CN110950945B - 一种蝠蛾昆虫的抗冻蛋白及其编码基因和应用 - Google Patents
一种蝠蛾昆虫的抗冻蛋白及其编码基因和应用 Download PDFInfo
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Abstract
本发明公开了一种蝠蛾昆虫的抗冻蛋白及其编码基因和应用,属于基因工程技术领域,蝠蛾昆虫的抗冻蛋白为由SEQ ID NO:1所示的氨基酸序列组成的蛋白质,或在SEQ ID NO:1所示的氨基酸序列中经取代、缺失或插入一个或几个氨基酸序列所获得的具有同等活性的蛋白质。本发明涉及的YPK_AFP1001抗真菌基因及蛋白具有更高,更广谱的抗真菌活性。本发明的HaAFP71647蛋白的氨基酸序列,有一定的规律性,只是和以往报道的昆虫抗冻蛋白为TCT开头的以12个氨基酸为一个循环不用,该蛋白以8个氨基酸为一个循环单位,且大多为TQT,THT,TPT,TRT等为主,以往未有报道。跟以往的抗冻蛋白没有同源性。
Description
技术领域
本发明属于基因工程技术领域,涉及一种蝠蛾昆虫的抗冻蛋白及其编码基因和应用。
背景技术
抗冻蛋白AFPs(antifreeze proteins)是具有提高生物抗冻能力的蛋白质类化合物的总称。这些蛋白质能结合到冰晶的表面,因此又被称为冰结合蛋白IBPs(ice-bindingproteins)或者冰结构蛋白ISPs(ice-structuring proteins)。抗冻蛋白主要有热滞活性(thermal hysteresis activity)和修饰冰晶形态活性(ice crystal habitmodification)等。
抗冻蛋白由于有热滞活性和修饰冰晶形态活性等特性。可以广泛地应用于精子、卵细胞、胚胎、各种食品和肉制品等的低温保存。美国的FDA已批准可以添加到食品中,中国也已获批可以在食品中添加。但是,以往的研究或者抗冻蛋白热滞活性低,或者有专利保护限制了其使用。
发明内容
本发明的目的在于提供一种蝠蛾昆虫的抗冻蛋白及其编码基因和应用。本发明主要集中在新疆高纬度、高海拔的山区,以新疆特有种昆虫——阿尔泰蝠蛾(Hepialusaltaicola)为研究对象,从其体内获取的新的抗冻蛋白,与以往报道的抗冻蛋白没有同源性。之所以选择阿尔泰蝠蛾为研究对象,因为该昆虫为新疆虫草菌(Ophiocordycepsgracilis)的寄主昆虫,新疆虫草是我国新疆阿尔泰山上特有的虫草资源,为新疆传统哈萨克民族药;关键是发现该昆虫的幼虫在体内结冰后还可以存活,因为我们在测完该昆虫的过冷却点(提示此时昆虫已结冰)后,该昆虫的存活率还可以达到100%(n>100),我们据此很感兴趣它体内的抗冻蛋白是怎样的,怎么能让其结冰后而不死亡。我们通过对零下低温诱导后的阿尔泰蝠蛾进行了转录组测序,获得了大量的转录基因,虽然没有注释为抗冻蛋白的基因,但是根据我们以往的研究经验,根据结构规律性分析,我们从中筛选了感兴趣的Haafp71647号基因,通过密码子优化,构建到pET32a表达质粒上,重组质粒pET32a-Haafp71647如图1所示,通过大肠杆菌(Escherichia coli)进行原核表达和纯化后发现该蛋白具有热滞活性,热滞值为0.3℃;并且具有修饰冰晶形态的功能。
其具体技术方案为:
一种蝠蛾昆虫的抗冻蛋白,由SEQ ID NO:1所示的氨基酸序列组成的蛋白质,或在SEQ ID NO:1所示的氨基酸序列中经取代、缺失或插入一个或几个氨基酸序列所获得的具有同等活性的蛋白质。
一种蝠蛾昆虫的抗冻蛋白的编码基因,其核苷酸序列如SEQ ID NO:2所示。
本发明所述蝠蛾昆虫的抗冻蛋白在生物医学样品低温保存过程中的应用。
本发明所述蝠蛾昆虫的抗冻蛋白在食品生产过程中的应用。
一种本发明所述蝠蛾昆虫的抗冻蛋白的诱导与纯化方法,包括蛋白的诱导表达和检测和重组蛋白的纯化,具体步骤如下:
(1)、蛋白的诱导表达和检测方法
1).将测序正确的重组质粒大肠杆菌BL21(DE3)阳性克隆菌株(20ul)接种于20mLLB液体培养基中(含100mg/mL氨苄青霉素40ul),37℃震荡培养过夜(转速170rpm/min)。
2).次日将过夜培养菌液按1:100的比例,转接含氨苄抗性的培养基。一般转接6mL过夜培养物于600mL(含100mg/mL氨苄青霉素600μl)LB液体培养基中,37℃震荡培养至OD600=0.6-0.8(约需3-4h)。
