CN1276834A - 新的修饰的MSP－1核酸序列以及增加细胞***中mRNA水平和蛋白质表达的方法 - Google Patents
新的修饰的MSP－1核酸序列以及增加细胞***中mRNA水平和蛋白质表达的方法 Download PDFInfo
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Abstract
本发明提供了修饰的重组核酸序列(优选的是DNA)以及增加疟原虫表面蛋白MSP－1的mRNA水平和蛋白质表达的方法,已知该蛋白质在细胞培养***、哺乳动物细胞培养***或转基因动物中难以表达。使用本发明重组技术表达的优选的“困难”蛋白质候选物是MSP－1蛋白质,它是由相对天然MSP－1基因含有降低的总AT含量或富AT区和/或mRNA不稳定基序和/或稀有密码子的DNA编码序列表达的。
Description
本发明的背景发明领域
本发明涉及异源基因表达。更具体地，本发明涉及在较高等的真核细胞***中表达疟原虫(malaria)基因。
相关现有技术综述
在体外细胞培养***或体内重组生产***中进行特定的异源基因产物的重组生长通常是困难的。例如，许多研究人员发现，在与蛋白质原始来源的细胞不同的细胞培养***，特别是哺乳动物细胞培养***中难以表达来自细菌、寄生生物和病毒的蛋白质。难以由哺乳动物细胞生产的医疗上重要蛋白质的一个例子是疟原虫裂殖子表面蛋白(malaria merozoite surface protein)(MSP-1)。
疟疾在热带国家是严重的健康问题。对现存药物的抗性迅速发展，因而急需疫苗。在恶性疟原虫(P.falciparum)生长周期中表达的多种抗原中，MSP-1是研究最深入的并且看来是最成功的接种候选。暴露于恶性疟原虫的个体产生抗MSP-1抗体，研究表明在对MSP-1的天然获得性免疫反应和减低的疟疾发病率之间有相关性。在一系列研究中，用纯化的天然MSP-1或该蛋白的重组片段免疫可以诱导至少部分对该寄生生物的防护(Diggs等，(1993)Parasitol Today9：300-302)。因此MSP-1是开发抗恶性疟原虫疫苗的重要靶。
MSP-1是一种190-220kDA的糖蛋白。C-末端区域已经是可用作疫苗的重组产物的焦点。然而，开发MSP-1用作疫苗的一个主要问题是在细菌或酵母表达***难以获得与亲和纯化的天然蛋白质具有同等免疫学能力的重组蛋白(Chang等，(1992)免疫学杂志(J.Immunol.)148：548-555)以及使疫苗生产可行的足够量的重组蛋白。
增强足够量MSP-1表达的改进的方法是有利的。
本发明的概述
本发明提供了改进的重组DNA成分和增加疟原虫表面抗原MSP-1在细胞培养***、哺乳动物细胞培养***或转基因动物中的mRNA水平和蛋白质表达的方法。在本发明的表达***表达的优选蛋白候选物是相对重组表达***而言具有降低的AT含量和消除了mRNA不稳定基序以及稀有密码子的DNA编码序列的MSP-1的C-末端衍生物。因而，第一个方面，本发明提供了从序列2衍生的DNA序列。此衍生序列显示于序列1。
第二个方面，本发明提供了制备本发明修饰核酸的方法，包括降低编码MSP-1的天然基因的总AT含量，消除所有mRNA不稳定基序以及使用乳腺组织偏爱密码子置换所有稀有密码子(均采用乳腺组织识别的、优选的为偏爱的并且编码与所置换的密码子同样的氨基酸的密码子来置换天然基因中的特定密码子)等步骤。本发明的这一方面进一步包括依据本发明方法制备的修饰核酸。
第三个方面，本发明亦提供了含有本发明修饰MSP-1核酸和山羊β-酪蛋白启动子以及信号序列的载体，以及用本发明核酸转化的宿主细胞。
第四个方面，本发明提供了种系含有本发明核酸的转基因的非人哺乳动物。
第五个方面，本发明提供了含有依据本发明的修饰MSP-1基因的DNA疫苗。
图的说明
图1描绘了依据本发明修饰的MSP-142的cDNA序列[序列1]，其中置换了306个核苷酸以降低AT含量并消除了mRNA不稳定基序同时保持MSP-142的相同蛋白质氨基酸序列。大写字母表示核苷酸置换。
图2描绘了编码“野生型”或天然MSP-142序列的核苷酸序列[序列2]。
图3是野生型MSP-142(在表中命名为“MSP-1wt”)和新修饰的MSP-142基因(在表中命名为“编辑的MSP”)以及几种乳蛋白质基因(来自山羊和小鼠的酪蛋白基因)的密码子使用表。每一栏中的数目表示在列出的各基因中特定密码子出现的实际次数。新的MSP-142合成基因是通过首先选择给定氨基酸的富GC密码子结合选择在乳蛋白中最常使用的氨基酸从***特异密码子用法中产生的。
图4a-c分别描绘了如实施例中所述的MSP-142构建体GTC479，GTC564和GTC627。
图5A栏是Northern检测，其中构建体GTC627含有依据本发明修饰的新MSP-142基因，GTC479是含有天然MSP-142基因的构建体，而GTC469是阴性对照DNA。
图5B栏是亲和纯化后洗脱组分的Western检测。数字是收获的级分。结果表明来自修饰MSP-142合成基因构建体GTC679的级分与MSP-1多克隆抗体反应而阴性对照GTC479则不反应。
图6描绘了实施例中所述的OT1[序列3]、OT2[序列4]、MSP-8[序列5]、MSP2[序列6]、MSP-1[序列7]的核酸序列。
图7图示质粒BC574。
图8图示BC620。
图9图示BC670。
图10图示转基因乳汁中的MSP的Westen印迹。
图11图示MSP42-2的核苷酸序列[序列8]。
图12图示BC718。
图13图示在转基因乳汁中BC718的表达的Westen印迹。
优选实施方案的详细描述
本专利和此处引用的科学文献建立了本技术领域熟练技术人员可利用的知识。颁布的美国专利、允许的申请、公开的外国申请以及此处引用的文献，通过参考文献形式并于此处。解决这些文献和本公开之间的任何冲突均以本发明为准。
