CN114150007B - 一种适用于家兔乳腺特异性表达去氨普酶的编码基因及其应用 - Google Patents
一种适用于家兔乳腺特异性表达去氨普酶的编码基因及其应用 Download PDFInfo
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Abstract
本发明涉及一种适用于家兔乳腺特异性表达去氨普酶DSPAα1的编码基因,应用所述编码基因来制备去氨普酶DSPAα1,表达量高,生产成本低。本发明还涉及一种从兔乳中提取、纯化去氨普酶rDSPA的方法,通过联用苯甲醚亲和层析、阳离子交换层析和活性蓝亲和层析进行分离纯化,操作简便、产品纯度高,可达98%。
Description
技术领域
本发明涉及遗传工程,尤其涉及编码蛋白的基因及其应用,更涉及采用编码基因制备重组蛋白。
背景技术
血栓性疾病是危害人类生命和健康的主要疾病之一,目前研究的溶栓药物不是直接溶解血栓,而是通过激活纤溶酶原***将血栓溶解,因此在溶解血栓时消耗纤维蛋白原和纤溶酶原的数量,引起明显的凝血因子的减少从而引发出血倾向。临床上用于治疗该类疾病溶栓药物以天然人组织纤溶酶原激活剂(tPA)为代表,然而tPA在血中的半衰期甚短(3-5分钟),使得在临床治疗中为维持其有效浓度而必须大剂量静脉滴注,从而增加了循环***出血的风险。
去氨普酶(DSPA)是从南美洲一种吸血蝙蝠唾液中分离的纤溶酶原激活剂,主要包括四种蛋白质:DSPAα1、DSPAα2、DSPAβ和DSPAγ。经研究发现,其中DSPAα1具有最好的生物化学和药理学性质,与其他纤溶酶原激活剂相比较,DSPAα1最大的特点是对纤维蛋白具有高度专一性,与目前广泛应用组织纤溶酶原激活剂(tPA)相比,在纤维蛋白存在条件下,DSPAα1的酶活性增加102100倍,而tPA仅增加550倍;在纤维蛋白与纤维蛋白原分别存在的条件下,DSPAα1两种酶活性比值为12900,而tPA仅为72;在临床上,DSPAα1用药量少,出血少,且不引起神经毒性,表现出比tPA更优的药用价值。DSPA作为一种正在开发的新型溶栓药,DSPA适用于陈旧性血栓,具有治疗窗口期宽(9小时),半衰期长达2.3小时,能直接降解纤维蛋白,减少出血及毒副作用等特点,将拥有广阔的发展前景。虽然重组DSPA能够在原核细胞表达,但表达产物没有活性,需进行复性,由此增加了生产环节。即使通过复性后产生活性,但其比活性也偏低。由此造成生产成本增加,致使其价格昂贵临床治疗费用高。此外,原核产品与人体相融性差,易相生不良反应,也降低节药物的安全性,增加了临床应用风险。而利用动物乳腺生物反应器技术生产药用重组蛋白,具有很高的商业应用价值。家兔具有泌乳量高、妊娠期短、每胎产仔数多等优点。到目前为止,应用兔乳腺生物反应器生产重组DSPA尚未见报道。
因此,设计并研制延长半衰期,降低***出血倾向,提高比活性,降低价格成为开发新型DSPA药物的重要目标,这类新药的开发对于保障人类的生命健康具有重要的实际意义。
由于对转基因动物乳液分离纯化目的蛋白的研究的历史还很短暂,因此国内外均无成熟的纯化技术。虽然纯化乳腺生物反应器生产的重组蛋白的基本原理与纯化一般蛋白相似,均是利用不同蛋白的理化及生物学差异性进行分离纯化的,但是哺乳动物乳汁成分比较复杂,有多种乳蛋白、球蛋白,还含有丰富的脂肪、糖类、维生素、矿物质,及动物组织脱落的碎片等。并且在重组蛋白提取过程中还要保持其活性结构及功能,这些均提高了目的蛋白的分离与纯化的要求。一般需要结合多种纯化技术来分离纯化某种重组蛋白,对于方案的选择要参考重组蛋白的表达***,因为表达***决定了重组蛋白的性质及杂蛋白的种类。
发明内容
本发明所要解决的技术问题是提供一种适用于家兔乳腺特异性表达去氨普酶DSPAα1的编码基因。
本发明所要解决的另一个技术问题是提供一种应用所述编码基因来制备去氨普酶DSPAα1的方法。
本发明所要解决的技术问题还包括,提供从兔乳中提取、纯化去氨普酶DSPA的方法。
为了解决上述技术问题,本发明采用如下技术方案:
适用于家兔乳腺特异性表达去氨普酶的编码基因,其特征在于,所述基因的核苷酸序列如SEQ ID NO.1所示。
该基因具有如下特点:(1)编码序列的密码子兼容家兔个体内表达的特征;(2)重组编码序列符合在兔乳腺组织特异性高效表达的要求。
由所述基因编码的去氨普酶DSPAα1重组蛋白,所述重组蛋白的氨基酸序列如SEQID NO.2所示。
所编码的DSPAα1重组蛋白具有如下特点:(1)该一级结构N端有助于翻译后多肽在细胞内加工为成熟蛋白;(2)重组的DSPA一级结构具备折叠或化学键形成而形成功能性蛋白,从而产生溶解血栓的活性。
所述编码基因在制备去氨普酶中的应用。
进一步的,所述的应用是通过家兔乳腺生物反应器来制备去氨普酶。
进一步的,所述制备的方法如下:
(1)构建乳腺特异性表达去氨普酶基因载体;
(2)制备乳腺特异性表达去氨普酶基因兔;
(3)兔乳汁中去氨普酶的检测分析,兔配种分娩后,收集乳汁;
(4)对乳汁中的rDSPA重组蛋白进行提取和纯化。
优选的,所述的去氨普酶为DSPAα1。
优选的,所述的方法联用苯甲醚亲和层析、阳离子交换层析和活性蓝亲和层析进行分离纯化。
进一步的,所述的方法包括如下步骤:
(1)兔乳通过超速离心、55%硫酸铵沉淀初步分离兔乳中的rDSPA,得兔乳前处理样品;
(2)苯甲醚亲和层析:
A.用平衡液平衡层析柱;平衡液为20mmol/L甘氨酸溶液,pH8.75;
B.平衡后以流速3mL/min上样,上样结束后用6倍柱体积平衡液洗涤层析柱至紫外吸收线UV280归零;
C.用3倍柱体积的淋洗液(20mmol/L磷酸盐缓冲液+0.5mol/L NaCl,pH7.5)淋洗,洗脱条件设置为0.1mol/L甘氨酸,pH3.0;
(3)阳离子交换层析:
A.平衡液平衡层析柱,流速5mL/min,待紫外吸收值UV280与电导值稳定后再平衡200mL;平衡液为50mmol/L磷酸盐缓冲液,pH8.0;
B.用6倍柱体积平衡液(20mmol/L甘氨酸溶液,pH8.75)洗层析柱至紫外吸收线(UV280)归零,洗脱条件设置为20m mol甘氨酸+0.31mol氯化钠;
(4)蓝柱亲和层析:
A.平衡液平衡,流速3mL/min,待紫外吸收值UV280与电导值稳定后再平衡30mL;所述平衡液为50mmol/L磷酸盐缓冲液,pH8.0;
B.20mmol/L磷酸盐缓冲液pH7.5稀释至500mL后上样,流速3mL/min;
C.20mmol/L磷酸盐缓冲液pH7.5+0.15mol/L氯化钠洗脱液丢弃;20mmol/L磷酸盐缓冲液pH7.5+0.4mol/L氯化钠洗脱液可回收重复用;20mmol/L+1.0mol/L氯化钠收集;
(5)脱盐成为rDSPA纯品。
