CN103981204A - 一种有活性的金鱼Tgf2转座子重组转座酶蛋白的表达方法 - Google Patents
一种有活性的金鱼Tgf2转座子重组转座酶蛋白的表达方法 Download PDFInfo
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Abstract
本发明涉及一种有活性的金鱼Tgf2转座子重组转座酶蛋白的表达方法。具体地,构建了包含金鱼Tgf2转座子的gfTP1转座酶读码框的表达载体,转入大肠杆菌Rosetta1(DE3)后,培养表达菌株至菌液在OD600下的吸光度达到0.3-0.4,加入IPTG,在21-22℃下150-200rpm震荡培养,诱导表达出可溶性的重组蛋白,并纯化获得了有功能活性的转座酶。本发明的诱导表达条件使得重组转座酶蛋白的表达量显著升高,取得了预料不到的技术效果,通过斑马鱼的转基因实验,证实了诱导表达的转座酶可获得良好的转座效果。本发明为鱼类及其它脊椎动物转基因和基因组插入提供一种高效的方法。
Description
【技术领域】
本发明涉及蛋白表达技术,具体地说,涉及一种有活性的金鱼Tgf2转座子重组转座酶蛋白的表达方法。
【背景技术】
DNA转座子是一种可在生物基因组中移动的遗传元件,以“切离-粘帖(cut-and-paste)” 机制转座,在生物基因组的遗传修饰方面有着重要的价值。转座子转座需要两个必要的元件参与:(1)供特异性识别的末端反向重复序列;(2)活性转座酶。自主性DNA转座子通常能够转录和翻译出活性转座酶来催化转座子转座。然而,绝大部分脊椎动物转座子在进化过程中会因宿主的纵向钝化(vertical inactivation)而导致转座酶编码区的突变失活。即便如此,根据失活转座子的转座酶保守序列,采用生物信息方法仍能重建出转座酶编码区,如人工构建的鲑科鱼类睡美人(Sleeping beauty)和青蛙王子(Frog Prince)转座系统就可在动物胚胎或细胞中实现转座。
在鱼类中,近十几年来已陆续发现若干具有自主转座活性的转座子。例如,来自青鳉的Tol2 转座子,归属hAT(hobo-Ac-Tam3) 转座子超家族,是脊椎动物中发现的第一例有天然活性的转座子。体外转录的Tol2 转座酶mRNA能够介导转座元件高效插入,在包括斑马鱼、爪蟾、鸡和哺乳动物在内的脊椎动物细胞内中均可实现转座。金鱼Tgf2 转座子也属于hAT 家族天然有活性的转座子,该转座子在金鱼、斑马鱼和其它鲤科鱼类胚胎中均能实现高效转座(文献1:X.Y. Jiang, X.D. Du, Y.M. Tian, R.J. Shen, C.F. Sun, S.M. Zou, Goldfish transposase Tgf2 presumably from recent horizontal transfer is active, FASEB J. 26 (2012) 2743-2752.文献2:L.D. Cheng, X.Y. Jiang, Y.M. Tian, J. Chen, S.M. Zou, The goldfish hAT-family transposon Tgf2 is capable of autonomous excision in zebrafish embryos, Gene (2013) http://dx.doi.org/10.1016/j.gene.2013.11.084.)。另外,来自比目鱼的Tc1-like 转座子Passport 在脊椎动物细胞中也具有转座活性。