本发明提供了改进的重组DNA成分以及增加疟原虫表面抗原MSP-1在细胞培养***、哺乳动物细胞培养***或转基因动物中mRNA水平和蛋白质表达的方法。在本发明的表达***表达的优选蛋白候选物是相对重组表达***而言具有降低的AT含量和消除了mRNA不稳定基序以及稀有密码子的DNA编码序列的MSP-1的C-末端衍生物。因而，第一个方面，本发明提供了从序列2衍生的DNA序列。此衍生序列显示于序列1。
在优选实施方案中，本发明核酸在哺乳动物细胞培养***或转基因动物中能够以天然基因在相同条件下于哺乳动物细胞培养***或转基因动物中表达水平的至少25％，优选的是50％，更优选的至少为100％或更高的水平表达MSP-1。
此处使用的术语“表达”是指导致蛋白质表达的mRNA转录。可通过本技术领域已知的一系列技术测定表达，包括使用对目的蛋白特异的抗体。“天然基因”或“自然基因”是指编码野生型MSP-1的和作为修饰核酸来源的基因序列或其片段(包括天然出现的等位基因变异)。“偏爱(优选)密码子”是指修饰的MSP-1要在其中表达的细胞或组织类型例如***组织更常用的密码子。并非所有在此描述的密码子改变都是变成偏爱密码子，只需置换的密码子至少能被小鼠***组织识别。此处使用的术语“减少的AT含量”是指由于在不改变靶蛋白的氨基酸序列的前提下使用小鼠***组织识别的核苷酸或密码子置换含A或T的核苷酸位点或含A和/或T的密码子而比天然MSP-1基因具有较低的含A(腺嘌呤)T(胸腺嘧啶)碱基的核苷酸的总百分数。
第二个方面，本发明提供了制备修饰核酸的方法，包括降低编码MSP-1的天然基因的总AT含量，消除所有mRNA不稳定基序以及使用乳腺组织的偏爱密码子置换所有稀有密码子(均采用乳腺组织识别的、优选地为偏爱的并且编码与所置换的密码子的相同的氨基酸的密码子来置换天然基因中的特定密码子)。描述重组DNA技术的一般原理的标准参考书包括Watson，J.D.等，基因的分子生物学(MolecularBiology of the Gene)卷I和卷II，the Benjamin/Cummings PublishingCompany，Inc.publisher，Menlo Park，CA(1987)Darnell，J.E.等，分子细胞生物学(Molecular Cell Biology)，Scientific AmericanBooks，Inc.，Publisher，New York，NY(1986)；Old，R.W.等，基因操作原理：基因工程入门(Principle of Gene Manipulation：AnIntroduction to Genetic Engineering)，第二版，University ofCalifornia Press，publisher，Berkeley CA(1981)；Maniatis，T.等，分子克隆：实验室手册(Molecular Cloning：A Laboratory Manual)，第二版，冷泉港实验室(Cold Spring Harbor Laboratory)，publisher，冷泉港(Cold Spring Harbor)，NY(1989)和分子生物学通用流程(Current Protocols in Molecular Biology)，Ausubel等，Wiley Press，New York，NY(1992)。本发明的这一方面进一步包括依据本发明方法制备的修饰核酸。
不局限于任何原理，以前的研究表明GM-CSF mRNA的3’非翻译区的一段保守AU序列(AUUUA)介导选择性mRNA降解(Shaw，G.和Kamen，R.细胞(Cell)46：659-667)。过去的焦点在于基因非翻译区的这些不稳定基序的存在。本发明是第一个意识到消除MSP-1基因编码区的不稳定序列的益处。
第三个方面，本发明还提供了含有本发明修饰MSP-1核酸和山羊β-酪蛋白启动子及信号序列的载体，以及用本发明核酸转化的宿主细胞。
第四个方面，本发明还提供了种系含有本发明核酸的转基因非人哺乳动物。本技术领域已知产生转基因动物的一般原理。参看例如Hoganet al.，小鼠胚胎操作：实验室手册(Manipulating the Mouse Embryo：A Laboratory Manual)，冷泉港实验室(Cold Spring HarborLaboratory)，(1986)；Simons et al，Bio/Technology 6：179-183，(1988)；Wall et al.，Biol.Reprod.32：645-651，(1985)；Buhleret al.，Bio/Technology 8：140-143(1990)；Ebert et al.，Bio/Technology 9：835-838(1991)；Krimenfort et al.，Bio/Technology 9：844-847(1991)；Wall et al.，细胞生物化学杂志(J.Cell.Biochem.)49：113-120(1992)。将外源DNA序列导入哺乳动物及其生殖细胞的技术最初是在小鼠中开发的。参看例如，Gordon et al.，美国国家科学院院刊(Proc.Natl.Acad Sci.USA)77：7380-7384，(1980)；Gordon and Ruddle科学(Science)214：1244-1246(1981)；Palmiter and Brinster，细胞(Cell)41：343-345，1985；Brinster et al.，美国国家科学院院刊(Proc.Natl.Acad.Sci.USA)82：4438-4442(1985)and Hogan et al.(出处如上)。随后将这些技术修饰为用于大型动物包括牛和羊。直至最近，产生转基因小鼠或家畜的最广泛应用的方法是将感兴趣的上百个线性DNA分子以转基因表达构建体的形式注射到受精卵的一个前核。将DNA注射到受精卵的细胞质亦有广泛应用。最近，能够注射到未受精卵的完整转基因细胞系的克隆已获得成功(KHS Campbell et al.，自然(Nature)380 64-66，(1996))。