9.根据权利要求8所述提取和纯化rDSPA重组蛋白的方法,其特征在于,所述的方法还包括:将纯化的rDSPA应用阴离子交换层析去除纯化产物中的内毒素。所述家兔为乳腺特异性表达去氨普酶基因兔。
与现有技术相比,本发明的有益技术效果体现在:
(1)采用本发明基因,通过家兔乳腺生物反应器,可高效表达DSPA;
(2)获得乳腺高效特异性表达具有生物活性的DSPA转基因兔,纤维蛋白溶解活性高于其它表达***(细菌、真菌及CHO细胞);
(3)生产的成本低、产量扩大简单;
(4)重组DSPA蛋白可获得翻译后修饰和正确折叠,为成熟蛋白。
(5)本发明提取、纯化工艺,操作简便、产品纯度高(可达98%)。
附图说明
图1:pUC57+DSPA的质粒图谱;
图2:pCL25+rhPA的质粒图谱;
图3:pCL25/DSPA构建流程图;
图4:重组质粒Xho I酶切图;
图5:序列分析鉴定重组质粒pCL25/DSPA中***片段的正反向;
图6:兔受精***期显微注射;
图7:整合DSPA基因兔的PCR扩增电泳图
图8:转基因兔乳汁中rDSPA的ELASA检测图;
图9:DSPA转基因兔乳清的FAPA分析;
图10:苯甲醚亲和层析柱纯化rDSPA转基因兔乳洗脱峰图;
图11:FAPA检测苯甲醚亲和层析柱纯化过程中样品纤溶活性;
图12:强阳离子交换层析柱线性梯度洗脱峰图;
图13:FAPA检测强阳离子交换层析柱线性梯度洗脱样品纤溶活性;
图14:强阳离子交换层析柱线性梯度洗脱样品SDS-PAGE检测;
图15:活性蓝亲和层析柱纯化rDSPA转基因兔乳峰图;
图16:FAPA检测活性蓝亲和层析柱纯化过程中样品纤溶活性
图17:HiTrap Capto Q柱洗脱峰图;
图18:内毒素检测结果。
核苷酸和氨基酸序列说明
SEQ ID NO.1:适用于家兔乳腺特异性表达去氨普酶DSPAα1的编码基因;
SEQ ID NO.2:本发明去氨普酶DSPAα1的氨基酸序列;
SEQ ID NO.3:引物DSPA-S的核苷酸序列;
SEQ ID NO.4:引物DSPA-A的核苷酸序列。
具体实施方式
下面结合实施例对本发明的技术方案做进一步的说明。
实施例1乳腺特异性表达去氨普酶(DSPA)基因载体的构建
1.载体:
pCL25+tPA为本实验室构建的乳腺特异性表达载体,是以山羊β-酪蛋白为表达调控序列,鸡β-珠蛋白为隔离原件,CMV为增强子;此载体已经在山羊和兔的细胞及个体水平上得到验证。重组DSPAα1cDNA序列由生工生物工程(上海)股份有限公司合成,并***至pUC57载体的多克隆位点(MSC)中;质粒pUC57购自上海圻明生物科技有限公司;宿主菌Trans10购自北京全式金生物。pCL25质粒由PBC1与CMV序列拼接而成,详见发明专利:“高表达水平的转基因动物乳腺特异性载体的构建方法”,发明人:成勇,专利号:ZL200610040436.9;PCL25+rhPA载体的构建参考2016年宋绍征博士毕业论文:“转基因兔乳腺行异性表达重组人纤溶酶原激活剂(rhPA)及其药效学研究”(中国知网,硕博论文数据库)。
pUC57+DSPA和pCL25+rhPA的质粒图谱如图1和图2所示。
2.材料
2.1主要仪器及器材
DNA扩增仪(美国Bio-Rad公司);DNA电泳仪和电泳槽(南京大学生物技术开发公司);数码凝胶成像***(上海天能科技有限公司);隔水式电热恒温培养箱(PYX-DHS-35×40–B-H,上海跃进医疗器械厂);电热恒温水槽(上海精宏实验设备有限公司);制冰机(日本SANYO公司);电子天平(上海精密天平制造有限公司);PH电极(梅特勒-托利多集团上海分公司);恒温振荡器(IS-RDV-1,INCUBATOR SHAKER);自动双重纯水蒸馏器(上海申立玻璃有限公司);低温高速离心机(德国Eppendorf公司);台式冷冻高速离心机(德国Eppendorf公司);真空抽干机(Speed Vac Plus SC110,美国);超净台(苏州净化仪器设备厂);普通冰箱(青岛海尔公司);超低温冰箱(日本SANYO公司);核酸蛋白定量分析仪(One DropTM OD-1000+,基因集团,上海仪涛生物仪器有限公司);实验室级超纯水器(EPED-20TF,南京易普易达科技发展有限公司);超声波清洗器(KQ-300DE,昆山市超声仪器有限公司)。
2.2主要药品与试剂
Xho I、Sal I、Not I等限制性内切酶,DL2000、λ-HindⅢ等Marker购自TaKaRa公司;Endo-Free Plasmid Mini Kit II D6950(无内毒素质粒小量提取试剂盒II)购自美国OMEGA公司;DNA回收纯化试剂盒购自QIAGEN公司,氨苄青霉素购自北京索莱宝,其它化学试剂均为国产分析纯。
3.方法
3.1合成重组DSPA功能基因
在NCBI中搜索DSPA cDNA序列,选取成熟肽序列后在其5’端加上goat BLG sig-peptide序列,组成重组DSPA功能基因,由生工生物工程(上海)股份有限公司合成并连接至pUC57载体中。将收到的pUC57+DSPA甘油菌少量培养鉴定其活力后,以50%甘油(3):菌液(7)的比例混合保种以备用,同时准备构建重组质粒。
在NCBI中收索出DSPA的mRNA序列,名称及编号如下所示:
Desmodus rotundus plasminogen activator alpha 1mRNA
GenBank:M63987.1
对cDNA序列进行修改后得到的序列见SEQ ID NO.1。
3.2构建重组DSPA质粒
将合成的DSPA cDNA序列***至本实验室保存的含有上述表达元件的乳腺特异性表达载体(pCL25)中,新构建成的乳腺特异性表达载体命名为pCL25/DSPA。
3.2.1提取质粒DNA及纯化回收DNA片段
本实验用无内毒素质粒小量提取试剂盒II(OMEGA)制备pCL25+tPA质粒DNA及pUC57+DSPA质粒DNA,实验步骤如下:
(1)将两种质粒分别接种于含有AMP的50mL LB培养液中,37℃摇床培养12h;
(2)室温5,000g离心5min收集细菌;
(3)弃掉LB培养液,加入500μl含RNA酶的Solution I混和液,振荡使之完全悬浮;
(4)将溶液转移到新的2mL离心管,加入500μl Solution II,轻轻颠倒混匀,将混合液室温放置2~3min;Note:加入Solution II后裂解时间不应超过5min;密封保存;
(5)加入事先预冷的N3 Buffer 250μl,轻柔翻转颠倒离心管7-8次,直至管内产生白色絮状物;
(6)室温12000g离心10min,吸取上清液至洁净灭菌指心管内;
(7)加入(6)中管内液体总体积的0.1×的ETR Solution,翻转颠倒7-8次,冰浴静置10min;