目前,通常采用带转座子左右臂的供体质粒和转座酶mRNA共同注射入鱼类1-2个细胞期受精卵,转座酶mRNA进入受精卵后首先需要进行转座酶的翻译,进而介导目标基因或元件插入鱼类基因组。转座酶mRNA需要体外转录,一般鱼类养殖场不具备合成和低温保存的条件。另外,转座酶翻译需要耗费一定的时间,通常会产生转基因嵌合体(T. Shibano, M. Takeda, I. Suetake, K. Kawakami, M. Asashima, S. Tajima, M. Taira, Recombinant Tol2 transposase with activity in Xenopus embryos, FEBS Lett. 581 (2007) 4333-4336.),而直接注射有活性的转座酶蛋白则可有效避免嵌合体的发生。
文献1:X.Y. Jiang, X.D. Du, Y.M. Tian, R.J. Shen, C.F. Sun, S.M. Zou, Goldfish transposase Tgf2 presumably from recent horizontal transfer is active, FASEB J. 26 (2012) 2743-2752,在金鱼中发现了一个有活性的hAT超家族Tgf2转座子,它有7种mRNA转录本,可编码686aa(gfTP1)、650aa(gfTP2-3)和577aa(gfTP4-7)3种不同长度的转座酶。目前关于有活性的金鱼Tgf2转座子重组转座酶蛋白还未见报道。
重组蛋白是应用了重组DNA或重组RNA的技术而获得的蛋白质,其获得途径可以分为体外方法和体内方法,两种方法的前提都是应用基因重组技术,获得连接有可以翻译成目的蛋白的基因片段的重组载体,之后将其转入可以表达目的蛋白的宿主细胞从而表达特定的重组蛋白分子。重组蛋白表达过程中通常会遇到不能表达、表达量低、表达产物无活性或活性不高等问题。影响重组蛋白表达量和活性的因素较多,如基因水平上对目的基因的改造、启动子的选择等,在菌体发酵方面诱导剂的浓度、诱导温度、诱导时间等。重组蛋白的生产是一个理论加实际的过程,为获得一种最佳的构建、表达、纯化方案,需要解决的问题是很多的。
【发明内容】
本发明的目的是针对现有技术中的不足,提供一种金鱼gfTP1转座酶蛋白表达质粒。
本发明的再一的目的是,提供一种金鱼gfTP1转座酶蛋白表达菌株。
本发明的另一的目的是,提供一种金鱼gfTP1转座酶蛋白表达方法。
为实现上述目的,本发明采取的技术方案是:
一种金鱼gfTP1转座酶蛋白表达质粒,所述的表达质粒是通过以下方法构建的:将SEQ ID NO.5所示的核苷酸序列插入到pET-28a的BamHI 和 XhoI位点之间。
为实现上述第二个目的,本发明采取的技术方案是:
一种金鱼gfTP1转座酶蛋白表达菌株,所述的金鱼gfTP1转座酶蛋白表达菌株是大肠杆菌Rosetta 1(DE3)且转化有如上所述的金鱼gfTP1转座酶蛋白表达质粒。
为实现上述第三个目的,本发明采取的技术方案是:
一种金鱼gfTP1转座酶蛋白表达方法,包括以下步骤:培养如上所述的金鱼gfTP1转座酶蛋白表达菌株至菌液在OD600下的吸光度达到0.3-0.4,加入IPTG,在21-22℃下150-200rpm震荡培养。
优选地,包括以下步骤:培养如上所述的金鱼gfTP1转座酶蛋白表达菌株至菌液在OD600下的吸光度达到0.35,加入IPTG,在22℃下150-200rpm震荡培养。
优选地,所述的培养时间为4-6小时。
优选地,所述的IPTG在培养液中的浓度为0.6-1.0mM。