乳腺表达***具有高表达水平、低成本、正确加工和可及性的优点。多名研究人员已经在泌乳转基因动物中产生了已知蛋白质，诸如牛和人α-乳清蛋白。(Wright et al.，Bio/technology9：830；834(1991)；Vilotte et al，欧洲生物化学杂志(Eur.J.Biochem.)，186：43-48(1989)；Kochi et al.，Mol Reprod，And Devel.33：160-164(1992)；Soulier et al.，FEBS Letters 297(1，2)：13-18(1992))，并且该***显示可以产生高水平的蛋白质。
第五个方面，本发明提供了含有依据本发明的修饰MSP-1基因的DNA疫苗。这种DNA疫苗可以不进行胶囊化给药，或可作为脂质体的部分给药，或作为病毒基因组的部分给药。通常以足够使修饰MSP-1基因表达并在接受DNA疫苗的动物体包括人体诱发抗体反应的量使用这种疫苗。随后至少在第一次给药后一星期之后可再次给药以增强抗体反应。优选的给药途径包括经粘膜以及肠胃外给药。
实施例
制造新的修饰MSP-142基因
提供了编码MSP-1C-末端片段的新的修饰核酸。图1显示了能够在本发明哺乳动物细胞中表达的编码MSP-1 42kD C-末端片段(MSP-142)的本发明新的修饰核酸。天然MSP-142基因(图2)不能够在哺乳动物细胞培养或转基因小鼠中表达。对天然MSP-142基因的分析表明其多种特征与哺乳动物基因有区别。首先，它有高达76％的AT含量。其次，在此1100bp的DNA片段(图2)中，mRNA不稳定基序AUUUA出现10次。为了针对这些区别，设计了新的MSP-142基因。向天然MSP-142基因的306个位点引入了沉默核苷酸置换将总AT含量降至49.7％。同样经过密码子用法的改变消除了天然基因中的10个AUUUA mRNA不稳定基序。为了改变密码子用法，使用多种小鼠和山羊***特异蛋白质制出了***组织特异性密码子使用表图3a。将该表用于指导如上所述修饰MSP-142基因的密码子用法选择。例如图3a中的表中所示，在天然基因中，65％(25/38)的亮氨酸由TTA编码，它在乳腺中是稀有密码子。在修饰的MSP-142基因中，100％的亮氨酸由CTG编码，而后者是乳腺中亮氨酸的优选密码子。
使用修饰的MSP-142基因构建了表达载体，其是通过将山羊β-酪蛋白的前端26个氨基酸融合到修饰MSP-142基因的N-末端和将携带融合基因的SalI-Xho I片段亚克隆到表达载体pCDNA3的XhoI位点而进行的。将His6标记融合到MSP-142基因的3’末端使基因产物能够亲和纯化。这样制备了质粒GTC627(图4c)
为了比较天然MSP-142基因构建体和本发明修饰的MSP-142核酸，亦构建了天然MSP-142基因的表达载体并将该基因加到哺乳动物细胞培养物和注射到小鼠中形成转基因小鼠，如下所述：
构建天然MSP-142表达载体
为了分泌截短的恶性疟原虫裂殖子表面蛋白-1(MSP-1)，将编码MSP-1 42kD C-末端部分(MSP-142)的野生型基因融合到编码山羊β-酪蛋白的前15或前26个氨基酸的DNA序列中。这是通过首先用引物MSP1和MSP2(图6)PCR扩增MSP-1质粒(来自Dr.David Kaslow，NIH)，然后将PCR产物克隆到TA载体(Invitrogen)获得的。将PCR产物的BglII-XhoI片段与寡聚物OT1和OT2(图6)接进入表达载体pCDNA3。这样产生了质粒GTC564(图4b)，它编码15个氨基酸的β-酪蛋白信号肽和成熟山羊β-酪蛋白的前11个氨基酸，随后是天然MSP-142基因。使用寡聚物MSP-8和MSP-2(图6)通过PCR扩增MSP-1质粒，然后将产物克隆到TA载体。切下XhoI片段并克隆到表达载体pCDNA3的XhoI位点产生质粒GTC479(图4a)，它编码融合到野生型MSP-142基因的15个氨基酸的山羊β-酪蛋白信号肽。在GTC564和GTC479中的MSP-142基因3’末端加上了组氨酸6(His6)标记。
在COS-7细胞中未表达天然MSP-142基因
通过瞬时转染检测测定了培养的COS-7细胞中天然MSP基因的表达。按照厂商手册使用脂质转染胺试剂(Gibco-BRL)将GTC479和GTC564质粒DNA导入COS-7细胞。转染两天后从COS细胞分离总细胞RNA。转染两天后通过将35S甲硫氨酸加入培养物中代谢标记新合成的蛋白质10个小时。
为了测定COS细胞中的MSP mRNA表达，用来自GTC479的32P标记DNA片段探测Northern印迹。在GTC479或GTC564转染体中未测到MSP RNA(未发表资料)。延长的曝光揭示有残余水平的降解的MSPmRNA。用抗MSP的多克隆抗体免疫沉淀35S标记的培养物上清和裂解物。免疫沉淀实验显示GTC479或GTC564转染细胞的裂解物或上清中没有表达(未发表资料)。这些结果显示天然MSP-1未在COS细胞中表达。
在转基因小鼠乳腺中没有天然MSP-142基因的表达
将编码15个氨基酸的山羊β-酪蛋白信号肽，山羊β-酪蛋白的前11个氨基酸以及天然MSP-142基因的GTC479 SalI-XhoI片段克隆到载体BC350中表达的β-酪蛋白XhoI位点。这样产生质粒BC574(图7)。将BC574的SalI-NotI片段注射到小鼠胚胎产生转基因小鼠。建立了15个株系的转基因小鼠。收集雌性建立者小鼠的乳汁并使用抗MSP多克隆抗体进行Western检测。检测的7个小鼠乳汁中均未发现表达MSP-142蛋白质。为了进一步确定在乳腺是否有MSP-142的mRNA表达，从第11天的泌乳转基因小鼠提取总RNA并进行Northern印迹检测。检测的任何一个BC574株系均未测到MSP-142 mRNA。因而，MSP-142转基因未在转基因小鼠的乳腺中表达。总的来说，这些实验提示天然寄生生物的MSP-142基因不能在哺乳动物细胞中表达，其阻断是mRNA丰度的水平的。
