CN114686487A - 一种猫犬白细胞介素11(il-11)在毕赤酵母中的高效表达方法及其应用 - Google Patents
一种猫犬白细胞介素11(il-11)在毕赤酵母中的高效表达方法及其应用 Download PDFInfo
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Abstract
本发明提供了一种猫/犬白细胞介素11(IL‑11)在毕赤酵母中的高效表达方法。涉及了猫/犬IL‑11重组核苷酸序列(如SEQ ID NO:3所示)、含有该核苷酸序列的载体和含有前述载体的工程细胞。本发明基于猫/犬IL‑11基因的天然序列,对其密码子进行不了优化,使其更适合于在毕赤酵母细胞中表达。将经过优化的编码猫/犬IL‑11的DNA序列克隆至毕赤酵母表达载体pPIC9K中,转化毕赤酵母表达菌株GS115,经过G418加压筛选,得到高效表达猫/犬IL‑11的克隆株,表达水平可达108mg/L。本发明的方法所表达的重组蛋白与天然猫/犬IL‑11一致,生物学活性好、表达效率高、操作过程简单,适宜于大规模工业化生产。
Description
技术领域
本发明属于生物技术领域,具体涉及一种全新编码的猫/犬白细胞介素11,及其核苷酸序列,以及在毕赤酵母中的高效表达方法。
背景技术
Paul等人(1990)鉴定并克隆了一种新的基质细胞衍生的淋巴细胞生成和造血细胞因子的基因,被称为白细胞介素-11。McKinley等人(1992)确定IL-11的基因组序列长7kb,由5 个外显子和4个内含子组成。Yang-Feng等人(1991)通过原位杂交证实IL-11的cDNA定位于 19q13.3-q13.4。
用基因预测法Gnomon对一个基因组序列(NC_018737.3)进行分析,得到猫IL-11的DNA序列和氨基酸序列,其前体蛋白由199个氨基酸组成,其中前21个氨基酸是信号肽,成熟蛋白由178个氨基酸组成,分子质量约21.3kDa,等电点pI为11.3。经过序列比对发现,猫IL-11与犬IL-11成熟蛋白的氨基酸序列完全相同,故基于此蛋白序列所表达的重组蛋白可用于猫和犬。
现有技术中已经证明IL-11具有以下几种重要的生物学作用
(1)对于造血系统的作用。IL-11为重要的造血调节因子,对造血系统具有以下作用。第一,促进造血干细胞的生长和分化:与IL-3、IL-4、IL-7和粒细胞刺激因子(Granular-cell stimulating factor,GSF)等协同作用于干细胞,缩短细胞周期,促进干细胞的扩增,并与造血微环境中其他细胞因子一起促进干细胞分化。第二,促进巨核细胞及血小板生成:IL-11与IL-3等干细胞生长因子(Stem cell factor,SCF)协同作用对巨核细胞系、红细胞系、粒细胞系和血小板生成的不同阶段都有刺激作用。IL-11对巨核祖细胞增殖、成熟和分化均有作用,与IL-3等细胞因子协同作用可缩短早期造血祖细胞G0期,使骨髓巨核系集落体积增大、数量增多,同时巨核细胞高倍体也增多。第三,促进淋巴细胞生成:SCF,IL-4 共同作用可促进B淋巴细胞生成,该过程可能手T细胞的调节,且IL-11、IL-4在体外可以逆转IL-3对早期B淋巴细胞产生过程中的抑制作用,故可诱导体液免疫。第四,IL-11对骨髓纤维母细胞的生长等亦有调节作用,已被用于减轻肿瘤化疗药物的骨髓抑制副反应,临床显示IL-11能显著减少造血功能受抑制肿瘤患者的血小板输注率,缩短血小板的恢复时间。
