CN110317821A - 一种融合蛋白thg及其应用 - Google Patents
一种融合蛋白thg及其应用 Download PDFInfo
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- CN110317821A CN110317821A CN201910520601.8A CN201910520601A CN110317821A CN 110317821 A CN110317821 A CN 110317821A CN 201910520601 A CN201910520601 A CN 201910520601A CN 110317821 A CN110317821 A CN 110317821A
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Abstract
发明公开了一种渔用口服或浸泡型重组融合蛋白THG及其制备方法和应用,利用基因工程技术将透皮增强肽基因序列(TDZ)、渔用生长激素基因(HG)和细胞穿膜肽基因序列(TAT)进行柔性结合,获得THG融合基因,转化大肠杆菌,筛选获得了一株可生产THG融合蛋白的基因工程菌。该菌株为大肠杆菌BL21(DE3)hsdS gal pET‑28a‑THG;CCTCC NO:M 2018014,该培养物已于2018年01月09日由中国典型培养物保藏中心收到,并登记入册。将该基因工程菌株进行诱导表达和THG融合蛋白的纯化,获得THG融合蛋白。该融合蛋白能降低机体生物屏障,能有效地促进细胞同化作用,加快鱼类生长,可减少鱼类养殖中的粪便排放,环境和生态效益好,在渔业生产上具体明显的经济价值和应用前景。
Description
技术领域
本发明涉及基因生物工程技术领域,具体涉及一种重组融合蛋白THG的菌株,还涉及一种重组融合蛋白THG的序列信息,还涉及一种能表达重组融合蛋白THG的制备方法,以及一种重组融合蛋白THG在促进鱼类生长、提高鱼类饲料利用率等方面中应用。
背景技术
鱼类生长激素(growth hormone,GH)是由鱼脑垂体远端的嗜酸性细胞分泌与合成的一类单链多肽。它在鱼的生长、能量的固定、性腺的发育等方面都具有重要的调节作用,也是水产养殖中一直备受研究者关注的领域。鱼类生长激素能够促进鱼类生长发育,提高饵料利用率,减少饵料的损耗,减少化学类药物的使用,降低对水环境的污染程度,对促进水产业的发展具有重要的经济和社会意义(王 伟,汪亚平,朱作言,人工合成草鱼生长激素cDNA在大肠杆菌中的表达,遗传学报,2001,28(4):306~312)。生长激素在鱼类之间具有很高的保守性,不同种鱼的 GH 都可促进某一种鱼类的生长,表明一种外源生长激素可以对多种鱼类起作用。早期的研究发现,将动物的脑垂体匀浆后,加入饵料中,投喂给鱼,鱼体生长加快,似乎鱼体的肠道能够吸收具有生物活性的生长激素,经研究证实,鱼体的后肠上皮细胞,通过泡饮作用,可以吸收完整的大分子蛋白,具有生物学活性的生长激素可以进入鱼体血液中,从而促进鱼体生长(王 伟,孙永华,汪亚平,朱作言,草鱼生长激素在毕赤酵母中的高效分泌表达,遗传学报,2003,30 (4):301~306)。多肽生长激素具有调节和促进生长的作用,是脊椎动物(包括鱼类)体内固有的成分,并可在动物体内自动降解而不影响人类食用。现行的鱼用生长激素,常用注射的方式对鱼类有效,口服或浸泡等方式,效果有限,也就鲜有报道。原因在于,生长激素蛋白分子缺乏穿膜机制。
透皮增强肽,也称为蛋白转导域,是目前最强、最快的跨膜转运分子,具有水性、低裂解性并通过非吞噬作用进入各种细胞膜等特点,具有很强的跨膜转运能力,能够携带比其相对分子质量大100 倍的外源性大分子进入细胞。同时,现在研究最多、应用范围最广的细胞穿膜肽(Trans-activating transcriptional activator, Tat) Tat蛋白中存在3个功能结构域,分别是:位于N一端的酸性区,主要起反式激活的功能;位于第22-37位氨基酸之间的DNA结合区,该区域富含半胱氨酸;位于第47-60位氨基酸之间的碱性区,是主要的蛋白转导结构域,参与Tat蛋白的细胞内化作用。Schwarze发现位于Tat蛋白中第47-57位之间的11个氨基酸YGRKKRRQRRR 不仅能够独立穿过细胞膜,而且其穿膜效率比全长的Tat蛋白还要高,这段多肽即是TAT 穿膜肽。本专利就是将透皮增强肽、鱼用生长激素和分子穿膜肽等三个基因进行柔性融合,获得有穿膜活性的生长激素蛋白,解决了生长激素口服,鱼体不能吸收,效果差的问题。而在这方面,尤其是在多基因片段之间柔性融合方面,更未见报道。