哺乳动物细胞中MSP的表达
进行了瞬时转染实验以评价本发明修饰的MSP-142基因在COS细胞中的表达。将GTC627和GTC479的DNA导入COS-7细胞。转染后48小时分离总RNA进行Northern检测。用32P标记GTC627的SalI-XhoI片段探测固定化RNA。观察到GTC479和GTC627的显著不同。如前面显示，GTC479转染细胞中未检测到MSP-142mRNA，而在GTC627中表达了丰富的MSP-142mRNA(图5，A栏)。GTC469用作阴性对照并且含有克隆到商品化克隆载体PU19的GTC564的***片段。使用35S甲硫氨酸的代谢标记实验以及随后的使用抗MSP的多克隆抗体的免疫沉淀(由NIH的D.Kaslow NIAID提供)显示转染的COS细胞合成了MSP-142蛋白质(图5，B栏)。此外，在转染的COS细胞上清中检测到MSP-142，表明MSP-142蛋白质亦分泌出来。此外，使用Ni-NTA柱，从GTC627转染的COS上清中亲和纯化了MSP-142。
这些结果证明寄生性MSP-142基因的修饰导致MSP的mRNA在COS细胞中表达。随后，哺乳动物细胞合成和分泌MSP-142产物。
亦可使用本技术领域熟知的方法制备本实验中使用的多克隆抗体(抗体：实验室手册(Antibodies：A Laboratory Manual)，Ed Harlowand David Lane，eds.冷泉港实验室(Cold Spring HarborLaboratory)，publishers(1988))。MSP血清抗体的产生亦描述于Chang et al.，感染和免疫(Infection and Immunity)(1996)64：253-261 and Chang et al.，(1992)美国国家科学院院报(ProcNatl.Acad.Sci.USA)86：6343-6347。
这些检测表明与根本不表达的天然蛋白质相比，本发明的修饰MSP-142核酸可以高水平表达。这些结果代表了降低基因中的AT％导致MSP基因在异源***中表达的第一个实验证据，同时代表了从MSP编码区消除AUUUA mRNA不稳定基序导致MSP蛋白质在COS细胞中表达的第一个证据。图5A栏Northern的结果(即天然基因无RNA而本发明的修饰DNA序列有合理的水平的RNA)很可能解释了蛋白质产生的增加。
下面的实施例描述了MSP1-42作为天然未融合(并且未糖基化)蛋白质在转基因小鼠乳汁中的表达。
MSP转基因的构建
为了将MSP1-42与15个氨基酸的β-酪蛋白信号肽融合，将编码15个氨基酸酪蛋白信号和结束于Cla I位点的MSP1-42的前5个氨基酸的寡聚物对MSP203和MSP204(MSP203：ggccgctcgacgccaccatgaaggtcctcataattgcctgtctggtggctctggccattgcagccgtcactccctccgtcat，MSP204：cgatgacggagggagtgacggctgcaatggccagagccaccagacaggcaattatgaggaccttcatggtggcgtcgagc)与编码其余的MSP1-42基因的BC620的CLA I-Xho I片段(图8)连接进入表达载体pCDNA3的Xho I位点。然后将这个质粒(GTC669)的Xho I片段克隆到乳汁特异性表达载体BC350的Xho I位点产生B670(图9)。
MSP1-42在转基因小鼠乳汁中的表达
从质粒BC670制备Sal I-Not I片段并微注射到小鼠胚胎产生转基因小鼠。通过从尾巴活组织检查提取小鼠DNA随后使用寡聚物GTC17和MSP101(寡聚物的序列：GTC17，GATTG ACAAG TAATA CGCTG TTTCCTC寡聚物MSP101，GGATTCAATAGATACGG)进行PCR检测确认转基因小鼠。在哺乳第7和9天收集雌性建立者转基因小鼠的乳汁，并使用多克隆抗-MSP抗体和单克隆抗MSP抗体5.2(Dr.David Kaslow，NIH)通过western检测确定MSP-1-42的表达水平。结果表明转基因小鼠乳汁中MSP1-42的表达水平是1-2毫克/毫升(图10)。
构建MSP1-42糖基化位点负突变体
我们对乳汁产生的MSP检测揭示转基因MSP蛋白质是N-糖基化的。为了消除MSP1-42基因中的N-糖基化位点，用谷氨酰胺(Q)取代了181和262位点的天冬酰胺(N)。通过设计与MSP1相应区域退火的携带AAC到CAG突变的DNA寡聚物导入了置换。然后将这些寡聚物用作PCR引物产生编码N至Q置换的DNA片段。
为了导入N262-Q突变，使用一对寡聚物MSPGYLYCO-3(CAGGGAATGCTGCAGATC AGC)和MSP42-2(AATTCTCGAGTTAGTGGTGGTGGTGGTGGTGATCGCAGAAAATAC CATG图11)PCR扩增含有合成的MSP1-42基因的质粒GTC627。将PCR产物克隆到pCR2.1载体(Invitrogen)。这样产生了质粒GTC716。
为了导入N181-Q突变，使用寡聚物 MSPGLYCO-1(CTCCTTGTTCAGGAACT TGTAG GG和MSPGLCO-2(GTCCTGCAGTACACATATGAG图4)PCR扩增质粒GTC627。将PCR产物克隆到pCR2.1载体。这样产生了质粒GTC700。