(2)对于上皮细胞的作用。上皮细胞对辐射较敏感,其存活和再生最终影响机体的存亡,故保护上皮细胞是抗辐射损伤的重要方面,在呼吸道合胞病毒感染时肺泡及支气管上皮细胞合成大量的IL-11,说明其参与了肺部炎症。有人认为IL-11对干细胞的保护作用可能与其抑制巨噬细胞肿瘤坏死因子,IL-1、IL-12防止上皮细胞凋亡有关。在体外,IL-11可延迟肠上皮细胞进入S期,同时抑制Rb蛋白磷酸化,诱导肠上皮细胞缩短生长停滞,从而起到保护上皮细胞的作用。
(3)对于免疫系统的作用。IL-11可诱导体液免疫,刺激浆细胞增殖和分化,对骨髓纤维母细胞的生长等亦有调节作用。同时,IL-11具有多种活性,它可诱导急性期反应物的产生,能抑制脂肪形成及调节细胞外基质的代谢等。
(4)IL-11亚家族的细胞因子参与了骨细胞增殖和分化的调控。Takuchi等人(2002)报告 IL-11对成骨细胞和骨形成有正面作用。在转基因小鼠中,人IL-11基因的过表达可刺激骨形成,增加长骨皮质厚度和强度。
此外,IL-11的生物学作用广泛,除对细胞和组织有保护作用外,尚能抑制肥胖、调节神经元分化,刺激金属蛋白酶抑制因子的产生和激活,可抑制细胞组织产生巨噬细胞肿瘤坏死因子,还具有一定的抗炎作用。Schafer等人(2017)的研究表明IL-11在纤维化中起着重要作用。
而IL-11的临床作用一般包括:
(1)肿瘤化疗后血小板减少症。化疗常会引起骨髓抑制,使白细胞和血小板下降,尤其是后者,严重时会引起出血而被迫终止化疗,影响系统化疗的连续性和完整性。IL-11具有升高血小板的功能。Saitoh等观察了单用IL-11及其联用3种抗癌药物时对肺癌细胞增殖的影响,结果发现IL-11可成功防止血小板减少而不改变化疗药物的抗癌活性。Reynolds的一个多中心、随机Ⅲ期临床实验也证实,IL-11可有效防止化疗后血小板减少,有效减少血小板输注量,并保证化疗可连续进行不需调整剂量。IL-11对肝硬化导致的血小板减少症同样有效。
(2)前期再生障碍性贫血的血小板减少。随着免疫再生障碍性贫血的治疗取得了较满意的疗效。而前期再生障碍性贫血仅表现为一系或两系血细胞减少,伴均一性巨核细胞减少,因而血小板减少,增加了出血的危险。商安芳等选用IL-11治疗再生障碍性贫血并发血小板减少患者6例,结果显效3例,良好1例,进步1例,无效1例,有效率为83.3%,效果良好。
(3)与肾上腺皮质激素合用于特发性血小板减少性紫癜 (Thrombocytopenicidiopathic purpura,TIP)。TIP是因免疫机制使血小板破坏增多的临床综合症,发病机理尚未完全阐明,目前肾上腺皮质激素仍是首选治疗药物,但起效较慢。
另外,对于慢性肝病,IL-11可以提高早期肝病患者的血小板计数,有利于治疗肝病时引起的血小板减少;IL-11对肠黏膜损伤也具有保护作用,能有效地促进肠上皮细胞增殖,降低肠源性感染,对放化疗引起的肠上皮细胞凋亡具有明显的抑制作用,从而加速肠黏膜损伤再生修复的进程;对于乳腺癌及其他肿瘤患者,IL-11可以安全地减轻放化疗引起的血小板减少症,保证放化疗顺利进行。它是目前全世界唯一己用于临床的促血小板生成药。其它适应症如肿瘤化疗引起的口腔粘膜炎、节段性回肠炎的治疗也正处于临床阶段,具有较好的临床应用价值。
由于IL-11在生物体内的含量极低,无法从天然材料中提取。在人医临床上使用的IL-11 产品均是用基因工程方法生产的,技术门槛较高。到目前为止,尚检索不到有关猫/犬IL-11克隆、表达的相关科学文献,也检索不到相关的发明专利。在宠物临床上,国内外尚无猫/犬IL-11 相关产品上市。近年来,随着宠物市场的发展,IL-11的临床需求强烈。开发猫/犬IL-11产品具有较好的应用前景。
