸡胚额骨成骨细胞以3×104个/mL 接种100 µL于96 孔板培养,分别培养72 h后,加入重组胰岛素样生长因子突变体IGF1M蛋白。试验各分8组,每组6个重复,各组中胰岛素样生长因子突变体IGF1M蛋白分别加入:0(对照组)、10 ng、20 ng、40 ng、80 ng。培养 24 h、72 h 后,换无血清培养液,并于每孔加入30 µL(2 mg/mL)MTT,4 h 后吸尽培养基,各加 150 µL DMSO,振荡10 min,分别于UV570 测光吸收值如图3所示。结果表明与野生型胰岛素样生长因子IGF1相比,突变后的IGF1M能够显著提高对鸡胚额骨细胞增殖的促进作用,且随浓度的升高促进作用更强。
对比例
为了进一步证明本发明的胰岛素样生长因子突变体IGF1M的细胞增殖效果,本发明制备了另一种胰岛素样生长因子突变体IGFM2,所述的IGFM2相对于家燕(Hirundo rustica)的野生型胰岛素样生长因子IGF1的序列发生了3个位点的突变,突变部位为E9D,F16T,C18S 。参照实施例8进行实验,胰岛素样生长因子突变体IGFM2的鸡胚额骨细胞增殖结果见图3,结果表明IGF1M对鸡胚额骨细胞增殖的促进作用显著高于IGFM2。
Claims (8)
1.一种胰岛素样生长因子突变体IGF1M,其特征在于,所述的胰岛素样生长因子突变体IGF1M的氨基酸序列如SEQ ID NO. 1所示。
2.根据权利要求1所述的胰岛素样生长因子突变体IGF1M,其特征在于,所述的SEQ IDNO. 1的N端连接信号肽序列SEQ ID NO.2。
3.编码权利要求1-2任一项所述的胰岛素样生长因子突变体IGF1M的核酸。
4.根据权利要求3所述的核酸,其特征在于,所述核酸的序列为SEQ ID NO.3或与SEQID NO.3具有90%以上同源性的序列。
5.一种包括权利要求3或4所述的核酸的重组载体。
6.根据权利要求5所述的重组载体,其特征在于,所述的载体是质粒、噬菌体和病毒中的一种。
7.包括权利要求5-6任一项所述的重组载体的细胞。
8.包含权利要求1或2所述的胰岛素样生长因子突变体IGF1M的细胞培养物或提取物。
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