本发明优点在于:本发明构建了包含金鱼Tgf2转座子的gfTP1转座酶读码框的表达载体pET-28a-gfTP1,在原核表达菌Rosetta 1(DE3)中诱导表达出可溶性的重组蛋白,并纯化获得了有功能活性的转座酶,本发明的诱导表达条件使得重组转座酶蛋白的表达量显著升高,取得了预料不到的技术效果。通过斑马鱼的转基因实验,证实了诱导表达的转座酶可获得良好的转座效果。本发明为鱼类及其它脊椎动物转基因和基因组插入提供一种高效的方法。
【附图说明】
附图1是pET-28a载体图谱。
附图2是金鱼gfTP1转座酶重组表达载体的构建。(a) Tgf2 转座子的结构图(全长4720 bp)。ITR:末端反向重复序列;STR:亚末端重复序列;IIR:内部重复序列。黑线对应Tgf2转座子的内含子,橘色区域对应Tgf2转座子的外显子。(b) 带有6×His 标签的重组gfTP1转座酶的结构图。虚框表示缺失氨基酸残基。
附图3是不同诱导条件下产生的重组gfTP1转座酶的SDS-PAGE电泳图。带有质重组粒的Rossetta 1 (DE3)在22℃条件下培养,诱导剂IPTG终浓度为0.8mM时,分别在早期生长阶段(OD600=0.3-0.4)和对数生长阶段(OD600=0.6-0.7)诱导表达蛋白。M1:标准分子质量Marker;泳道0:诱导含有空质粒的菌体裂解液;泳道1:未经诱导的含表达载体质粒的菌体裂解液;泳道2-4:在OD600=0.3~0.4条件下,含有重组质粒经过诱导4h、6h、9h的菌体裂解液;泳道5-7:在OD600=0.6~0.7条件下,含有重组质粒经过诱导4h、6h、9h的菌体裂解液。箭头显示重组酶带。
附图4是重组gfTP1转座酶的纯化。(a) 重组转座酶纯化过程中的SDS-PAGE电泳分析图,含有重组质粒的Rossetta 1 (DE3)在LB培养基中诱导表达,表达条件为:22°C,对数生长早期(OD600=0.3-0.4),终浓度为0.8 mM的IPTG。M1:标准分子质量Marker;泳道0:未诱导的含表达载体质粒的菌体裂解液;泳道1:经诱导含表达载体质粒的菌体裂解液;泳道2:经过镍柱亲和层析柱纯化标签蛋白质的咪唑洗脱峰。(b)Western blotting分析。M2:预染分子量Marker。泳道0,未经诱导的含重组质粒的菌体裂解液;泳道1,在OD600=0.3~0.4条件下经诱导的含重组质粒的菌体裂解液。
附图5是金鱼gfTP1重组转座酶的MALDI-TOF/TOF串联质谱鉴定。(a)是通过表达质粒pET28a-gfTP1推测出的gfTP1重组转座酶的序列,垂直箭头标记的是胰蛋白酶切产生的相关肽段如图5中(b)与(d);下划线标记的1-34个氨基是由质粒产生的;虚线标记的序列是酶切的片段;与串联质谱结果相一致。在胰蛋白酶水解后,gfTP1重组转座酶溶液注射进入质谱仪,不同的肽段经过分类,然后进一步分析。(b–d)通过串联质谱测序产生的gfTP1重组转座酶肽段。(b) GSSHHHHHHSSGLVP R,(c) VFGVENNDIETEAR和(d) VLDQDDGFEFQLPK。标记出来的b-、y-代表峰值。
附图6是重组gfTP1转座酶的DNA结合活性EMSA检测。金鱼Tgf2 转座子左臂和右臂末端50bp序列使用生物素标记作为探针,通过EMSA实验验证结合活性,通过非变性聚丙烯酰胺凝胶电泳检测,电泳条件为80V,1.0h。泳道1:左(右)臂生物素标记探针;泳道2:未经诱导的细胞上清液;泳道3:生物素标记的冷探针对照与纯化的重组gfTP1转座酶共同温育电泳;泳道4:Tgf2 转座子左(右)臂生物素标记探针与纯化的重组gfTP1转座酶共同温育电泳;泳道5:Tgf2 转座子左(右)臂生物素标记探针与纯化的10×重组gfTP1转座酶共同温育电泳。