通过下面三个步骤构建了MSP双糖基化突变体：首先将BC670的Xho I-Bsm I片段和GTC716的BSm I-Xho I片段连接导入pCR2.1载体的Xho I位点。这样产生了含有具N262-Q突变的MSP1-42基因的质粒。然后将这个质粒中的EcoN I-Nde I片段用GTC716质粒的EcoN I-NdeI片段取代，引入第二个突变N181-Q。最后将这个质粒的Xho I片段克隆到BC350产生BC718(图12)。
非糖基化MSP1的转基因表达
BC718具有下列特征：它携带处于β-酪蛋白启动子控制下的MSP1-42基因因而可在转基因动物哺乳期乳腺中表达。此外，它编码直接与MSP1-42融合的15个氨基酸的β-酪蛋白前导序列，这样在N-末端没有任何多余氨基酸的MSP1-42可以分泌到乳汁中。最后，由于N-Q置换，转基因动物乳汁中此构建体产生的MSP不会被糖基化。总而言之，在乳汁中由BC718产生的转基因MSP与寄生生物MSP1是相同的。
从质粒BC718制备了SalI/XhoI片段并微注射到小鼠胚胎产生了转基因小鼠。如前面所述鉴定转基因动物。收集雌性建立者的乳汁，并使用抗体5.2进行Western印迹检测。结果示于图13，表明非糖基化MSP1以0.5至1毫克/毫升的浓度表达。
Claims (8)
1.序列1所示的修饰的MSP-1核酸序列。
2.制备修饰的MSP-1核酸的方法，包括降低编码MSP-1天然基因的总AT含量，消除所有的mRNA不稳定基序以及用小鼠乳腺偏爱密码子置换所有稀有密码子等步骤。
3.用权利要求2的方法制备的修饰的MSP-1核酸。
4.含有权利要求1的修饰的MSP-1核酸和山羊β-酪蛋白启动子及信号序列的载体。
5.用权利要求4的载体转化的宿主细胞。
6.含有权利要求4的载体的转基因非人哺乳动物。
7.含有权利要求1的修饰的MSP-1核酸的转基因非人哺乳动物。
8.含有权利要求1的修饰的核酸的DNA疫苗。
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Families Citing this family (35)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001520048A (ja) | 1997-10-20 | 2001-10-30 | ジェンザイム トランスジェニックス コーポレイション | 新規な改変されたＭＳＰ−１核酸配列並びに細胞システムにおいてｍＲＮＡレベル及び蛋白質発現を増大させる方法 |
| US7022830B2 (en) * | 2000-08-17 | 2006-04-04 | Tripep Ab | Hepatitis C virus codon optimized non-structural NS3/4A fusion gene |
| CN1176945C (zh) * | 2001-02-01 | 2004-11-24 | 中国人民解放军第二军医大学 | 疟原虫融合抗原及其制法和用途 |
| ATE291925T1 (de) * | 2001-06-05 | 2005-04-15 | Curevac Gmbh | Stabilisierte mrna mit erhöhtem g/c-gehalt und optimierter codon usage für die gentherapie |
| DE10162480A1 (de) | 2001-12-19 | 2003-08-07 | Ingmar Hoerr | Die Applikation von mRNA für den Einsatz als Therapeutikum gegen Tumorerkrankungen |
| WO2003059381A2 (en) * | 2002-01-18 | 2003-07-24 | Curevac Gmbh | Immunogenic preparations and vaccines on the basis of mrna |
| EP1490494A1 (en) * | 2002-04-01 | 2004-12-29 | Walter Reed Army Institute of Research | Method of designing synthetic nucleic acid sequences for optimal protein expression in a host cell |
| WO2003084472A2 (en) * | 2002-04-01 | 2003-10-16 | Walter Reed Army Institute Of Research | Recombinant p. falciparum merozoite protein-142 vaccine |
| US20050197496A1 (en) * | 2004-03-04 | 2005-09-08 | Gtc Biotherapeutics, Inc. | Methods of protein fractionation using high performance tangential flow filtration |
| US20060130159A1 (en) * | 2004-12-09 | 2006-06-15 | Nick Masiello | Method of purifying recombinant MSP 1-42 derived from Plasmodium falciparum |
| GB0510763D0 (en) * | 2005-05-27 | 2005-06-29 | London School Hygiene & Tropical Medicine | Malaria vaccines |