(8)事先调整恒温水浴锅至42℃,待(7)中冰浴结束即刻转移至水浴锅,孵育5min;
(9)室温12,000g离心3min,吸取上层无色透明液体转至洁净灭菌指心管内,加入管内液体总体积的0.5倍的无水乙醇,翻转颠倒7-8次,20℃静置2min;
(10)吸取不超过700μL的(9)中液体,转移至含2mL收集管的DNA结合柱中,12000g离心1min,弃掉收集管中的废液;
(11)重复(10),直至全部过柱;
(12)最后一次弃掉废液,加入HBC Buffer 500μl,12000g离心1min,弃废液;
(13)加入DNA洗涤缓冲液700μl,12000g离心1min,弃废液,重复2-3次;
(14)12000g空离2min;
(15)丢弃收集管,将DNA结合柱倒置,待酒精味散去转置于无菌指心管中,加灭菌双蒸水60μl,室温静置2min,12000g离心1min;
(16)将收集到的两种滤液即质粒,分别-20℃保存备用;
将上述两种质粒分别用XhoI限制性内切酶酶切,37℃水浴锅孵育过夜(约8h),酶切体系如下表1:
表1 XhoI酶切反应体系
准备连接所需的DNA片段回收,所用纯化试剂盒为Axygen公司生产,具体步骤如下:
(1)用1×的TAE电泳缓冲液配制0.8%的琼脂糖凝胶,对两种DNA酶切产物分别进行电泳;
(2)电泳条件设置为恒压90V,并观察条带是否跑歪,随时调整,3-4h后结束电泳;
(3)用纸巾吸尽未染色凝胶表面液体,切下凝胶左侧宽约为1cm的长条形胶块,用EB染色20s,脱色5min后,在紫外灯下找到目的DNA的条带并做好标记,以此为参照,切下未染色凝胶中含有目的DNA的琼脂糖凝胶;
(4)计算凝胶质量,先将指心管去皮,称取的凝胶质量不超过300mg,按照说明书计算所加试剂的体积,如:1mg=1μL;
(5)加入(4)中凝胶量的3倍体积的DE-A缓冲液,剧烈上下颠倒后,置于70℃水浴锅内熔化,每隔2min,上下颠倒7-8次,直至管内胶块全部熔化,液体呈均匀透明粉红色;
(6)加入(5)中DE-A缓冲液的0.5倍体积的DE-B缓冲液,颠倒混匀至液体呈均匀透明黄色;
(7)吸取(6)中的黄色液体,转移至含2mL收集管的DNA制备管内,12,000g离心1min,弃去收集管内的废液,重复直至(6)中液体全部过柱;
(8)管内加500μL W1缓冲液,12,000g离心0.5min,倒掉收集管内废液;
(9)管内加750μL W2缓冲液,12,000g离心0.5min,倒掉收集管内废液,重复3次;
(10)12,000g空离2min;
(11)丢弃收集管,将DNA制备柱倒置,待酒精味散去转置于无菌指心管中,加入事先加热至50℃的灭菌双蒸水40μl,室温静置10min,12000g离心1min,-20℃保存备用。
3.2.2重组DSPA与pCL25载体连接
使用Promega公司生产的T4 DNA连接酶,制备pCL25/DSPA质粒,操作步骤如下:
(1)按照下面的公式分别计算出已纯化的DSPA片段和乳腺特异性表达载体pCL25片段,连接时所需的ng,并换算成体积;
(1)经One Drop测定,纯化所得的DSPA片段浓度为191.3ng/μL,纯化所得的pCL25载体部分的片段浓度为148.9ng/μL。根据T4 DNA连接酶的说明书及试验实际情况,进行了调整,反应体系如下(表2):
表2 T4 DNA连接酶反应体系
将上述反应体系放置4℃冰箱内过夜连接,或15℃反应4–18h。
3.2.3重组pCL25/DSPA质粒的转化
重组pCL25/DSPA质粒转化至Trans 10感受态细胞步骤如下:
(1)缓缓吸取50μL冰浴上融化的Trans 10感受态细胞,置于1.5mL离心管中,再轻轻加入10μL上述的连接产物,轻柔的混匀并在冰浴中静置30min;
(2)实验前先将水浴锅精确调节到42℃,用温度计确认后水浴热激45s,然后快速将离心管快速转移到冰水混合物中,冰浴静置2min,切忌转移过程中摇晃离心管;
(3)向离心管中加入500μL无菌的不含氨苄(AMP)的LB液体培养基,轻轻混匀后置于37℃,200rpm/min的摇床上,培养1h,使细胞复苏;
(4)根据实验要求,5000rpm/min离心5min,弃掉300μL上清液,重悬细胞后吸取100μL已转化的感受态细胞加到含AMP的LB固体琼脂培养基上,均匀涂开后,将培养基正放于37℃的恒温培养箱中,20min后倒置LB培养基,37℃过夜培养(12h),于第二天上午取出放置4℃冰箱,准备晚上挑取克隆扩增。
3.2.4 pCL25/DSPA质粒的酶切鉴定及序列分析
取出置于4℃冰箱内的LB固体培养基,挑取单个菌落接种至7mL LB液体培养基(含有AMP,终浓度为50μg/ml)中,一次共挑取48个单个菌落,做好标记并置于37℃,200rpm/min的摇床上过夜培养。摇菌12-14h后使用SDS碱裂解法对每管各提取2ml菌液的质粒,同时将32μl Ribonuclease A(10mg/ml)加入至1568μl DW,保存4℃备用;每管pCL25/DSPA质粒各用30μl的含RNAse的DW溶解,混合均匀后于37℃反应1小时,取2μl质粒提取液用Xho I酶切体系反应2h(酶切反应体系见表3),取5μl反应液电泳,筛选出有且只有1398bp和16341bp两个条带的质粒(即获得***目的基因的重组质粒);将筛选出的样品送上海生工生物工程有限公司测序,鉴定DSPA DNA***的正反向及有无突变情况。通过MegAlign软件比对理论序列与实际测序结果,最终获得正确连接的pCL25/DSPA质粒及菌种,将其置于-20℃保存备用。其中,pCL25/DSPA乳腺特异性表达载体的构建流程见图3。
表3 XhoI酶切反应体系
4.设计要求和鉴定分析
4.1 pCL25/DSPA质粒的酶切鉴定及序列分析结果
本研究用Xho I酶小切48个菌液样本的质粒,随机挑选了23个酶切产物进行琼脂糖凝胶电泳,结果筛选获得9个符合条件的重组质粒,电泳结果见图4;同时从中挑选7个条带较明显的样品送生工测序,经序列分析筛选到3个正向连接且无突变情况的pCL25/DSPA重组质粒,见表4,S6、S25、S27序列与理论序列比对结果一致,见图5。
表4重组pCL25/DSPA质粒连接情况数据
实施例2乳腺特异性表达DSPA基因兔的制备
1.材料
1.1实验动物
本实验所选用的动物均为普通级新西兰白兔,购自江苏省扬州市施桥镇运河种兔场。所有实验用兔自购买14天后方可进行实验,其中,雌兔5-12月龄,雄兔6-12月龄,体重2.5-3.5kg/只。饲养条件:笼养并配备自动冲水***;室内配备空调,全年温度控制在18-25℃,且通风良好;相对湿度在40%-45%;每日光照处理12h;饲喂颗粒饲料早晚各1次,饮水自由。本研究涉及到的所有实验操作均严格按照实验动物使用标准操作规程和伦理道德。