发明内容
基于现有技术存在的问题,本发明的第一目的在于提供一种猫/犬IL-11,本发明的第二目的在于提供含有编码该猫/犬IL-11DNA序列的载体;本发明的第三目的在于提供含有上述载体的工程细胞;本发明的第四目的在于提供猫/犬IL-11在毕赤酵母中的高效表达方法,方法表达的蛋白接近天然,表达效率高、操作过程简单,适宜于大规模工业化生产。
本发明的上述目的是通过以下技术方案来实现的:
本发明提供一种猫/犬IL-11,所述猫/犬IL-11包含如下氨基酸序列或与其具有85%以上同源性的氨基酸序列:SEQ ID NO:2所示的氨基酸序列。
上述的猫/犬IL-11中,编码该猫/犬IL-11的DNA序列如SEQ ID NO:3所示。
本发明还提供一种载体,所述载体含有上述编码该猫/犬IL-11的DNA序列。
本发明还提供一种工程细胞,所述工程细胞含有上述的载体。
本发明还提供一种猫/犬IL-11在毕赤酵母中的高效表达方法,包括以下步骤:
1、菌株、质粒和培养基:在DNA操作、克隆和测序过程中,使用本实验室保存的大肠杆菌JM109菌株,毕赤酵母GS115菌株(his4,Mut+,His-,aox1+,aox2+)pPIC9K载体质粒,大肠杆菌重组子在LB固体培养基平板(含100μg/ml氨苄青霉素)37℃过夜培养。使用含0.25~5mg/ml G418的MD平板进行His+转化子筛选,然后接着在含有不同浓度G418(0~5mg/ml)的YPD平板筛选含有多拷贝表达载体的重组子。毕赤酵母GS115菌株重组子在BMGY/BMMY培养基上培养,进行高拷贝重组子的筛选,然后将此重组子用于蛋白质大量表达纯化。
2、猫/犬IL-11基因表达载体的构建:
猫/犬IL-11基因(GenBank号:XM_003997398)的DNA编码序列(如SEQ ID NO:1 所示)经过优化后,使用SnaB I和Not I酶切位点,将合成的基因克隆至酵母表达载体pPIC9K中,得到pPIC9K-IL-11质粒,转化大肠杆菌JM109,37度培养过夜,然后挑挑选单个菌落接种于5ml含Amp的LB培养液中,37℃振荡培养10~14h,于Ep管中收集5ml菌液, 13,000r/min离心1~3min后弃上清。将菌体沉淀加入250μl溶液I(加RNAse),并充分使其悬浮;加入250μl溶液II(裂解液),温和地上下翻转混合6~10次,使菌体充分裂解,室温放置2min直至溶液变得清亮。加入300μl溶液III(中和液),立即轻柔地上下翻转6~10次,室温放置5min,13,000r/min离心15min。将离心后的上清液转移到DNA结合管中,13,000r/min 离心1min,弃去滤液,加入700μl洗涤缓冲液,13,000r/min离心1~3min,重复漂洗一次后空柱13,000r/min离心2min,室温敞口放置5~10min,除去残留的乙醇。最后用50μl 50~ 70℃预热的无菌去离子水洗脱质粒DNA。然后将此质粒测定浓度,用于线性化实验。
(1)重组质粒线性化:
100μl体系中分别加入:
37℃酶切过夜。第二天采用酚/氯仿/异丙醇的方法将线性化的质粒进行纯化。
a)配置酚/氯仿/异丙醇,配好后混匀。将100μl酶切产物+100μl ddH2O+200μl酚/氯仿 /异丙醇溶液,涡漩混合后,13000r/min离心10~20min.