3).取出3ml菌液移至4mLEP管中,作为诱导前(不加诱导剂)对照,12000rpm/min离心1min,弃上清、留沉淀菌体;再加入3mlPBS重悬菌体后,2000rpm/min离心1min,弃上清、留沉淀菌体,标记后-20℃冻存备用。
4).剩余培养基中加入IPTG(1M/L)至终浓度1mmol/L,20℃继续诱导培养8h(转速130rpm/min)。取出3ml菌液移至4mLEP管中,12000rpm/L离心1min,弃上清、留沉淀菌体再加入3mlPBS重悬菌体后,2000rpm/min离心1min,弃上清、留沉淀菌体,标记后-20℃冻存备用。HaAFP71647蛋白经IPTG诱导前后的表达图谱如图2所示。
5).于上述样品中各加入40μl PBS后混匀,再分别加入10μl蛋白上样缓冲液(5XLoading Buffer)继续混匀。
6).将上述样品一起煮沸10min,12000rpm/min离心3min,取10-15ul上清用于SDS-PAGE分析。
(2)、重组蛋白的纯化方法
1).将按照上述蛋白诱导表达的方法得到的菌体,加入裂解液(含20mM磷酸二氢钠,500mM NaCl,20mM咪唑,pH7.4),涡旋振荡重悬菌体后,在冰浴条件下超声破碎(超5s停5s,功率50%,持续1h),离心30min(4℃,12000rpm/min)去除细胞碎片和非可溶蛋白,收集上清备用。
2).层析柱的准备:将Ni-Agarose Resin填料混匀后加入层析柱,室温静置10min,待凝胶与溶液分层后,把底部的出口打开,让乙醇溶液通过重力缓慢流出。向装填好的柱中加入5倍柱体积的去离子水将乙醇冲洗干净,再用10倍柱体积Binding Buffer平衡柱子。
3).上清样品通过0.45μm滤膜,以10-15mL/h(10倍住体积)流速上Ni-AgaroseResin柱。
4).洗脱杂蛋白:用含有20mM咪唑的PBS,以10-15mL/h(15倍住体积)流速洗柱,洗去杂蛋白。
5).洗脱目标蛋白:用含有500mM咪唑的PBS,以10-15mL/h(15倍住体积)流速洗柱,洗脱目的蛋白。
6).将洗脱的目的蛋白溶液,装入合适大小的透析袋中,用PBS透析;用超滤管超滤后,用于蛋白活性分析。
与现有技术相比,本发明的有益效果:
本发明的HaAFP71647蛋白的氨基酸序列,有一定的规律性,只是和以往报道的昆虫抗冻蛋白为TCT开头的以12个氨基酸为一个循环不用,该蛋白以8个氨基酸为一个循环单位,且大多为TQT,THT,TPT,TRT等为主,以往未有报道。跟以往的抗冻蛋白没有同源性。
附图说明
图1是重组质粒pET32a-Haafp71647示意图;
图2是HaAFP71647蛋白经IPTG诱导前后的表达图谱;
图3是没有抗冻蛋白时,溶液的冰晶随温度下降的生长状态图,为圆形,(400×);
图4是第一次在有HaAFP71647蛋白存在时,冰晶的生长状态图,(400×);
图5是第二次在有HaAFP71647蛋白存在时,冰晶的生长状态图,(400×)。
具体实施方式
下面结合具体附图和实施方案对本发明的技术方案作进一步详细地说明。
一、融合蛋白修饰冰晶作用的观测步骤
用纳升渗透仪Nanolitre Osmometer(Otago Osmometers Ltd,New Zealand)在蔡司显微镜AX10(Zeiss,German)下,观察抗冻蛋白对冰晶的形态修饰作用和抑制重结晶作用。具体方法是,用0.50μL的微量进样器,将待观测样品(0.1mM,pH=7.4)加入纳升渗透压仪控温台上的样品孔中,然后将控温台置于显微镜的载物台上先快速降温到-20℃~-30℃将样品快速冷冻,然后缓慢升高温度,观察冰开始熔化的温度,直至视野中只有单个(或少数几个)冰晶存在,再更加缓慢地升温,观测冰晶熔化时的形态变化;或者看到单冰晶后,缓慢地降温,观测冰晶生长时的情况。
二、融合蛋白的热滞活性测定步骤
用差示扫描量热仪DSC 200F3(Maia,German)测定抗冻蛋白的热滞活性。具体做法是,先用移液器量取约1μL的抗冻蛋白溶液,添加在铝盘中央,并加盖密封,以密封后的空白铝盘做参照,分别放置在样品池中的两个测量台上。首先以1℃/min的降温速率降温至-30℃,然后以1℃/min的升温速率升至25℃,再以1℃/min的降温速度降至-30℃,测量样品的熔化温度(Tm)。随后,根据样品的熔化温度(Tm),设定样品的保留温度(Th)(样品体系为固液混合物的状态时的温度,保留温度略低于熔化温度),并将样品由-30℃以1℃/min的速率升温至保留温度(Th),停留2min,再将温度以1℃/min的降温速度降至-30℃,测定样品开始结晶的温度(T0)。之后,重复以上测试过程,并分别在不同的保留温度下停留2min,以测定在不同的保留温度下,样品体系开始冻结的温度(T0)。保持温度(Th)和重结冰温度(To)之差即为THA值,即THA=Th-To。