附图7是pTgf2-zfβ-actin-eGFP转基因供体质粒图谱。
附图8是pTgf2-zfβ-actin-eGFP供体质粒与重组金鱼gfTP1转座酶蛋白共同注射入斑马鱼受精卵12h的图片。左边为荧光拍摄图片,显示荧光全身性高强度表达;右边为正常白光拍摄图片。
【具体实施方式】
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1 重组金鱼gfTP1转座酶蛋白的表达和验证
1材料和方法
1.1材料
金鱼胚胎取自上海海洋大学青浦鱼类育种试验站。引物由上海生工生物公司合成,镍柱亲和层析柱购自GE医疗保健公司,超滤管购自美国Millipore公司,限制性酶、DNA聚合酶和T4 DNA连接酶购自Takara公司。pET-28a载体、DH5a、BL21(DE3)和Rosetta 1(DE3)宿主菌购自默克公司。电泳迁移率变动实验试剂盒购自碧云天生物研究所。其余的试剂购自上海生工生物有限公司,均为分析纯或以上纯度。
1.2方法
1.2.1 金鱼gfTP1转座酶开放阅读框的分子克隆
取受精后24hr的琉金金鱼(拉丁学名Carassius auratus Ryukin)胚胎10个,以TRIZOL(Invitrogen)试剂提取总RNA。取1μg总RNA使用TaKaRa RNA PCR Kit(AMV)Ver. 3.0中的MMLV逆转录酶和oligo-dT反转为单链cDNA。根据金鱼Tgf2转座子gfTP1转座酶(GenBank JN886586)的编码框序列设置一对引物,上游引物为5’-CGCGGATCCATGTTCATTGGTCCTTTGG-3’(SEQ ID NO.1),下游引物为5’-CCGCTCGAGCTACTCAAAGTTGTAAAACCT-3’ (SEQ ID NO.2),分别包含BamHI和 XhoI酶切位点。以单链cDNA为模板,采用上述引物以Pfu DNA聚合酶进行PCR扩增,PCR反应程序为:94℃ 5min;94℃ 30s,55℃ 30s,72℃ 60s,35个循环;72℃延长5min。扩增产物经1.2%琼脂糖凝胶,以TBE为缓冲液,在5V/cm的电压下电泳分离后,割胶回收目的片段,将回收产物连接至pMD-19T载体(TaKaRa)中,并转入大肠杆菌DH5α,挑取阳性克隆,接种到氨苄青霉素含量为50 μg/mL的液体LB培养基中,37℃振荡过夜培养,取200μl菌液甘油保存,并送往上海生工生物公司进行双向测序,并提取获得pMD-19T-gfTP1质粒。
1.2.2 金鱼gfTP1转座酶表达质粒的构建
对pMD-19T-gfTP1质粒进行BamHI、XhoI双酶切,酶切产物经1.2%琼脂糖凝胶,以TBE为缓冲液,在5V/cm的电压下电泳分离后,割胶回收目的片段,获得双酶切gfTP1产物。对pET-28a(载体图谱见图1)用限制性内切酶BamHI 和 XhoI双酶切,酶切产物经1.2%琼脂糖凝胶电泳分离后,割胶回收目的片段,获得双酶切pET-28a产物。获得的pET-28a和gfTP1双酶切产物用T4 连接酶连接获得原核表达载体pET28a-gfTP1。然后将质粒pET28a-gfTP1分别转化入大肠杆菌BL21和大肠杆菌Rosetta 1(DE3)感受态细胞中,通过PCR扩增、限制性酶切和DNA测序方法鉴定阳性克隆。
1.2.3 金鱼gfTP1转座酶的原核表达