| US20080213318A1 (en) * | 2005-07-05 | 2008-09-04 | Hawaii Biotech, Inc. | Malaria MSP-1 C-terminal enhanced subunit vaccine |
| US7531632B2 (en) * | 2006-02-16 | 2009-05-12 | Gtc Biotherapeutics, Inc. | Clarification of transgenic milk using depth filtration |
| JP2009106163A (ja) * | 2007-10-26 | 2009-05-21 | Kyushu Univ | 核酸配列、ベクター、形質転換体、製造方法、及び、核酸配列プライマー |
| EP2141177A1 (en) * | 2008-07-04 | 2010-01-06 | Bujard, Hermann | MSP-1 protein preparations from Plasmodium |
| GB0909689D0 (en) * | 2009-06-05 | 2009-07-22 | Trinity College Dublin | Fluorescent proteins |
| WO2012019168A2 (en) | 2010-08-06 | 2012-02-09 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
| EP2622064B1 (en) | 2010-10-01 | 2019-05-29 | Modernatx, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
| CN116585504A (zh) | 2010-12-30 | 2023-08-15 | 法国化学与生物科技实验室 | 作为病原体灭活剂的二元醇 |
| US8710200B2 (en) | 2011-03-31 | 2014-04-29 | Moderna Therapeutics, Inc. | Engineered nucleic acids encoding a modified erythropoietin and their expression |
| US9464124B2 (en) | 2011-09-12 | 2016-10-11 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
| HRP20220250T1 (hr) | 2011-10-03 | 2022-04-29 | Modernatx, Inc. | Modificirani nukleozidi, nukleotidi i nukleinske kiseline, te njihove uporabe |
| EP2791160B1 (en) | 2011-12-16 | 2022-03-02 | ModernaTX, Inc. | Modified mrna compositions |
| US9283287B2 (en) | 2012-04-02 | 2016-03-15 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of nuclear proteins |
| US9572897B2 (en) | 2012-04-02 | 2017-02-21 | Modernatx, Inc. | Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins |
| US9878056B2 (en) | 2012-04-02 | 2018-01-30 | Modernatx, Inc. | Modified polynucleotides for the production of cosmetic proteins and peptides |
| EP2834259A4 (en) | 2012-04-02 | 2016-08-24 | Moderna Therapeutics Inc | MODIFIED POLYNUCLEOTIDES |
| ES2921623T3 (es) | 2012-11-26 | 2022-08-30 | Modernatx Inc | ARN modificado terminalmente |
| MX2015010427A (es) | 2013-02-13 | 2016-03-17 | Lab Francais Du Fractionnement | Anticuerpos anti-tnf-alfa altamente galactosilados y sus usos. |
| US10034921B2 (en) | 2013-02-13 | 2018-07-31 | Laboratoire Français Du Fractionnement Et Des Biotechnologies | Proteins with modified glycosylation and methods of production thereof |
| US8980864B2 (en) | 2013-03-15 | 2015-03-17 | Moderna Therapeutics, Inc. | Compositions and methods of altering cholesterol levels |
| JP2016532100A (ja) | 2013-07-05 | 2016-10-13 | ラボラトワール・フランセ・デュ・フラクシオンマン・エ・デ・ビョテクノロジーＬａｂｏｒａｔｏｉｒｅ Ｆｒａｎｃａｉｓ Ｄｕ Ｆｒａｃｔｉｏｎｎｅｍｅｎｔ Ｅｔ Ｄｅｓ Ｂｉｏｔｅｃｈｎｏｌｏｇｉｅｓ | アフィニティークロマトグラフィーマトリックス |