1.2主要仪器
体视显微镜(厦门麦克奥迪公司;TMS,NIKON,Japan);荧光倒置显微镜和显微操作仪(Leica公司,德国;Eppendorf TransferMan NK2公司;IX-71,Olympus,日本);拉针仪(MODEL P-97,INSTRUMENT,USA);磨针仪(BV-10Micropipette SUTTER;Beveler SUTTER);CO2培养箱(MCO-18M ThermoForma);DNA扩增仪(S1000TM Thermal Cycler BiO-RAD公司;PTC-200,基因公司,美国);单人双面净化工作台(苏州净化设备有限公司);DNA电泳仪、电泳槽(南京大学生物技术开发公司);数码凝胶成像仪(GIS-1000,上海天能科技有限公司);制冰机(SIM-F124,日本三洋公司);隔水式电热恒温培养箱(GSP-9080MBE,中国上海);电子天平(FA1604,上海精密天平);PH检测仪(HA405-K2,梅特勒托利多上海公司);电恒温干燥箱(MICRO-4HYBAID);台式高速冷冻离心机(Biofuge公司,德国;Eppendorf公司,德国);低速台式离心机(80-2型,上海医疗器械有限公司手术器械厂);垂直电泳***(POWER-P,厦门市三鹏科技发展有限公司);PVDF膜(美国Pall公司);酶标仪(深圳Rayto有限公司);DNA浓缩仪(Speed Vac Plus SC110,USA);超纯水仪(南京易普易达科技发展有限公司);手提式压力蒸汽灭菌锅(上海***实业有限公司医疗设备厂);恒温水浴锅(北京市长风仪器仪表公司;DK-600型,上海精宏实验设备有限公司);移液器(Eppendorf公司,德国);普通冰箱(青岛海尔公司);超低温冰箱(日本SANYO公司);真空冷冻干燥机(FDU-2100,EYELA,TOKYORIKAKIKAI CO.,LTD,日本);立式恒温振荡器(IS-RDV1,南京畅翔仪器设备有限责任公司);核酸蛋白定量分析仪(One DropTM OD-1000+,基因集团,上海仪涛生物仪器有限公司);荧光定量PCR仪(ABI StepOneTM Plus)。
1.3主要试剂及溶液配制
限制性内切酶Not I、Sal I(宝日医生物技术有限公司TAKARA)及质粒无内毒素提取试剂盒(美国OMEGA);DNA胶纯化回收试剂盒(美国OMEGA);FSH(宁波三生药业有限公司);HCG(江苏省苏北医院生殖科);陆眠宁注射液(吉林华牧动物保健品有限公司);M2、M16培养基(美国Sigma);生理盐水(四川科伦药业有限公司);无水乙醇(上海苏懿化学试剂有限公司);苯酚、蛋白酶K(北京索莱宝科技有限公司);三氯甲烷(国药集团化学试剂有限公司);PCR检测引物由上海生工生物工程股份有限公司合成,其它涉及的固体试剂购自美国Sigma;相关溶液配制如表5。
表5相关试剂配制表格
Table 2-1 The preparation table of related reagents
2.方法
2.1显微注射用基因片段的准备
通过Sal1和Not1双酶切pCL25/DSPA载体(酶切体系如下表6),OMEGA胶回收试剂盒回收大小约17.74kb的转基因片段(如图6),TE稀释到2.5ng/uL后用于显微注射。
2.2供体兔超数***与供受体兔同期发情处理
每组手术挑选2-3只未发情的5-12月龄的母兔作为供体,挑选的特征为母兔的外***无肿胀且颜色泛白越接近惨白更好。挑选的供体用Marker笔在耳内写上笼号以作标记。采用FSH+HCG结合注射方案对供体母兔进行肌注FSH以及耳缘静脉注射HCG,其中每只供体母兔的FSH注射总量为60IU,HCG注射总量为100IU。
在最后一针FSH的当天6:30pm,每组手术选取4-5只受体,挑选的特征为母兔外***肿胀潮红,有明显的湿润度,且伴有特殊气味,与其它母兔一同放置地上时,常发生爬跨现象。挑选的受体用Marker笔在耳内写上笼号以作标记。受体母兔与供体母兔同时耳缘静脉注射HCG 100IU/只,供体母兔准备合笼配种,受体母兔按标记放回笼内,取下食槽作饥饿处理。
2.3收集受精卵
供体兔注射hCG后18~20h,常规输卵管伞部回收受精卵,在体视显微镜下捡取受精卵,计数并移至M16培养液中,置于38℃、5%CO2、饱和湿度培养箱中培养。
2.4显微注射兔受精卵
事先预热操作台,将受精卵转移至高压灭菌的硅化玻璃的M2液滴中,同时准备hold针和注射针,其中hold针的外径约80μm,内径约25μm,注射针的制备则按拉针仪的设定程序。取出分装的DSPA注射用片段,12000r/min离心2分钟,转移至注射针内,各项准备就绪后开始注射,注射部位为细胞核(雄原核)见图7;显微注射结束后将重构卵用M16洗8-10遍,转移至新的M16方杯中,置于恒温培养箱中培养30min,准备胚胎移植手术。
2.5胚胎移植
常规胚胎移植,移植前需观察显微注射卵的存活率,剔除死卵和状态不好的卵。输卵管和卵巢,观察卵巢是否有***点或者滤泡即可移植。胚胎移入输卵管,在输卵管壶腹部注入受精卵。一般情况下,母兔怀孕30天左右会自然分娩,常规母兔产后护理及仔兔哺乳。
2.6出生仔兔基因组DNA的提取
苯酚-氯仿抽提法:
(1)仔兔出生后的第二天,将仔兔编号并用消毒的灭菌眼科剪剪取一小块尾巴组织,放入2mL灭菌管中剪碎,每管内加入组织裂解液750μl及蛋白酶k 7.5μl,用封口膜密封后,置于55℃杂交炉中旋转消化,至组织消化呈透明澄清状为止;
(2)取出消化好的组织液,撕去封口膜,管内加入750μl苯酚,颠倒轻缓混匀,12000r/min离心8min,取上层澄清黏液于2ml灭菌管内;
(3)管内加入750μl 1:1的苯酚-氯仿混合液,颠倒轻缓混匀,12000r/min离心8min,取上层澄清黏液于2mL灭菌管内;
(4)管内加入750μl氯仿,颠倒轻缓混匀,12000r/min离心8min,取上层澄清黏液于2mL灭菌管内;
(5)管内加入事先预冷的异丙醇750μl,多次颠倒混匀沉淀DNA;
(6)取洁净的灭菌指心管,每管内加入70%乙醇1ml,用灭菌枪头挑取白色DNA团,置于此管内,12000r/min离心3min,去上清,重复洗涤一次;
(7)弃上清,12000r/min离心1min,用枪头吸去残余液体,室温下晾干且无酒精味,根据基提取到的DNA团块大小加入灭菌双蒸水50-100μl,37℃,2h溶解或4℃,过夜溶解,-20℃保存。
2.7 PCR整合检测
以乳腺特异性表达载体pCL25/DSPA为模板设计引物,用PCR方法检测出生仔兔是否转入外源基因,电泳结果出现目的条带,则该PCR产物送测序,以进一步验证检测结果。(1)引物设计及合成:以构建载体pCL25/DSPA为模板,应用Primer5.0软件设计一对引物,上游引物在CMV上,下游引物在DSPA编码区上。引物扩增产物大小为722bp,由上海生工生物工程股份有限公司合成。引物如表6:
表6引物信息
Table6 The information of Prime
(2)通过PCR法对出生仔兔基因组DNA进行外源基因整合检测,分别设灭菌双蒸水为空白对照、正常兔基因组DNA为阴性对照、显微注射片段为阳性对照。PCR反应体系如表7:
表7 PCR反应体系
Table 7 The reaction System of PCR
PCR扩增条件为:94℃预变性5min;94℃变性30s,55℃退火30s,72℃延伸50s,重复33个循环;72℃孵育7min;4℃保存。
(3)PCR产物经1%的琼脂糖凝胶电泳检测,完成电泳后用EB染色2min,脱色10min后,置于凝胶成像仪中观察结果,若出生仔兔基因组DNA经PCR反应能扩增出大小约为722bp的条带,则初步证明该仔兔整合了外源基因CMV+DSPA基因,并将该PCR产物送测序,淘汰未整合的仔兔。
(4)通过PCR及序列分析获得的转DSPA基因重点培养,断奶后打耳标进行编号并饲养成熟以备后续实验。
3.技术效果判定和DSPA表达检测
3.1出生仔兔外源基因整合检测
获得的转DSPA基因兔,编号后采样整合检测。结果见图7,其中D4、D7、D9出现整合条带,D13为阳性对照M:DL-2000Marker。
3.2转基因兔乳汁中DSPA检测及活性检测
3.2.1转基因兔乳清的ELISA检测
(1)包被:在酶标条中每孔加入100μL乳清和100μL包被液,4℃过夜;同时加入阿替普酶作为阳性对照,正常兔乳清作为阴性对照,PBS作为空白对照。
(2)洗涤:弃去包被液,PBS-T洗涤3次,每次5min,拍干。
(3)封闭:每孔加入200μL封闭液,37℃水浴2h。
(4)洗涤:弃去封闭液,PBS-T洗涤3次,每次5min,拍干。
(5)加一抗:每孔加入100μL经抗体稀释液以1:2000比例稀释的抗体,37℃水浴2h。
(6)洗涤:弃去一抗液,PBS-T洗涤3次,每次5min,拍干。
(7)加酶标二抗:每孔加入100μL经抗体稀释液以1:2000比例稀释的羊抗鼠IgG-HRP,37℃水浴2h。
(8)洗涤:弃去酶标二抗液,PBS-T洗涤3次,每次5min,拍干。
(9)显色:每孔加入50μL的显色液,37℃避光孵育20min。
(10)终止:每孔加入50μL 2M H2SO4的终止液,终止显色反应。
(11)结果判定:眼观颜色变化,同时应用酶标仪测定OD450值,并拍照、记录。
结果参见图8,所有乳样品均用PBS稀释5倍。A1-A8:阳性标准品(2000,1000,500,250,125,62.5,31.25,0μg/mL);B1-B8,C1-C6:分别是D20、A1、A2、A3、C1、C12、C13、C15、C16、C17、C20、C22、C26、C28转基因兔乳;C7-C8:野生型兔乳汁。
3.2.2转基因兔乳清的Western Blot检测
(1)正确安装垂直电泳槽。
(2)制备12%的分离胶:取事先高压灭菌过的10mL离心管,依次加入3.5mL灭菌重蒸水、4mLA液、2.5mL B液、50μL 10%(NH4)2S2O8和10μL TEMED;快速充分混匀后加到间隙为1.5mm的玻璃板内,直至距离待插梳齿下缘约1cm处停止;加入1mL灭菌重蒸水使之与空气隔绝和起到胶面平整的作用。室温静置待其凝固后,弃掉重蒸水,并吸去剩余水分。
(3)制备4%的浓缩胶:取事先高压灭菌过的10mL离心管,依次加入2.3mL灭菌重蒸水、0.67mL A液、1.0mL C液、30μL 10%(NH4)2S2O8和5μL TEMED;快速充分混匀后加到12%的分离胶上方,直至玻璃板上边缘停止;轻轻***梳齿,室温静置待其凝固后,轻轻拔掉梳齿。
(4)煮样及加样:将30μL样品和6μL 6×电泳loading Buffer用枪吹打使之充分混匀,放入沸水中煮7-8min,使混合液中的蛋白质充分变性;取出后,用小离心机离心5min,去除杂质,将样品依次加入凝胶孔中,每孔加入30-40μL,加样量一般在5-50μg;其中正常兔乳清为阴性对照组,阿替普酶为阳性对照组,添加合适的蛋白质Marker作为参照标准。
(5)电泳:分两次电压进行电泳,首先70V恒压至溴酚蓝染料迁移到浓缩胶末端结束,再120V恒压至溴酚蓝色染料迁移到分离胶底部结束,关闭电源。
(6)SDS-PAGE电泳结束后,轻缓取出凝胶,用重蒸水冲洗3-5次。
(7)按照凝胶胶的大小,裁剪0.45μm PVDF膜,并浸没于无水甲醇中处理45s。
(8)将凝胶、PVDF膜、滤纸等浸泡于转印缓冲液中,平衡10-20min。
(9)在夹子上的放置顺序依次为:滤纸垫、滤纸、PVDF膜、凝胶、滤纸、滤纸垫,轻轻赶走各层之间的气泡之后关闭夹子(尤其重要的是赶走PVDF膜与凝胶之间的气泡,并做好标记),凝胶靠近阴极侧,PVDF膜靠近阳极侧,确保凝胶与PVDF膜完全浸没于转印液中,盖上盖子,开启电源。
(10)以300mA恒流膜转印1.5-2h后结束,取出PVDF膜,观察是否有鲜明的Marker,若有则进行后续操作,若无或淡,则需查明情况后重复该实验。
(11)用灭菌重蒸水冲洗3次,每次5-10min。将PVDF膜充分浸没于10mL封闭液中,4℃过夜或37℃,2h。
(12)取出膜,用TBS-T洗3次,每次5-10min,将膜浸没于10mL经抗体稀释液以1:000比例稀释的Mouse-Anti human t-PA抗体,37℃杂交炉孵育1.5h。
(13)取出膜,用TBS-T洗3次,每次5-10min。将膜浸没于10mL经抗体稀释液以1:1000比例稀释的Goat-Anti Mouse IgG/HRP,37℃杂交炉孵育1.5h。
(14)取出膜,用TBS-T洗3次,每次5-10min,同时预冷仪器,准备显色。
(15)ECL显色:于暗室配制显色液,吸取适量A液B液1:1充分混匀,将膜放入显色液中,显色1min,调整曝光时间,拍照记录。
3.2.3 FAPA溶纤活性检测
采用FAPA和FAPA加热法,进行DSPA转基因兔乳腺表达产物溶解纤维蛋白机理的初步探索。其中,若乳腺表达产物通过FAPA能产生透明溶圈,则表明其具有体外纤溶活性;若表达产物在FAPA加热平板上产生透明圈,且阿替普酶组未产生溶圈,则表明表达产物可以直接降解纤维蛋白。
(1)称量:称取0.15g琼脂糖置于三角烧瓶中,0.015g纤维蛋白原置于2mL离心管中,-20℃取出15U的凝血酶,分别加入适量生理盐水(约20mL,考虑加热蒸发)。
(2)加热:三角烧瓶微波加热后制成1.0%琼脂糖凝胶溶液;同时,纤维蛋白原和凝血酶放入37℃恒温水浴锅溶解预热。