发明内容
本发明的主要目的是提供了一种THG融合蛋白,其序列为SEQ ID NO.2所示。本发明首次将透皮增强肽(TDS)、鱼生长激素(GH)、细胞穿膜肽(TAT)与融合表达,获得的融合蛋白可以顺利穿过鱼体鳃部细胞和肠道膜细胞,进入鱼体循环***中,可直接发挥其促生长作用。
本发明的另一个目的在于提供了一种THG融合蛋白的制备方法,大肠杆菌Escherichia coli BL21(DE3)pET28a-THG,工程菌大量表达该融合蛋白。表达量大且可溶,易于工业化生产,成本低,安全性高。
本发明的另一个目的是提供了一种大肠杆菌基因工程菌株,Escherichia coliBL21(DE3) hsdS gal pET28a-THG,大肠杆菌BL21(DE3) hsdS gal pET28a-THG,CCTCC NO:M2018014。该菌株可以表达含透皮增强肽TDS、鱼生长激素(GH)、细胞穿膜肽TAT等融合蛋白THG,使其能顺利穿过细胞膜进入胞内产生作用,该菌株能大量表达THG蛋白,且操作简便快捷,可更好的应用于工业化生产中,为制备口服促生长制品提供良好的基础。
本发明还有一个目的是提供了一种THG表达蛋白在促进草鱼生长中的应用,通过选取大小不同的幼鱼,用一定剂量的融合蛋白分别以剂量对其口服喂食,观察其促生长效果的差异性,可得出最优化的饲养方式。
为了实现上述的目的,本发明采用以下技术措施。
A.TDS-GH-TAT融合基因的制备。
人工合成TDS基因、GH基因和TAT序列,利用融合PCR技术,实现三段基因序列的柔性融合。先是TDS基因和GH基因柔性融合(图1)再是;基因和GH基因,与TAT序列的柔性融合(图2)。
B.TDS-GH-TAT融合基因表达载体的构建。
用BamH1对TDS-GH-TAT序列和质粒pET28a(+)进行酶切,并用T4连接酶连接,得到重组质粒pET28a-THG(图3)。
C.大肠杆菌基因工程菌的制备。
将质粒pET28a-THG转化到表达菌株Escherichia coli BL21 (DE3)(由Novagen公司购得,实验室保种)中,PCR鉴定筛选出阳性转化子,所得阳性克隆即为本发明涉及的能够表达THG的大肠杆菌基因工程菌株Escherichia coli BL21(DE3) hsdS gal pET28a-THG。
申请人于2018年1月9日将该菌送至中国典型培养物保藏中心进行保藏,保藏地址:中国武汉,武汉大学,分类命名:大肠杆菌BL21(DE3) hsdS gal pET28a-THG, CCTCCNO:M2018014,通过上述方法获得了一种大肠杆菌基因工程菌株Escherichia coli BL21(DE3)pET28a-THG。
D.基因工程融合蛋白的制备。
将上述基因工程菌Escherichia coli BL21 (DE3) pET28a-THG转接到LB培养基中,经IPTG诱导,融合蛋白THG以包涵体的形式表达,且表达效率和纯化的蛋白产率高,如图4,图5所示,其氨基酸序列为SEQ ID NO.2 所示。
E.一种THG融合蛋白促进草鱼生长中的应用,其应用过程是。
将大肠杆菌基因工程菌株发酵所得目的蛋白纯化并溶于PBS(pH 7.4,含1.0-2.0mM的L-精氨酸)中,并对蛋白进行定量检测,得到目的蛋白含量为1.0 mg/mL的THG融合蛋白,对草鱼生长和免疫机理的检测是饲养5周后,对每一周每一组的草鱼进行测量体重、体长,根据测算的数据,求算出草鱼的增长率、增重率、肥满度和生存率,以此为依据对草鱼生长情况进行分析,实验结果表明,THG蛋白,无论是浸泡还是口服,对鱼类生长均有较好的促进作用(图6,图7)。
与现有技术相比,本发明具有以下优点。