| EP3052106A4 (en) | 2013-09-30 | 2017-07-19 | ModernaTX, Inc. | Polynucleotides encoding immune modulating polypeptides |
| EP3052521A1 (en) | 2013-10-03 | 2016-08-10 | Moderna Therapeutics, Inc. | Polynucleotides encoding low density lipoprotein receptor |
| CA2925021A1 (en) | 2013-11-01 | 2015-05-07 | Curevac Ag | Modified rna with decreased immunostimulatory properties |
Family Cites Families (31)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3751873T2 (de) | 1986-04-09 | 1997-02-13 | Genzyme Corp | Genetisch transformierte Tiere, die ein gewünschtes Protein in Milch absondern |
| EP0832981A1 (en) * | 1987-02-17 | 1998-04-01 | Pharming B.V. | DNA sequences to target proteins to the mammary gland for efficient secretion |
| US4873316A (en) * | 1987-06-23 | 1989-10-10 | Biogen, Inc. | Isolation of exogenous recombinant proteins from the milk of transgenic mammals |
| US5225534A (en) | 1987-09-08 | 1993-07-06 | Hoffmann-La Roche Inc. | Recombinant malarial polypeptides |
| US5194587A (en) | 1987-12-30 | 1993-03-16 | Behringwerke Aktiengesellschaft | Malaria-specific DNA sequences, expression products thereof, and the use thereof |
| NZ230375A (en) | 1988-09-09 | 1991-07-26 | Lubrizol Genetics Inc | Synthetic gene encoding b. thuringiensis insecticidal protein |
| US5231168A (en) * | 1988-09-16 | 1993-07-27 | Statens Seruminstitut | Malaria antigen |
| GB8905857D0 (en) | 1989-03-14 | 1989-04-26 | Hoffmann La Roche | Plasmodium merozoite rhoptries antigen |
| US5646247A (en) | 1989-04-05 | 1997-07-08 | New York University | Merozoite antigens localized at the apical end of the parasite |
| ATE140027T1 (de) | 1989-12-01 | 1996-07-15 | Pharming Bv | Herstellung rekombinanter polypeptide durch rinder und transgene methoden |
| GB9012580D0 (en) | 1990-06-06 | 1990-07-25 | Univ Nijmegen | Novel protein |
| DE4041836A1 (de) | 1990-12-24 | 1992-06-25 | Behringwerke Ag | Protektive plasmodium falciparum hybridproteine, die teilsequenzen der malaria-antigene hrpii und serp enthalten, ihre herstellung und verwendung |
| US5831141A (en) | 1991-01-11 | 1998-11-03 | United States Of America As Represented By The Department Of Health And Human Services | Expression of a heterologous polypeptide in mammary tissue of transgenic non-human mammals using a long whey acidic protein promoter |
| US5766597A (en) * | 1991-03-07 | 1998-06-16 | Virogenetics Corporation | Malaria recombinant poxviruses |