b)将上层液体吸出,加入10%(NaAc 3mol/L),3倍体积无水乙醇,涡漩混合后,-20℃放30min后,13000r/min离心10~20min。
c)底部见一白色沉淀,将上清吸掉。加1ml 70%乙醇(冷),涡漩混合后,13000r/min离心10~20min。
d)加1ml 70%乙醇(冷),涡漩混合后,13000r/min离心10~20min,将上清吸掉。置于56℃金属浴中,使多余乙醇挥发(1~5min)。
e)加12μl ddH2O溶解DNA,涡漩混合后,用金属浴加热1~5min,吸1~2μl稀释测浓度。
(2)电转酵母GS11菌株多拷贝重组子筛选:
a)取80μl GS115感受态细胞+5μl线性化的重组质粒(约7μg),轻轻混合后,冰上放置5min。
b)将其转到2cm预冷的电转杯中,用电穿孔仪电转化,电转结束后,往细胞中加入1ml 1~3mol/L的山梨醇溶液。
c)将其转入15ml无菌BD管中,30℃孵育1~5h后,1000r/min离心5~10min,随后将其涂在MD平板上,置于28℃培养箱中,倒置培养48~72h,以筛选His+转化子。用1~ 2ml无菌水将每个MD平板长出的菌落吹起。将细胞悬液转移到无菌50ml离心管中混匀。使用分光光度计测细胞密度(1OD600=5×107细胞/ml)。之后在含有不同浓度G418(0.25~ 5.0mg/ml)的YPD平板上28℃培养,需要2~5天长出菌落。
为了筛选高表达IL-11的克隆,从MD平板上选取10个克隆以及含5.0mg/ml G418的YPD平板上的所有克隆,在含有20ml BMGY培养基的100ml三角瓶中,28℃、230r/min培养,当培养物OD600达到22~24(约18h)时,将细胞重悬于含有20ml BMGY培养基的100ml 三角瓶中,让其在同样条件下生长。每24h往培养瓶中加入纯甲醇,浓度为1~5%(v/v),以维持诱导。用同样处理的空载体转化的毕赤酵母进行作为阴性对照。诱导96h后,12000g 离心5min,收集培养物,用SDS-PAGE分析上清,选择目的蛋白表达最高的转化子用于后续的生产。
3、表达条件的优化:使用原始的培养条件(28℃诱导温度、pH 6.0、每24h添加0.5%甲醇、生物量积累用BMGY/诱导用BMMY、在100ml摇瓶中用20ml培养基、230r/min),优化了摇瓶的大小对rIL-11产量的影响。在此,我们选用单因素法,以便能够独立地控制每个参数。研究了温度(22、24、26、28、30℃)、pH(4.0、4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0) 和甲醇浓度(0.5%、1.0%、1.5%、2.0%)、甲醇诱导时间(0、24、48、72、96、120、144h) 对选定克隆在诱导期蛋白表达量的影响。并评估了在诱导期开始时添加氨基酸(1g/L Ala、 His、Gly、Glu、Leu和Lys)、酪蛋白氨基酸(0.5%~2.0%)、油酸(0.01%~0.1%)、抗坏血酸(10mmol/L)和EDTA(12mmol/L)对表达量的影响。在诱导96h后取样,测定OD600,然后,对所取样品12000g离心5min,取上清进行SDS-PAGE分析。
4、样品分析:使用UV/Vis分光光度计(Ultrospec 2100pro,GE,USA)测量OD600,取10ml 培养物,12000g、4℃离心10min,用去离子水洗涤沉淀2次,称重。在变性条件下,在聚丙烯酰胺凝胶上分析蛋白浓度,使用BCA法测定总蛋白浓度,使用质谱仪 (SciexTripleTOF 6600)进行鉴定。
本发明的有益效果:
(1)将编码猫/犬IL-11的DNA序列经过优化后,可以在毕赤酵母GS115株中高效表达,经质谱鉴定是猫/犬IL-11。在摇瓶中的表达量可达108mg/L(图2第3泳道);而经同样处理的未经优化的野生型序列的表达量仅为15mg/ml(图2第2泳道)。
(2)目前,国内外尚无猫/犬IL-11相关产品上市,本发明所生产的猫/犬IL-11有效填补了猫/犬生物制品的空白。
(3)密码子优化的优势在于能够提高目的蛋白在相应宿主细胞中更有效的表达,但序列优化随机性很大,能找到一种好的优化的序列,意义还是很大的。
附图说明
图1为pPIC9K载体的图谱;
图2序列优化前后II-L-11表达的SDS-PAGE图谱;
图3为表达产物的质谱鉴定图。
具体实施方式
除非另外定义,否则本文所用的所有科学和技术术语都具有与本发明所属领域普通技术人员通常所理解的相同含义。此外,本发明实施例中所采用的原料若无特殊说明均为市售获得。