A.将透皮增强肽TDS、鱼生长激素(GH)和细胞穿膜肽TAT进行柔性融合,获得融合蛋白,活性强,穿膜效果好,可以顺利穿过鱼体鳃部和肠道膜细胞,进入鱼体循环***中发挥其促生长作用,比单纯的TAT-GH融合效果要强。
B.选取了大肠杆菌作为表达***,比酵母表达***或其他植物***,其操作简单,生长周期短,产量高,成本低,适用于大规模的工业化生产中。同时,本研究中所用的融合基因序列已经过了优化,表达效率显著高于未改造前。
C. 本方法生产的口服型和浸泡型渔用生长激素,给药方式比传统的注射方法,省事省力。
附图说明。
图1为TDS与GH融合图。
图1中:M,DL2000分子标准;1,TDS序列片段;2,GH基因片段;3,TDS-GH融合片段。
图2为TDS与GH,和TAT序列的融合图。
图2中,M,DL2000分子标准;1,TDS-GH融合片段;2,TAT序列片段; 3,THG(TDS-GH-TAT)基因片段。
图3为融合表达载体pET28a-THG的构建。
在图3中,M,DL5000分子标准;1,THG基因片段;2,pET28a-THG质粒经BamHI酶切。
图4为pET28a-THG原核诱导融合示意图。
在图4中,M, 蛋白分子标准。1, 诱导前的总蛋白;2, 诱导后的总蛋白;3,菌体超声处理后上清中的总蛋白;4, 菌体超声处理后下部分的蛋白。
图5为Western blot验证纯化后的THG融合蛋白。
在图5中,M, 蛋白分子标准;1,纯化的THG融合蛋白经Western blot检测。
图6为THG融合蛋白的口服拌料投喂后,草鱼的增重率。
图7为THG融合蛋白的浸泡草鱼后,草鱼的增重率情况。
具体实施方式
本实验中所涉及的分子生物学方法为常规方法,为本领域人员所熟悉。本发明中未详细阐述的请参见《分子克隆实验指南》 J.萨姆布鲁克,D.W. 拉塞尔等主编。
实施例一:融合基因THG的制备。
1) TAT核苷酸片段的生成。
上游引物TDF1一条,下游引物TDR一条,一次聚合合成。
TDF1:GGATCCATGGCCTGCAGCTCGAAGAAATCCAAGCACTGCGGCGGCAGCGGCAGCGGC。
TDR:GATGACTGCGTTGTTGAAGAGCCGCTGGTTCTCTGACATGCTGCCGCTGCCGCTGCC。
先将两条引物分别配成10 μM/L混合后,95 ℃加热10 min后,室温冷却l.0 h后,生成TDS核苷酸片段,4 ℃保存备用。其生成的细胞穿膜肽TDS及柔性联系序列为:ggatccatggcctgcagctcgaagaaatccaagcactgcggcggcag。
2)TAT与TDS和GH的柔性融合。
以TAT和人工合成的TDS和GH为模板,经PCR扩增得THG融合基因,在引物中加入BamH 1酶切位点,以利于后续的重组质粒构建工作,设计的特异引物序列如下。
TATF:CTGGGCGGCTCCGGCAGCGGCGGCTCCGGCAGCGGCGGCTCCGGCAGCTACGCGCGCGC。
TATR:GCTCATCTTTCTCAGACGCGCGGGCCTGGCGGGCCGCGGCGCGCGCGTAGCTGCC。
PCR反应的总体积为50 μL,PCR扩增条件为:热启动(94℃,5min),继续进行35个循环,每一循环的程序为94℃,30 sec;56 ℃,30sec;72 ℃,30 sec,最后一个延伸反应(72℃,5.0 min)取扩增产物5.0 μL在1.2%琼脂糖凝胶上电泳,EB染色后,紫外灯下观察,拍照,扩增产物用PCR纯化试剂盒纯化。
实施例二:THG融合基因的表达载体构建和工程菌株的获得。
用BamH1酶切 pET28a并进行末端磷酸化处理,纯化并回收 pET28a载体,同时用BamH1酶切 THG片段,将目的片段THG与开环pET28a载体,在16 ℃连接过夜,转化大肠杆菌TP10感受态细胞;其步骤和方法,按照《分子克隆实验指南》进行;选取5个克隆进行测序,鉴定方向和序列信息正确的菌株,提取质粒,并和Escherichia coli BL21(DE3)感受态细胞冰浴30 min。42 ℃水浴中热激90sec,速移至冰水中2-3 min,无菌下,加入 600 μ1液体LB培养基,37 ℃,100 rpm轻摇,温浴45 min使细胞复苏。取上述菌液150u l ,用无菌三角玻棒均匀涂布在50 μ g/ml Kan的LB平板上,正向放置1-2 h,直至液体被全部吸收,倒置于37℃培养箱中培养过夜。选取5个克隆进行测序,鉴定THG***到pET-28a载体中的方向和序列信息,分析编码框的完整性。由此法可得到Escherichia coli BL21(DE3)hsdS galpET28a-THG菌落。