| AU663101B2 (en) * | 1991-08-13 | 1995-09-28 | Wisconsin Milk Marketing Board | DNA sequence encoding bovine alpha-lactalbumin and methods of use |
| US5643578A (en) * | 1992-03-23 | 1997-07-01 | University Of Massachusetts Medical Center | Immunization by inoculation of DNA transcription unit |
| US5856178A (en) * | 1993-08-30 | 1999-01-05 | Utah State University | DNA cassettes for expression of lytic peptides in mammalian cells and transgenic organisms containing same |
| GB9318207D0 (en) * | 1993-09-02 | 1993-10-20 | Sandoz Ltd | Improvements in or relating to organic compounds |
| US5827690A (en) | 1993-12-20 | 1998-10-27 | Genzyme Transgenics Corporatiion | Transgenic production of antibodies in milk |
| US6713662B1 (en) | 1994-07-27 | 2004-03-30 | Pharming Intellectual Property B.V. | Production of collagen in the milk of transgenic mammals |
| AUPM772494A0 (en) | 1994-08-30 | 1994-09-22 | Austin Research Institute, The | Improvements in production of proteins in host cells |
| US5795737A (en) * | 1994-09-19 | 1998-08-18 | The General Hospital Corporation | High level expression of proteins |
| WO1997026911A1 (en) | 1996-01-29 | 1997-07-31 | Georgetown University | Malaria vaccine based upon the addition of a msa1 peptide |
| FR2744723A1 (fr) | 1996-02-14 | 1997-08-14 | Pasteur Institut | Proteine recombinante contenant un fragment c-terminal d'une proteine msp-1 d'un plasmodium infectieux pour l'homme pour la production de vaccins anti-paludiques |
| FR2744724B1 (fr) | 1996-02-14 | 2002-08-02 | Pasteur Institut | Proteine recombinante contenant un fragment c-terminal de la proteine msp-1 d'un plasmodium infectieux pour l'homme pour la production de vaccins anti-paludiques |
| HRP970092A2 (en) * | 1996-02-22 | 1998-04-30 | Merck & Co Inc | Synthetic hiv genes |
| US6268487B1 (en) | 1996-05-13 | 2001-07-31 | Genzyme Transgenics Corporation | Purification of biologically active peptides from milk |
| US5956178A (en) * | 1996-09-02 | 1999-09-21 | Nikon Corporation | Keplerian variable magnification viewfinder |
| US6114148C1 (en) | 1996-09-20 | 2012-05-01 | Gen Hospital Corp | High level expression of proteins |
| DE19640817A1 (de) * | 1996-10-02 | 1998-05-14 | Hermann Prof Dr Bujard | Rekombinantes Herstellungsverfahren für ein vollständiges Malaria-Antigen gp190/MSP 1 |
| JP2001520048A (ja) | 1997-10-20 | 2001-10-30 | ジェンザイム トランスジェニックス コーポレイション | 新規な改変されたＭＳＰ−１核酸配列並びに細胞システムにおいてｍＲＮＡレベル及び蛋白質発現を増大させる方法 |
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