将过夜培养的阳性Rosetta 1(DE3)菌液4ml转接到100ml LB培养基(配方:胰蛋白胨(Tryptone) 10g/L、酵母提取物(Yeast extract) 5g/L、氯化钠(NaCl) 10g/L)中,在37℃下震荡培养,当培养菌液在OD600下的吸光度达到预定值(设置6个梯度,分别为0.3、0.35、0.4、0.6、0.65、0.7),加IPTG到终浓度为0.8mM诱导表达,在22℃下继续180rpm震荡培养5小时。将培养物在4℃,10000g离心力下离心10min,使用磷酸盐缓冲液(12mM磷酸盐,125mM NaCl,pH 8.0)重悬菌体。然后,在-80℃下冻结,解冻的细胞液使用超声破壁,然后在4°C 、12,000 g离心力下离心15min,细胞裂解液、上清液、沉淀通过SDS-PAGE电泳检测,然后使用考马斯亮蓝染色(参考U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 277 (1970) 680-685.),上清液通过镍亲和层析柱纯化。
1.2.4 重组金鱼gfTP1转座酶的纯化
从超声裂解液中收集上清液,通过AKTA纯化仪使用镍亲和层析柱纯化。用10个柱床体积的结合缓冲液平衡柱,洗脱缓冲液以1 mL/min流速线性梯度洗脱5个柱床体积,洗脱缓冲液:500 mM磷酸钠,0.5 M NaCl,500 mM咪唑,pH7.4。蛋白浓度在280nm吸光度下检测,洗脱峰收集液通过SDS-PAGE电泳检测,使用考马斯亮蓝染色,通过超滤管浓缩,纯化的gfTP1转座酶在20%的甘油中保存于-80℃下。
1.2.5 Western blotting
蛋白质凝胶电泳后在80V、1h条件下转到PVDF膜上,用5%的脱脂牛奶室温下封闭1h,用抗His标签的单克隆抗体孵育2h,使用60ml TBST缓冲液清洗三次,用羊抗兔IgG-HRP孵育膜1h,然后使用TBST清洗,用增强型化学发光试剂发光,使用X胶片曝光,然后拍照。
1.2.6 重组gfTP1转座酶的鉴定
重组gfTP1转座酶从SDS-PAGE凝胶上切割下来,用胰蛋白酶消化,通过MALDI-(TOF)/TOF串联质谱鉴定转座酶的多肽链。
1.2.7 重组gfTP1转座酶的DNA结合活性
通过电泳迁移率分析(EMSA)验证重组gfTP1转座酶的DNA结合活性,采用EMSA生物素标记试剂盒,将Tgf2转座子左右臂末端重复和亚末端重复序列共50bp用生物素标记,左右臂探针序列分别如下:
5’-CAGAGGTGTAAAAGTACTTAAGTAATTTTACTTGATTACTGTACTTAAGT-3’(SEQ ID NO.3)和5’-CAGAGGTGTAAAAGTACTAAAAATATTTACTAAAGTGAAAGTACAAGTAT-3’ (SEQ ID NO.4)。将探针和重组转座酶在25°C条件下孵育0.5-1h,将所有的样品在 4%的聚丙烯酰胺凝胶上以80V电压电泳0.5-1.5h。PAGE胶上的蛋白在80V的恒压下转到尼龙膜上,转膜时间为1.5h,膜在120 mJ/cm2的紫外灯下交联50s,用EMSA探针检测试剂盒孵育,用增强型化学发光试剂发光,使用X胶片曝光,然后拍照。
2结果和讨论
2.1 金鱼gfTP1转座酶重组表达载体的构建
金鱼hAT超家族Tgf2 转座子全长4720 bp,包含4 个外显子(图2a)。Tgf2转座子转座酶共有7种mRNA转录本(GenBank JN886586–JN886592),可编码gfTP1(686aa)、gfTP2-3(650aa)和gfTP4-7(577aa)3种不同长度的转座酶(图2b)。其中,gfTP1转座酶比gfTP4-7转座酶多109个氨基酸残基,而gfTP2-3转座酶则比gfTP4-7转座酶多73个氨基酸残基,其它氨基酸残基序列完全一致。本研究表达的gfTP1转座酶cDNA全长为2633 bp,其编码区长2058 bp,翻译686个氨基酸残基,是金鱼Tgf2转座子所编码的最长的一种转录本。