实施例三:基因工程融合蛋白THG的诱导表达和纯化。
重组基因工程菌株 Escherichia coli BL21(DE3) pET-28a -THG的单菌落接种到10 ml新鲜液体LB(含终浓度为50 ug/ml 卡那霉素)培养基中。37℃ 150 rpm 摇床培养过夜。无菌条件下,从培养过夜的菌中,取出0.5 ml 菌液接种到50 ml的新鲜液体LB中,37℃培养箱中 200 rpm 摇床培养4.0 h,再从培养液中,取出2.0 ml 菌液接种到200 ml的新鲜液体LB中,37℃,200 rpm 摇床培养4.0 h,取至OD600为0.8时(约4h)加入终浓度为0.5mM IPTG,37℃诱导4h后,离心6000 g, 4℃, 10 min收集菌体,用 10 mM PBS(pH 8.0)重悬超声波破壁,离心收集上清和沉淀并用SDS-PAGE电泳检测目的蛋白的表达。以优化后的条件扩大培养,超声波破壁后,离心12000 g, 4℃, 20 min收集沉淀,并用尿素变性液在4℃冰箱中溶解处理过夜,4℃离心12000 g, 30 min收集上清。融合蛋白THG的纯化按Ni-NTA说明书进行,用SDS-PAGE检测蛋白纯化结果。得到纯化的THG蛋白,其序列为SEQ ID NO.2所示。
实施例四:融合蛋白THG的抗体制备和Western blot鉴定。
纯化的THG融合蛋白与弗氏不完全佐剂等量混合,腹腔注射Bal/C小白鼠5只,间隔2周免疫1次,共三次,最后1次免疫后第7天,从小白鼠眼眶取少量的血,37℃温箱中作用2h后,4℃放置过夜,取上清采用ELISA检测效价。如达到1:10000,就可大量取血,同上制备血清。THG的鼠源抗血清,4℃保存备用。
纯化后的融合蛋白THG,经SDS-PAGE电泳,转膜,利用THG的鼠源抗血清进行Western blot鉴定,证明纯化后的融合蛋白THG是所需要的蛋白,且特异性好。
实施例五:分光光度法测定蛋白含量。
A.试剂准备。
1)将样品用蒸馏水做适当倍数的稀释。
2)取1.5 mL离心管,按照下表的比例分别加入各溶液,其中0号管为空白对照。
溶液管号 | 1 | 2 | 3 | 4 | 5 |
双蒸水 (μL) | 1000 | 900 | 800 | 750 | 700 |
1mg/mL BSA (μL) | 0 | 100 | 200 | 250 | 300 |
BSA浓度(μg/mL) | 0 | 100 | 200 | 250 | 300 |
B.具体步骤。
1)选取酶标板上7个孔,分为标准组和样品组。
标准组:5个孔,每个标准品设置1个重复,各孔分别加入150 μL相应浓度的标准蛋白质溶液;对应管编号为0、1、2、3、4、5。
样品组:剩余2孔,分为2个重复组,同一重复组编号相同。其中编号不同的两孔分别加入浓度不同的150 μL样品稀释液。
2)各酶标孔加入150 μL Bradord工作液,迅速混匀。
3)室温25-30 ℃,反应5 min后,以0号管为空白对照,在酶标仪上测各孔的A595值。
4)以标准组各孔A595平均值为纵坐标,对应的蛋白质浓度为横坐标,在MicrosoftExceL 软件中绘制标准曲线。
5)根据两个相同样品稀释液A595值的平均值,在标准曲线上计算出该样品经过稀释后的蛋白质浓度,选择合适稀释度的样品计算最终的样品蛋白浓度,再由稀释倍数计算原样品蛋白质浓度。
C.最终将目的蛋白溶于PBS(pH 7.4,含1.0—2.0 mM的L-精氨酸)中,并根据标准曲线,将THG融合蛋白浓度调整到1.0 mg/mL。
实施例六:鱼类口服THG蛋白促生长实验。