以下结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
猫/犬IL-11基因的合成:基于野生型猫/犬IL-11的基因序列(如SEQ ID NO:1)、(氨基酸序列如SEQ ID NO:2),用密码子优化软件(http://www.jcat.de)对猫/犬IL-11的DNA编码序列进行优化,获得本发明编码该猫/犬IL-11的DNA序列(如SEQ ID NO:3),经由上海生工合成全序列,由其演绎的猫/犬IL-11的氨基酸序列如SEQ ID No.2。同时,合成野生型IL-11 的基因序列(如SEQ ID NO:1),作为对照。
重组质粒的构建与鉴定:如图1所示,将合成的猫/犬IL-11DNA序列插入到SnaB I和Not I位点之间,在AOX1启动子的控制下表达,在N端与可被切割的酿酒酵母α因子分泌信号融合。5’AOX1:乙醇氧化酶1启动子区;α-factor:α因子分泌信号;3’AOX1(TT):乙醇氧化酶1转录终止区;HIS4:组氨酸基因;KanR2:卡那霉素抗性基因;SnaB I和Not I:插入子连接位点;Sal I:质粒线性化位点。
将猫/犬血清白重组蛋白的编码序列和毕赤酵母表达载体pPIC9K分别用SnaB I和Not I酶切。1%琼脂糖凝胶电泳分离、纯化回收,用T4 DNA连接酶连接过夜,次日用连接产物转化大肠杆菌JM109感受态细胞,涂布至含有Amp的LB平板上,37℃培养过夜。次日,从平板上挑取单菌落,扩增后提取重组质粒,经SnaB I和Not I双酶切鉴定,得到大小与目的DNA长度一致的条带,经测序验证,表达质粒构建正确。
将携带pPIC9K-IL-11质粒的大肠杆菌接种于100ml LB培养基中,37℃培养过夜,次日,用质粒提取试剂盒提取质粒,用NanoDrop定量后,取大约100μg质粒,用Sal I酶切过夜,用DNA提取试剂盒回收,得到线性化的pPIC9K-IL-11质粒,-20℃保存备用。
取适量线性化的pPIC9K-IL-11质粒,用电转仪转化GS115感受态细胞,用Sal I将pPIC9K-IL-11质粒线性化,取7μg线性化后的质粒(10μl),用电穿孔仪(Bio-Rad,CA,USA)电转化(25μF,200Ω,1.0kV)毕赤酵母GS115(80μl)感受态细胞。电穿孔后,将细胞与1ml预冷的山梨醇(1mol/L)混合,28℃培养2h。随后将其涂在MD平板上,并28℃倒置培养48~72h,以筛选His+转化子。
用1~2ml无菌水将每个MD平板长出的菌落吹起。将细胞悬液转移到无菌50ml离心管中混匀。使用分光光度计测细胞密度(1OD600=5×107细胞/ml)。之后在含有不同浓度G418(0.25~5.0mg/ml)的YPD平板上28℃培养,需要2~5天长出菌落。
为了筛选高表达IL-11的克隆,从MD平板上选取10个克隆以及含5.0mg/ml G418的YPD平板上的所有克隆,在含有20ml BMGY培养基的100ml三角瓶中,28℃、230r/min培养,当培养物OD600达到22~24(约18h)时,将细胞重悬于含有20ml BMGY培养基的100ml 三角瓶中,让其在同样条件下生长。每24h往培养瓶中加入纯甲醇,浓度为1%(v/v),以维持诱导。对携带野生型猫/犬IL-11DNA序列的克隆进行同样处理,作为对照。对用空载体转化的毕赤酵母进行同样的处理,作为阴性对照。诱导96h后,12000g离心5min收集培养物,用SDS-PAGE分析上清,选择目的蛋白表达最高的转化子用于后续的生产。
取诱导后的培养上清,进行SDS-PAGE,用考马氏亮蓝R-250染色。经序列优化的克隆,在分子质量约21kDa附近,可见两条与目的蛋白分子质量接近的条带(如图2第3泳道箭头所示),野生型猫/犬IL-11的克隆也可见两条与目的蛋白分子质量接近的条带(如图2第2泳道箭头所示),但表达量明显要低于序列优化的克隆。上面的条带分子质量稍大于目的蛋白的理论分子质量,下面的条带与目的蛋白的理论分子质量一致。图2中,1为蛋白Marker(自上至下依次为55、40、35、25、15、10kDa)。
从胶上切下两条目的条带,用Sciex TripleTOF 6600质谱仪进行鉴定,经数据库搜索,证明上、下两条带都是猫/犬IL-11(如图3所示),上面的条带分子质量比理论值稍大,可能是糖基化造成的。
蛋白表达量测定:经过DNA序列优化的克隆菌株在摇瓶中的表达量可达108mg/L(图 2第3泳道);而经同样处理的未经优化的野生型序列的表达量仅为15mg/ml(图2第2泳道)。可见DNA序列优化效果明显。