图3中,没有抗冻蛋白时(pET32a质粒的空载蛋白TrxA),溶液的冰晶随温度下降的生长图形,为圆形,(400×);
图4中,在有HaAFP71647蛋白存在时(TrxA-HaAFP71647蛋白),冰晶的生长为多边形,且有分层现象,正是由于该蛋白能结合在冰晶表面限制冰晶自由生长所致,(400×);
图5中,再一次附图显示在有HaAFP71647蛋白存在时,冰晶的生长为多边形,且有分层生长现象(见中央和左下角的冰晶),正是由于该蛋白能结合在冰晶表面限制冰晶自由生长所致,(400×)。
以上所述,仅为本发明较佳的具体实施方式,本发明的保护范围不限于此,本领域的技术人员可知蝙蝠蛾科蝠蛾属的一个物种,除了本发明采用的阿尔泰蝠蛾外,比如白蛾蝠蛾、虫草蝠蛾、白马蝠蛾、美丽蝠蛾、东隅蝠蛾、贡嘎蝠蛾、甲郎蝠蛾、金沙蝠蛾、康定蝠蛾、康姬蝠蛾、察隅蝠蛾、察里蝠蛾等,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
序列表
<110> 石河子大学
<120> 一种蝠蛾昆虫的抗冻蛋白及其编码基因和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 94
<212> PRT
<213> 阿尔泰蝠蛾(Hepialus altaicola )
<400> 1
Met His Ser Arg Thr Arg Thr Leu Ala Leu Thr His Ala Arg Thr His
1 5 10 15
Ser Arg Thr His Ala Arg Glu Arg Ser Gly Pro Gly Ala Gly Ile Gly
20 25 30
Gln Asn Ala Arg Thr His Thr His Ile His Lys Arg Thr Gln Thr His
35 40 45
Thr His Val Arg Thr His Ile His Thr His Thr Tyr Thr Arg Ala Tyr
50 55 60
Thr Tyr Lys Tyr Thr Pro Thr His Thr Asp Asn Tyr Thr Phe Thr His
65 70 75 80
Thr Ala Asn Pro Phe His Ser Tyr Pro Val Arg Ala Thr Cys
85 90
<210> 2
<211> 285
<212> DNA
<213> 阿尔泰蝠蛾(Hepialus altaicola )
<400> 2
atgcactcac gcacacgcac actggcactc acgcacgcac gcacgcactc acgcactcac 60
gcgcgagagc gctcgggccc gggcgcgggc ataggccaga atgcgcgcac gcacactcac 120
atacacaagc gcacgcaaac acacacacac gtacgcacgc atatacacac acacacttac 180
acacgcgcgt acacgtataa atatacacct acacacaccg ataactacac atttacacac 240
acagccaatc catttcattc gtaccccgtc cgtgcaactt gttaa 285
Claims (4)
1.一种蝠蛾昆虫的抗冻蛋白,其特征在于,由SEQ ID NO:1所示的氨基酸序列组成的蛋白质。
2. 一种权利要求1所述的蝠蛾昆虫的抗冻蛋白的编码基因,其特征在于,其核苷酸序列如SEQ ID NO:2所示。
3.一种权利要求1所述蝠蛾昆虫的抗冻蛋白在生物医学样品低温保存过程中的应用。
4.一种权利要求1所述蝠蛾昆虫的抗冻蛋白在食品生产过程中的应用。
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CN102177872A (zh) * | 2011-04-13 | 2011-09-14 | 舟山市普陀新展望水产饲料有限公司 | 昆虫抗冻蛋白源的获取方法 |
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