以琉金金鱼24 hpf胚胎总RNA为模板,扩增gfTP1转座酶cDNA的编码框,核苷酸序列如SEQ ID NO.5所示,克隆到表达载体pET-28a中,构建出原核表达重组质粒pET28a-gfTP1,此重组质粒包含编码34个氨基酸残基的6×His标签(图2b)。重组gfTP1转座酶的理论长度为720aa,通过ProtParam计算出表达的重组蛋白的分子量理论值为79.4 kDa。
2.2 金鱼重组gfTP1转座酶的表达
本研究的目标是获得一种有活性的金鱼gfTP1重组转座酶蛋白,因此在宿主菌中如何大量表达出可溶的而非包涵体形式的重组转座酶是研究成功的关键因素。为了克服真核基因在大肠杆菌中表达密码子偏爱的问题,本研究使用了Rossetta1(DE3)而不是BL21 (DE3) 作为宿主菌。重组质粒转化到BL21 (DE3)中,在所有表达条件下都不能检测到金鱼重组gfTP1转座酶。
为了克服外源蛋白在原核生物中表达量低的问题,在大肠杆菌Rossetta1(DE3)中开展了表达条件的优化,优化的表达条件包括IPTG的浓度、温度和菌液诱导浓度。我们发现IPTG浓度对可溶转座酶的表达量影响不大,而温度影响比较明显,在22℃下诱导能够产生大量的重组gfTP1转座酶。SDS聚丙烯胺酰胺凝胶(SDS-PAGE)电泳表明,大约80kDa处有明显的蛋白条带,和预测的重组gfTP1转座酶分子量一致(图3)。然而,研究发现诱导前的菌液浓度明显影响重组蛋白的表达量,在对数生长前期(OD600= 0.3-0.4)诱导比对数生长期(OD600 = 0.6-0.7)诱导能够产生更多的蛋白(图3)。因此,带有重组质粒的Rossetta 1 (DE3)在0.8mM IPTG终浓度诱导下,以22℃培养在对数生长前期(OD600=0.3-0.4)可产生大量可溶性重组gfTP1转座酶蛋白。
2.3 重组金鱼gfTP1转座酶的纯化
从培养菌液中收集的细胞在-80℃条件下冻结、解冻、超声破壁,然后离心,收集的上清液通过AKTA高效液相色谱系统过镍柱纯化,收集洗脱峰,用SDS-PAGE检测,电泳图上显示的重组gfTP1转座酶的分子量和理论计算的分子量一致。纯化获得了可溶性重组gfTP1转座酶,不含有杂质蛋白质(图4a)。
Western blotting实验使用抗His标签的特异性抗体,出现了与细胞溶解液中的重组gfTP1转座酶一致的单一条带(图4b)。
2.4 重组金鱼gfTP1转座酶的鉴定
将重组蛋白质从SDS-PAGE凝胶上切割下来,用胰蛋白酶消化,MALDI-TOF/TOF串联质谱分析证明了色谱纯化的蛋白确实是金鱼gfTP1重组转座酶。每三个肽段通过MS/MS光谱分析,结果如图5所示。通过Mascot软件检索,结果表明,其中的两个肽段与来自金鱼的gfTP1转座酶开放阅读框的序列相对应(图5-a, c, d)。
第一个肽段是胰蛋白酶消化获得的寡肽GSSHHHHHHSSGLVPR(SEQ ID NO.6),这与被预测包括载体质粒和6×His标签的Tgf2 转座酶的2-17肽段相同,其它两个肽段的分子量是1592.8 Da 和1650.9Da,串联质谱鉴定出它们的序列分别是VFGVENNDIETEAR(SEQ ID NO.7)和VLDQDDGFEFQLPK(SEQ ID NO.8)(图 5-c, d),它们分别对应重组gfTP1转座酶的326-339和366-349氨基酸残基,都位于第二个外显子中,这些片段前后由胰蛋白酶酶切产生四个切割位点,如图5-a可见。