SEQUENCE LISTING
<110> 泰州博莱得利生物技术有限公司
<120> 一种猫犬白细胞介素11(IL-11)在毕赤酵母中的高效表达方法及其应用
<130> 1
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 537
<212> DNA
<213> Felis catus
<400> 1
ccggggccac cgccgggccc cccacgggcc tccccagacc ctcgggccga gctggacagc 60
gccgtcctcc tgacccgctc tctcctggcg gacactcgac agcttgctgc gcagctgaga 120
gacaaatttc cagccgacgg agaccacaac ctggactctc ttcccacctt ggccatgagt 180
gcaggggcgc tgggagccct acagctccca ggtgtgctga cgcggctgcg ggcagacctg 240
ttctcctacc tgcggcacgt gcagtggctg cggcgggcgg gcggcccttc cttgcggacc 300
ctggagccgg agctgggcgc cctgcaggcc cggctggaca gactgctgcg ccggctgcag 360
ctcctgatgt cccgcctggc cttaccccaa gcacccccag acccaccagc tcccccactg 420
gcccccccag cctccgcctg ggggggcatc agggcagccc acgccattct tggggggttg 480
cacctgaccc ttgactgggc cgtgcggggc ttgctgctgc tgaagactcg gttgtga 537
<210> 2
<211> 178
<212> PRT
<213> Felis catus
<400> 2
Pro Gly Pro Pro Pro Gly Pro Pro Arg Ala Ser Pro Asp Pro Arg Ala
1 5 10 15
Glu Leu Asp Ser Ala Val Leu Leu Thr Arg Ser Leu Leu Ala Asp Thr
20 25 30
Arg Gln Leu Ala Ala Gln Leu Arg Asp Lys Phe Pro Ala Asp Gly Asp
35 40 45
His Asn Leu Asp Ser Leu Pro Thr Leu Ala Met Ser Ala Gly Ala Leu
50 55 60
Gly Ala Leu Gln Leu Pro Gly Val Leu Thr Arg Leu Arg Ala Asp Leu
65 70 75 80
Phe Ser Tyr Leu Arg His Val Gln Trp Leu Arg Arg Ala Gly Gly Pro
85 90 95
Ser Leu Arg Thr Leu Glu Pro Glu Leu Gly Ala Leu Gln Ala Arg Leu
100 105 110
Asp Arg Leu Leu Arg Arg Leu Gln Leu Leu Met Ser Arg Leu Ala Leu
115 120 125
Pro Gln Ala Pro Pro Asp Pro Pro Ala Pro Pro Leu Ala Pro Pro Ala
130 135 140
Ser Ala Trp Gly Gly Ile Arg Ala Ala His Ala Ile Leu Gly Gly Leu
145 150 155 160
His Leu Thr Leu Asp Trp Ala Val Arg Gly Leu Leu Leu Leu Lys Thr
165 170 175
Arg Leu
<210> 3
<211> 354
<212> DNA
<213> Artificial Sequence
<220>
<223> Arfificial seqence for IL-11
<400> 3
CCAGGTCCACCACCAGGTCCACCAAGAGCTTCTCCAGACCCAAGAGCTGA 50
ATTGGACTCTGCTGTTTTGTTGACTAGATCTTTGTTGGCTGACACTAGAC 100
AATTGGCTGCTCAATTGAGAGACAAGTTCCCAGCTGACGGTGACCACAAC 150