选取规格整齐的草鱼作为实验对象,先对实验鱼进行训养3周后,随机对鱼进行分组实验。对草鱼生长和免疫机理的检测是饲养5周后,对每一周每一组的草鱼进行测量体重、体长,根据测算的数据,求算出草鱼的增长率、增重率、肥满度和生存率,以此为依据对草鱼生长情况进行分析。实验分组、投喂添加剂量及投喂方式如下。
(1)对照组:A组、D组、F组,投喂普通饲料添加pET28a(载体)蛋白的0.02%。
(2)实验组,B组、C组、E组,THG拌喂组,THG蛋白先和少量饲料颗粒混匀,后再和大量饲料混合,最后,THG蛋白占饲料的0.02%。
A、B、C、D、E、F、G、H等6箱,每箱15尾鱼,充氧。每天上午9点投喂,投喂鱼体总重的3%,每天下午16点30分换水,每次换水量约为总水位的三分之一。每周测量一次鱼的体长和体重,并记录。结果如图6。
实施例七:鱼类浸泡THG融合蛋白后的生长实验。
选取规格整齐的草鱼作为实验对象,先对实验鱼进行训养3周后,随机对鱼进行分组实验。对草鱼生长和免疫机理的检测是饲养5周后,对每一周每一组的草鱼进行测量体重、体长,根据测算的数据,求算出草鱼的增长率、增重率、肥满度和生存率,以此为依据对草鱼生长情况进行分析。实验分组、投喂添加剂量及投喂方式如下。
(1)对照组:A组、D组、F组,浸泡pET28a(载体)蛋白占水体体积量的0.05%,浸泡时间30 min。
(2)实验组,B组、C组、E组,THG浸泡组,THG蛋白占水体体积量的0.05%,浸泡时间30min。
A、B、C、D、E、F、G、H等6箱,每箱15尾鱼,充氧。每天上午9点投喂,投喂鱼体总重的3%,每天下午16点30分换水,每次换水量约为总水位的三分之一。每周测量一次鱼的体长和体重,并记录。结果如图7。
序列表
<110> 鲁东大学
<120> 一种融合蛋白THG及其应用
<141> 2019-06-17
<160> 2
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<213> 口服型草鱼生长激素基因(Escherichia coli)
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ctggctgcag agatgatcaa cgacttcgag gacaacctgt tgcctgagga acgcagacag 180
ctgagcaaga tcttccctct gtccttctgc aactccgact ccatcgaggc gcccactggc 240
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ttcgaggact tctacctgac catgggggag agcagcctca gagagagctt ccgtcttctg 540
gcttgcttca agaaggacat gcacaaggtg gagacctacc tgagggtagc gaactgcagg 600
agatccctgg actcaaactg caccctgggc ggctccggca gcggcggctc cggcagcggc 660
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cta 723
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<212> PRT
<213> 口服型草鱼生长激素基因(Escherichia coli)
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Ile Arg Val Gln His Leu His Gln Leu Ala Ala Glu Met Ile Asn Asp
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Phe Glu Asp Asn Leu Leu Pro Glu Glu Arg Arg Gln Leu Ser Lys Ile
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Phe Pro Leu Ser Phe Cys Asn Ser Asp Ser Ile Glu Ala Pro Thr Gly