(3)配制溶液:将凝胶糖溶液倒入50mL离心管中待溶液温度降至40℃,依次加入纤维蛋白原、凝血酶,使其终浓度分别为1mg/mL和1U/mL,用枪头轻缓吹打,充分混匀后倒入事先高压灭菌的无色玻璃培养皿内,倾倒时保证凝胶的厚薄均一。
*在往琼脂糖凝胶溶液中加纤维蛋白原时,若发现有絮状物出现,则表明制备失败,需重新配制。
(4)制作纤维蛋白加热平板法:是在FAPA的基础上,将其置于70℃的恒温水浴锅内,加热40min,目的是使平板内的纤溶酶原失活。
(5)打孔封底:室温凝固2-3h,按照加样样品个数合理分散打孔,打孔后用烧热的螺丝钉封底,每个加样孔的直径约为7mm准备加样。
(6)加样:分别加入50μL/孔的待测样品组、阴性对照组、阳性对照组;37℃恒温培养箱培养12h;其中,阿替普酶药物为阳性对照组,正常兔乳清阴性对照。
(7)结果判定:首先观察是否产生纤溶透明圈,再测量透明圈直径(d)大小。若只有待测样品和阳性对照产生透明圈,而阴性对照未变化,则说明待测样品具有纤溶功能,透明圈面积越大则说明溶栓活性越高,拍照记录。结果见图9,其中1:正常公兔乳样品;2:正常母兔乳阴性对照组;3:D49号转基因兔第1d乳样品;4:D49号转基因兔第2d乳样品;5:药用阿替普酶药物(浓度为20μg/mL)。
实施例3从兔乳汁中分离、纯化DSPA
1.1实验仪器
主要实验仪器及设备见表8。
表8主要实验仪器及设备
Table 8 The main experimental instruments and equipment
1.2实验试剂及耗材
本实施例所用实验试剂及耗材相关情况如表9。
表9主要实验试剂及耗材
Table 9 The main experimental material reagents and consumables
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1.3常用试剂配制
CNBr-activated Bestarose 4FF-IgG配基偶联相关试剂相关试剂如表10:
表10 CNBr-activated Bestarose 4FF-IgG配基偶联相关试剂配制表
Table 10 CNBr-activated Bestarose 4FF-IgG ligand conjugation-relatedreagents
蛋白质纯化实验相关试剂配制如表11:
表11蛋白质纯化相关试剂配制表
Table 11 The preparation table of protein purification relatedreagents
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2.1兔乳前处理
rDSPA转基因兔配种分娩后,人工收集乳汁,并储存于-80℃冰箱。纯化兔乳前将保存在-80℃冰箱的转基因兔乳汁50mL于37℃快速融化,融化后4℃,35000g/min,离心30min。离心后的乳汁自上而下分为三层:乳脂、乳清和酪蛋白沉淀(图3-1)。小心地吸出乳清备用,这一步主要是分离乳清和去除酪蛋白。
3.2硫酸铵沉淀兔乳清蛋白
在0℃条件下,硫酸铵浓度55%的饱和硫酸铵进行盐析时比较合适,此时兔乳清中rDSPA几乎完全沉淀下来,除去上清便可除去部分杂蛋白。
3.3苯甲醚亲和层析法纯化转基因兔乳中rDSPA
打开全自动蛋白纯化仪,无菌双蒸水清洗仪器后,将Benzamidine Bestarose4FF(20.0×2.6cm)连接至纯化仪上。用平衡液(20mmol/L磷酸盐缓冲液+0.5mol/L NaCl,pH7.5)平衡层析柱,设置流速10mL/min,柱压上限0.35MPa,待紫外吸收值(UV280)与电导值平稳后再平衡200mL。将经饱和硫酸铵沉淀的兔乳样品用20m mol/L磷酸盐缓冲液(pH7.5)稀释至2L,再与20m mol/L磷酸盐缓冲液+3mol/L NaCl(pH7.5)的溶液以5:1的比例混合后上样,流速8mL/min,并收集穿透液待检测。上样结束后继续用3倍柱体积的平衡液(20mmol/L磷酸盐缓冲液+0.5mol/L NaCl,pH7.5)淋洗层析柱至紫外吸收线(UV280)归零,并再平衡200mL。用洗脱液(0.1mol/L甘氨酸,pH3.0)洗脱,收集洗脱液,立刻向洗脱液中加入碱性中和液(1mol/L Tris-HCL,pH9.0),将溶液pH调至7.5左右。纯化峰图如图10(其中,Peak 1:穿透峰;Peak 2:洗脱峰)。收集液FAPA溶纤活性检测、鉴定结果见图11(1-5:苯甲醚亲和层析洗脱液;6-9:苯甲醚亲和层析再生液;10:阿替普酶(20μg/mL);11:rDSPA转基因兔乳;12:苯甲醚亲和层析上样液;13:苯甲醚亲和层析穿透液;14:阴性兔乳清;15:空白;16:PB),验证了应用苯甲醚亲和层析可从兔乳清硫酸铵沉淀物中提取rDSPA。
3.4强阳离子交换层析法纯化转基因兔乳中rDSPA
打开全自动蛋白纯化仪,无菌双蒸水清洗仪器后,将强阳离子交换层析柱(Capto S ImpAct column,20mL,GE医疗生命科学,瑞典)连接至纯化仪上。用平衡液(20mmol/L甘氨酸溶液,pH8.75)平衡层析柱,设置流速7mL/min,柱压上限0.35MPa,待紫外吸收值(UV280)与电导值平稳后再平衡90mL。将兔乳前处理样品(调pH至8.75)与20mmol/L甘氨酸溶液(pH8.75)的溶液以1:3的比例混合后上样,流速3mL/min,并收集穿透液待检测。上样结束后继续用6倍柱体积平衡液(20mmol/L甘氨酸溶液,pH8.75)洗层析柱至紫外吸收线(UV280)归零。洗脱条件设置为20mM甘氨酸+0.31M氯化钠。强阳离子交换层析洗脱峰见图12,rDSPA主要在Peak 2中。活性检测见图13(1:强阳离子交换层析上样液;2:强阳离子交换层析穿透液;3-10:强阳离子交换层析洗脱液;11:强阳离子交换层析再生液;12:阴性兔乳清;13:阿替普酶(20μg/mL);14:PB),分离获得DSPA蛋白电泳图见图14,验证了应用强阳离子交换明显提高rDSPA纯度。/>
3.5活性蓝亲和层析法纯化转基因兔乳中rDSPA