TTGGACTCTTTGCCAACTTTGGCTATGTCTGCTGGTGCTTTGGGTGCTTT 200
GCAATTGCCAGGTGTTTTGACTAGATTGAGAGCTGACTTGTTCTCTTACT 250
TGAGACACGTTCAATGGTTGAGAAGAGCTGGTGGTCCATCTTTGAGAACT 300
TTGGAACCAGAATTGGGTGCTTTGCAAGCTAGATTGGACAGATTGTTGAG 350
AAGATTGCAATTGTTGATGTCTAGATTGGCTTTGCCACAAGCTCCACCAG 400
ACCCACCAGCTCCACCATTGGCTCCACCAGCTTCTGCTTGGGGTGGTATC 450
AGAGCTGCTCACGCTATCTTGGGTGGTTTGCACTTGACTTTGGACTGGGC 500
TGTTAGAGGTTTGTTGTTGTTGAAGACTAGATTGTAA
Claims (9)
1.一种猫/犬IL-11重组核苷酸序列,其特征在于:所述猫/犬IL-11重组核苷酸序列如SEQ ID NO:3所示。
2.一种猫/犬IL-11,其特征在于:该猫/犬IL-11是由权利要求1所述猫/犬IL-11重组核苷酸序列编码获得;
优选地,所述猫/犬IL-11包含如下氨基酸序列或与其具有85%以上同源性的氨基酸序列:SEQ ID NO:2所示的氨基酸序列。
3.一种载体,其特征在于:所述载体含有权利要求1中编码该猫/犬IL-11的DNA序列。
4.一种工程细胞,其特征在于:所述工程细胞含有权利要求3所述的载体。
5.一种重组猫/犬IL-11在毕赤酵母中的高效表达方法,其特征在于,包括以下步骤:
(1)将编码猫/犬IL-11的DNA序列和毕赤酵母表达载体pPIC9K分别用SnaB I和Not I酶切,琼脂糖凝胶电泳分离、纯化回收,经T4 DNA连接酶连接过夜,次日用连接产物转化大肠杆菌JM109感受态细胞,涂布至含有Amp的LB平板上,37℃培养过夜;次日,从平板上挑取单菌落,扩增后提取重组质粒;
其中,编码猫/犬IL-11的DNA序列如SEQ ID NO:3所示;
(2)将携带重组质粒pPIC9K-IL-11的大肠杆菌接种于培养基中,37℃培养过夜,用质粒提取试剂盒提取质粒,用NanoDrop定量后,用Sal I酶切过夜,用DNA提取试剂盒回收,得到线性化的pPIC9K-IL-11质粒,-20℃保存备用;
(3)取线性化的pPIC9K-IL-11质粒,用电转仪转化GS115感受态细胞,用Sal I将pPIC9K-IL-11质粒线性化,取7~10μg线性化后的质粒,用电穿孔仪电转化毕赤酵母GS115感受态细胞;电穿孔后,将细胞与1~3ml预冷的山梨醇混合,28℃孵育1~3h;随后将其涂在MD平板上,并28℃倒置培养48~72h,筛选His+转化子;
(4)用1~2ml无菌水将每个MD平板长出的菌落吹起;将细胞悬液转移到无菌50ml离心管中混匀,使用分光光度计测细胞密度,之后在含有不同浓度G418的YPD平板上25~30℃培养2~5天;
(5)筛选高表达猫/犬IL-11的克隆,从MD平板上选取10个克隆以及含5.0mg/mlG418的YPD平板上的所有克隆,在含有20ml BMGY培养基的100ml三角瓶中,28℃、230r/min培养,当培养物OD600达到22~24时,将细胞重悬于含有20ml BMGY培养基的100ml三角瓶中,让其在同样条件下生长,每24h往培养瓶中加入纯甲醇,以维持诱导,对用空载体转化的毕赤酵母进行同样的处理,作为阴性对照;诱导72~96h后,12000g离心5min收集培养物,用SDS-PAGE和Western blot分析上清,选择产蛋白最好的转化子,用于后续生产。
6.根据权利要求5所述的方法,其特征在于:所述预冷的山梨醇浓度为1~3mol/L;所述纯甲醇浓度为1~5%(v/v)。
7.根据权利要求5所述的方法,其特征在于:所述MD平板的成分包括20~30g/L的葡萄糖、2~4g/L不含氨基酸和硫酸铵的酵母氮源、300~500μg/L的生物素和10~30g/L的琼脂。
8.根据权利要求5所述的方法,其特征在于:所述不同浓度G418的浓度范围为0.25~5.0mg/ml。
9.根据权利要求5所述的方法,其特征在于:所述YPD平板的成分包括5~15g/L的酵母提取物、20~30g/L的胰蛋白胨、20~30g/L的葡萄糖和15~30g/L的琼脂。
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