Western blotting和 MS/MS质谱分析的结果表明,分子量为79.4kDa的重组蛋白在大肠杆菌中成功的表达,并确定这个蛋白质就是金鱼gfTP1重组转座酶。
2.5 重组金鱼gfTP1转座酶的DNA结合活性
转座酶是否可与转座子顺式元件结合关乎转座酶的活性。电泳迁移率变动分析(EMSA)能够鉴定出纯化的重组金鱼gfTP1转座酶是否具有结合Tgf2 转座子顺式元件的能力。本实验合成两个50bp的DNA探针,分别对应Tgf2 转座子5' 端、3' 端的末端序列,并用生物素标记。然后,在25°C条件下,标记好的探针与gfTP1转座酶一起温育0.5-1h。
结果显示,20倍浓度的gfTP1转座酶能与Tgf2 转座子探针温育组的电泳迁移速率明显慢于自由探针(图6,泳道4),而在仅左(右)臂生物素标记探针(图6,泳道1)和仅未诱导的细胞裂解产物(图6,泳道2)中未能检测到结合信号,在生物素标记的冷探针对照与纯化的重组gfTP1转座酶共同温育组中也未能检测到结合信号(图6,泳道3)。另外,重组gfTP1转座酶在10倍浓度下可形成低聚物,其与探针的复合物(图6,泳道5)的迁移速率明显慢于1倍浓度的转座酶与探针的复合物(图6,泳道4)。上述结果表明,重组gfTP1转座酶具有与金鱼Tgf2 转座子顺式DNA元件结合的活性。
实施例2 重组金鱼gfTP1转座酶蛋白表达的条件优化
根据实施例1的方法,将含有重组质粒pET28a-gfTP1的菌株Rossetta1(DE3)进行过夜培养,取4ml菌液转接到100ml LB培养基中,在37℃下震荡培养,当培养菌液在OD600下的吸光度达到预定值,加IPTG至一定浓度后诱导表达,在一定温度下继续200rpm震荡培养4小时。诱导gfTP1转座酶蛋白的表达。设置5个实验组(表1),每个实验组做5个重复。
表1 实验组设置
待培养结束后,按照实施例1的方法收集菌体,裂解细胞,离心收集上清液,上清液通过镍亲和层析柱纯化,蛋白浓度在280nm下检测吸光度,上清液及洗脱峰收集液通过SDS-PAGE电泳检测纯度,使用考马斯亮蓝染色后采用灰度扫描法计算纯度,通过超滤离心得到浓缩蛋白液。数据使用ANOVA检验,考察各实验组蛋白表达量的差异是否显著。
实验结果如表2所示。结果表明实验组1-3诱导表达的gfTP1转座酶蛋白总量均显著高于实验组4(P<0.05)和实验组5(P<0.05)。
表2 各实验组蛋白定量结果
实施例3 重组金鱼gfTP1转座酶蛋白的表达
根据实施例1的方法,将含有重组质粒pET28a-gfTP1的菌株Rossetta1(DE3)进行过夜培养,取4ml菌液转接到100ml LB培养基中,在36℃下震荡培养,当培养菌液在OD600下的吸光度达到0.35,加IPTG至0.8mM后诱导表达,在21℃下继续150rpm震荡培养6小时。
按照实施例2的方法测得转座酶占总蛋白的比例为:占菌体裂解上清液3.6%,占洗脱收集液35%。转座酶总量为642μg。
实施例4 重组金鱼gfTP1转座酶蛋白在斑马鱼中的转基因效果
斑马鱼的饲养:实验用斑马鱼饲养于循环水族箱中,雌雄分开暂养,每日饲喂两次,一次为卤虫,一次喂人工饲料,温度恒定于28℃,光照14h,黑暗10h。产卵前一晚将雌鱼与雄鱼配对于同一小水族箱中,中间用透明挡板隔开。实验当天早上抽去挡板让其自然产卵。