蛋白纯化仪开机,无菌超纯水清洗后,将Blue Sepharose 6Fast Flow(5.0×1.2cm,GE医疗生命科学,瑞典)连接至纯化仪上。用平衡液20mmol/L磷酸盐缓冲液(pH 7.5)平衡,流速3mL/min,待紫外吸收值(UV280)与电导值稳定后再平衡30mL。兔乳前处理样品用20mmol/L磷酸盐缓冲液(pH7.5)稀释至500mL后上样,流速3mL/min,并收取穿透液备后续检测。上样结束后继续用3倍柱体积的平衡液继续淋洗至紫外吸收(UV280)归零电导值平稳。20mM PB+0.15M氯化钠,丢弃;20mMPB+0.4M氯化钠,可回收重复用;20mMPB+1.0M氯化钠,收集,脱盐为纯品。纯化峰图如图15。收集的各管样品溶液用于FAPA溶纤活性检测,发现活性蓝亲和层析洗脱液中有明显透明溶纤圈(见图16,其中1:活性蓝亲和层析上样液;2:活性蓝亲和层析穿透液;3:活性蓝亲和层析洗脱峰1洗脱液;4:活性蓝亲和层析洗脱峰3洗脱液;5,6:活性蓝亲和层析洗脱峰4洗脱液;7,8活性蓝亲和层析洗脱峰5洗脱液;9,10:活性蓝亲和层析再生液;11:阴性兔乳清;12:PB;13:阿替普酶(20μg/mL)),验证了活性蓝亲和层析柱可纯化rDSPA。
3.6纯化产物去除内毒
打开全自动蛋白纯化仪,无菌双蒸水清洗仪器后,将阴离子交换层析柱(HiTrap Capto Q column,1mL,GE医疗生命科学,瑞典)连接至纯化仪上。用平衡液(20mmol/L Tris-HCL,pH8.0)平衡层析柱,设置流速1mL/min,柱压上限0.35MPa,待紫外吸收值(UV280)与电导值平稳后再平衡3-5倍柱体积。将3.5中的脱盐样品上样,流速1mL/min。用无内毒素试管收集穿透液。上样结束后继续用平衡液(20mmol/L Tris-HCL,pH8.0)洗层析柱至紫外吸收线(UV280)归零。然后用洗脱液(20mmol/L Tris-HCL+1mol/L NaCl,pH8.0)洗脱。峰图如图17所示。
纯化产物的内毒素检测结果如图18所示,A:除内毒素后的DSPA;B:阴性对照(无内毒素水);C:阳性对照(内毒素);纯化样品与阴性对照均是可流动的液态,未有凝胶固体形成,而阳性对照成了不可流动的凝胶固态。从而判定去除内毒素后的纯化样品中内毒素含量小于0.015EU/mL。
显然,上述实施例仅仅是为清楚地说明技术方案而作的举例,并非对本发明实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明要求的保护范围之内。
序列表
<110> 扬州大学
<120> 一种适用于家兔乳腺特异性表达去氨普酶的编码基因及其应用
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<170> SIPOSequenceListing 1.0
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ttcctgtgtg gggggatact gatcagttcc tgctgggtcc tgactgctgc ccactgcttc 780
caggagagct atcttcctga ccagcttaag gtggttttgg gcaggacata ccgggtgaaa 840
cctggagagg aagagcagac atttaaagtc aaaaaataca tcgtccataa ggaatttgat 900
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gcccaagaga gtgacagtgt ccgcgccatt tgtctccctg aagccaacct gcagctgccc 1020
gactggacag aatgtgagct gtctggctac ggcaagcata agtcatcttc tcctttctat 1080
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tttctgttta acaaaaccgt cacaaacaac atgctgtgtg ctggagacac ccggagcgga 1200
gagatctatc caaatgtgca cgatgcctgc cagggtgact caggaggccc cttggtgtgt 1260
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Met Lys Cys Leu Leu Leu Ala Leu Gly Leu Ala Leu Ala Cys Gly Ile
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Tyr Arg Arg Gln Glu Ser Trp Leu Arg Pro Glu Val Arg Ser Lys Arg
35 40 45
Val Glu His Cys Gln Cys Asp Arg Gly Gln Ala Arg Cys His Thr Val
50 55 60
Pro Val Asn Ser Cys Ser Glu Pro Arg Cys Phe Asn Gly Gly Thr Cys
65 70 75 80
Trp Gln Ala Val Tyr Phe Ser Asp Phe Val Cys Gln Cys Pro Ala Gly
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Tyr Thr Gly Lys Arg Cys Glu Val Asp Thr Arg Ala Thr Cys Tyr Glu
100 105 110
Gly Gln Gly Val Thr Tyr Arg Gly Thr Trp Ser Thr Ala Glu Ser Arg
115 120 125
Val Glu Cys Ile Asn Trp Asn Ser Ser Leu Leu Thr Arg Arg Thr Tyr
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Asn Gly Arg Met Pro Asp Ala Phe Asn Leu Gly Leu Gly Asn His Asn
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Tyr Cys Arg Asn Pro Asn Gly Ala Pro Lys Pro Trp Cys Tyr Val Ile
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Lys Ala Gly Lys Phe Thr Ser Glu Ser Cys Ser Val Pro Val Cys Ser
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Lys Ala Thr Cys Gly Leu Arg Lys Tyr Lys Glu Pro Gln Leu His Ser