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Lys Asp Glu Thr Gln Lys Ser Ser Met Leu Lys Leu Leu Arg Ile Ser
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Phe Arg Leu Ile Glu Ser Trp Glu Phe Pro Ser Gln Thr Leu Ser Gly
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Ala Val Ser Asn Ser Leu Thr Val Gly Asn Pro Asn Gln Ile Thr Glu
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Lys Leu Ala Asp Leu Lys Val Gly Ile Ser Val Leu Ile Lys Gly Cys
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Leu Asp Gly Gln Pro Asn Met Asp Asp Asn Asp Ser Leu Pro Leu Pro
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Leu
Claims (6)
1.一种合成的基因,其序列为SEQ ID NO.1所示,其由723个碱基构成,其中,包含一个独立的ORF阅读框,无内含子。
2.权利要求1所述基因编码的蛋白质,其序列为SEQ ID NO.2所示,由241个氨基酸组成。
3.含有权利要求1所述基因的基因工程菌:Escherichia coli BL21(DE3)hsdS galpET-28a-THG,大肠杆菌 BL21(DE3)hsdS gal pET-28a-THG;CCTCC NO: M2018014,该培养物已于2018年01月09日由中国典型培养物保藏中心收到,并登记入册。
4.权利要求2所述蛋白的制备方法,其步骤主要有菌株的三级培养,诱导和提取融合蛋白,采用含150—200 mM的咪唑溶液洗脱目的蛋白,经离心透析后,目的蛋白溶于PBS(pH7.4,含1.0—2.0 mM的L-精氨酸)中进行蛋白复性,即得一种活性的THG融合蛋白,其序列为SEQ ID NO.2所示。
5.权利要求1所述的基因或权利要求2所述蛋白在制备水产饲料添加剂或在促进鱼类生长方面的应用。
6.权利要求1所述的基因或权利要求2所述蛋白在制备鱼类生长调节药物中的应用。
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CN102250255A (zh) * | 2011-06-28 | 2011-11-23 | 广西南宁众达生物工程有限公司 | 抗对虾白斑综合症病毒工程蛋白tat-vp28-gh及制备和用途 |
CN102703483A (zh) * | 2012-06-08 | 2012-10-03 | 武汉凯肽来生物科技有限公司 | 罗非鱼重组口服蛋白tat-gh及制备方法和应用 |
CN102766645A (zh) * | 2012-06-08 | 2012-11-07 | 武汉凯肽来生物科技有限公司 | 一种促进黄鳝生长的口服重组蛋白tat-gh及制备方法和应用 |
CN102899342A (zh) * | 2012-06-08 | 2013-01-30 | 武汉凯肽来生物科技有限公司 | 鲇生长激素与穿肠膜肽tat融合蛋白及制备方法和应用 |
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CN111286511A (zh) * | 2019-12-20 | 2020-06-16 | 鲁东大学 | 一种生产人表皮生长因子与灵芝免疫调节蛋白方法和应用 |
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