显微注射:用0.3×Danieau buffer(含0.1%酚红)配制终浓度为50ng/μl的pTgf2-zfβ-actin-eGFP转基因供体质粒(载体图谱见图7)和纯化的1×实施例1的重组gfTP1转座酶蛋白的混合溶液。显微注射使用的是美国WPI公司的PV830 Pneumatic Pico Pump型号的显微注射仪,整套显微注射系统通过氮气气压驱动。显微注射用针为1mm玻璃毛细管经显微注射拉针器拉制而成,使用前用镊子去除玻璃针最尖端。注射时将转基因质粒溶液与重组gfTP1转座酶蛋白的混合溶液添加到显微注射针中,并注射到斑马鱼1~2细胞期胚胎的卵黄(靠近细胞部位)中,每个胚胎注射1nl溶液,转基因供体质粒的注射剂量各约为50 pg,重组gfTP1转座酶蛋白为1倍体积。注射完毕后,将受精卵放置于28℃的胚胎培养液中正常发育,定期(每隔6小时)在荧光显微镜下观察荧光表达情况。
结果一:pTgf2-zfβ-actin-eGFP供体质粒与重组金鱼gfTP1转座酶蛋白共同注射入斑马鱼受精卵12h的图片见图8,结果显示荧光全身性高强度表达;注射pTgf2-zfβ-actin-eGFP+重组金鱼gfTP1转座酶蛋白的胚胎36小时存活245颗,有绿色荧光201颗,荧光率为82.0%,荧光全身性高强度表达;6天后有荧光的斑马鱼仔鱼有112条,整合率为55.7%。结果二:在另一注射组胚胎36小时存活158颗,有绿色荧光87颗,荧光率为55.1%,荧光全身性高强度表达;6天后有荧光的斑马鱼仔鱼有44条,整合率为50.6%。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
SEQUENCE LISTING
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Claims (6)
1.一种金鱼gfTP1转座酶蛋白表达质粒,其特征在于,所述的表达质粒是通过以下方法构建的:将SEQ ID NO.5所示的核苷酸序列插入到pET-28a的BamHI 和 XhoI位点之间。
2.一种金鱼gfTP1转座酶蛋白表达菌株,其特征在于,所述的金鱼gfTP1转座酶蛋白表达菌株是大肠杆菌Rosetta 1(DE3)且转化有权利要求1所述的金鱼gfTP1转座酶蛋白表达质粒。
3.一种金鱼gfTP1转座酶蛋白表达方法,其特征在于,包括以下步骤:培养权利要求2所述的金鱼gfTP1转座酶蛋白表达菌株至菌液在OD600下的吸光度达到0.3-0.4,加入IPTG,在21-22℃下150-200rpm震荡培养。
4.根据权利要求3所述的金鱼gfTP1转座酶蛋白表达方法,其特征在于,包括以下步骤:培养权利要求2所述的金鱼gfTP1转座酶蛋白表达菌株至菌液在OD600下的吸光度达到0.35,加入IPTG,在22℃下150-200rpm震荡培养。
5.根据权利要求3或4所述的金鱼gfTP1转座酶蛋白表达方法,其特征在于,所述的培养时间为4-6小时。
6.根据权利要求3或4所述的金鱼gfTP1转座酶蛋白表达方法,其特征在于,所述的IPTG在培养液中的浓度为0.6-1.0mM。
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WO2015143930A1 (zh) * | 2014-03-24 | 2015-10-01 | 上海海洋大学 | 一种有活性的金鱼Tgf2转座子重组转座酶蛋白的表达方法 |
CN105409882A (zh) * | 2015-12-04 | 2016-03-23 | 上海海洋大学 | 一种筛选携带缺失型Tgf2转座子金鱼亲本以繁育高正品率后代的方法 |
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