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Thr Gly Gly Leu Phe Thr Asp Ile Thr Ser His Pro Trp Gln Ala Ala
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Ile Phe Ala Gln Asn Arg Arg Ser Ser Gly Glu Arg Phe Leu Cys Gly
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Gly Ile Leu Ile Ser Ser Cys Trp Val Leu Thr Ala Ala His Cys Phe
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Gln Glu Ser Tyr Leu Pro Asp Gln Leu Lys Val Val Leu Gly Arg Thr
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Tyr Arg Val Lys Pro Gly Glu Glu Glu Gln Thr Phe Lys Val Lys Lys
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Tyr Ile Val His Lys Glu Phe Asp Asp Asp Thr Tyr Asn Asn Asp Ile
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Ala Leu Leu Gln Leu Lys Ser Asp Ser Pro Gln Cys Ala Gln Glu Ser
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Asp Ser Val Arg Ala Ile Cys Leu Pro Glu Ala Asn Leu Gln Leu Pro
325 330 335
Asp Trp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys His Lys Ser Ser
340 345 350
Ser Pro Phe Tyr Ser Glu Gln Leu Lys Glu Gly His Val Arg Leu Tyr
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Pro Ser Ser Arg Cys Ala Pro Lys Phe Leu Phe Asn Lys Thr Val Thr
370 375 380
Asn Asn Met Leu Cys Ala Gly Asp Thr Arg Ser Gly Glu Ile Tyr Pro
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Asn Val His Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys
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Met Asn Asp Asn His Met Thr Leu Leu Gly Ile Ile Ser Trp Gly Val
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Gly Cys Gly Glu Lys Asp Val Pro Gly Val Tyr Thr Lys Val Thr Asn
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agggcactga cagacaaa 18
Claims (8)
1. 一种适用于家兔乳腺特异性表达去氨普酶的编码基因,其特征在于,所述基因的核苷酸序列如SEQ ID NO.1所示。
2. 一种由权利要求1所述基因编码的去氨普酶,其特征在于,所述去氨普酶的氨基酸序列如SEQ ID NO.2所示。
3.权利要求1所述编码基因在制备去氨普酶中的应用。
4.根据权利要求3所述的应用,其特征在于,通过家兔乳腺生物反应器来制备去氨普酶。
5.根据权利要求4所述的应用,其特征在于,制备的方法如下:
(1)构建乳腺特异性表达去氨普酶基因载体;
(2)制备乳腺特异性表达去氨普酶基因兔;
(3)兔乳汁中去氨普酶的鉴定及溶纤活性分析,兔配种分娩后,收集乳汁;
(4)对乳汁中的去氨普酶重组蛋白进行提取和纯化。
6.一种从兔乳中提取和纯化去氨普酶重组蛋白的方法,所述兔为乳腺特异性表达权利要求1所述的去氨普酶基因的兔,其特征在于,所述的方法联用苯甲醚亲和层析、阳离子交换层析和活性蓝亲和层析进行分离纯化。
7.根据权利要求6所述提取和纯化去氨普酶重组蛋白的方法,其特征在于,所述的方法包括如下步骤:
(1)兔乳通过超速离心、55%硫酸铵沉淀初步分离兔乳中的去氨普酶;
(2)苯甲醚亲和层析:
A.用平衡液平衡层析柱;平衡液为20mmol/L甘氨酸溶液,pH8.75;
B.平衡后以流速3mL/min上样,上样结束后用6倍柱体积平衡液洗涤层析柱至紫外吸收线UV280归零;
C.淋洗条件为20 mmol/L磷酸盐缓冲液+0.5 mol/L NaCl,pH7.5;洗脱条件设置为0.1mol/L甘氨酸,pH3.0;
(3)阳离子交换层析:
A.平衡液平衡层析柱,流速5mL/min,待紫外吸收值UV280与电导值稳定后再平衡200mL;平衡液为50 mmol/L磷酸盐缓冲液,pH8.0;
B.将淋洗条件设置为20 mmol/L甘氨酸溶液,pH8.75;洗脱条件设置为20m mol甘氨酸+0.31mol氯化钠;
(4)蓝柱亲和层析:
A.平衡液平衡,流速3 mL/min,待紫外吸收值UV280与电导值稳定后再平衡30 mL;所述平衡液为50mmol/L磷酸盐缓冲液,pH8.0;
B.20mmol/L磷酸盐缓冲液pH7.5稀释至500mL后上样,流速3mL/min;
C.20mmol/L磷酸盐缓冲液pH7.5+0.15 mol/L氯化钠洗脱液丢弃;20 mmol/L磷酸盐缓冲液pH7.5+0.4mol/L氯化钠洗脱液可回收重复用;20mmol/L +1.0mol/L氯化钠收集;
(5)脱盐成为去氨普酶纯品。
8.根据权利要求7所述提取和纯化去氨普酶重组蛋白的方法,其特征在于,所述的方法还包括:将纯化的去氨普酶应用阴离子交换层析去除纯化产物中的内毒素。
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