CN1325650C - 猪促生长素传递*** - Google Patents
猪促生长素传递*** Download PDFInfo
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- CN1325650C CN1325650C CNB998122564A CN99812256A CN1325650C CN 1325650 C CN1325650 C CN 1325650C CN B998122564 A CNB998122564 A CN B998122564A CN 99812256 A CN99812256 A CN 99812256A CN 1325650 C CN1325650 C CN 1325650C
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Abstract
本发明公开了用于传递外源多肽(如促生长素等生长激素)至宿主中的表达构建体。在本发明一个应用中,将表达构建体导入非宿主重组细胞,该细胞包封于半渗透膜中,用于植入宿主。半渗透膜抑制免疫监视和细胞排斥事件,从而可以使用非宿主的高表达重组细胞。
Description
发明领域
本发明涉及传递外源多肽至宿主中的表达构建体。本发明还涉及包括该表达构建体的重组细胞和包括该重组细胞的半渗透胶囊。
发明背景
在哺乳动物中,促生长素(生长激素)通常由脑垂体分泌。但是,已经证实向猪给予外源促生长素会提高摄食效率15-20%,增加日增重10-15%,降低身体脂肪10-20%,增加瘦肉含量5-10%,降低摄食量。令人遗憾的是,促生长素(它是190个氨基酸的小蛋白质)对胃酸和蛋白降解敏感,因此需要每日注射才会有效果。当前,福利和道德的考虑使得使用气动pST注射枪不受欢迎,每日用药的费用也限制了其大规模的使用。
基因治疗的最新进展已经发展出了新的策略,无需依赖自体靶细胞和免疫抑制治疗,而使用包封在半渗透藻酸盐-聚-L-赖氨酸-藻酸盐(APA)膜中的转染细胞。APA胶囊环境允许细胞存活和生长,因此转染细胞保持存活并在较长时间内分泌生长因子。APA对小蛋白是可通透的,因此基因表达可以通过外源机制控制。APA屏障抑制免疫监视和细胞排斥事件,从而可以在胶囊中使用非宿主、高表达细胞。APA屏障也可能阻止宿主受体中转染细胞不受控制的增殖。APA胶囊可以取出,可能还可以再次使用,从而打消对于食用转基因材料的担忧。而且,如果胶囊被严重的组织创伤损害,正常的宿主-移植物排斥就会摧毁植入的细胞。
发明概述
发明人现已发现,在基因序列导入宿主细胞中之前,胰岛素分泌信号与异源基因序列的连接导致外源基因产物分泌水平出乎意料的增长。这一发现已经导致开发出了改进的基因传递***,包括包封重组细胞植入宿主中。
因而,第一方面,本发明提供了一种表达盒,包括编码胰岛素分泌信号的序列,其与编码一种多肽的异源序列有效连接。
“异源序列”是指不是编码胰岛素的序列的序列。
“有效连接”是指胰岛素分泌信号序列与异源编码序列邻接并位于同一阅读框。
优选的胰岛素分泌信号是具有SEQ ID NO:1所示氨基酸序列的胰岛素分泌信号。但是,本领域技术人员会认识到,可以对分泌信号进行多种修饰而不会对信号的生物活性有不利影响。例如,这可以通过在肽序列中进行各种改变或通过保守或非保守的氨基酸***、缺失和取代(如D-氨基酸,脱氨基酸)实现,所述改变如硫酸化、磷酸化、硝化和卤化,只要这些改变不会对分泌信号的总体生物活性有不利影响。因此,在表达盒中包括已经以上述一种或多种方式修饰的胰岛素分泌信号被认为包括在本发明之中。
异源序列可以编码除胰岛素以外的目标多肽。例如,异源序列可以编码激素、细胞因子、受体激动剂或拮抗剂、信息素或酶。在优选实施方案中,异源序列编码生长激素。优选,生长激素是促生长素。
本发明第二方面提供了包括第一方面的表达盒的载体。该载体可以是将表达盒导入细胞的任何合适的载体。合适载体包括病毒载体和细菌质粒。
本发明第一方面的表达盒或第二方面的载体还可以包括一个或多个调控基因表达的元件。合适调控元件的实例包括褪黑素应答元件(MRE)(见Schrader et al.,1996,此处全文引入作为参考),和/或雷帕霉素介导转录因子(见Magari et al.,1997,此处全文引入作为参考)。在优选实施方案中,调控元件可以实现目标多肽的脉动表达。
本发明第三方面提供了一种重组细胞,它包括本发明第一方面的表达盒。
重组细胞可以是细菌、真菌、昆虫或哺乳动物细胞。在优选实施方案中,重组细胞是哺乳动物细胞。在进一步优选的实施方案中,细胞是大鼠成肌细胞(L6)。
本发明第四方面提供生产多肽的方法,包括在可以表达和分泌所述多肽的条件下培养第三方面的重组细胞,可选地包括分离该多肽。
本发明的重组细胞可以包封在半渗透基质中以传递或植入人体。
因此,本发明第五方面提供植入宿主的胶囊,该胶囊包括包封本发明第三方面的一种或多种重组细胞的半渗透膜。
在优选实施方案中,半渗透膜是藻酸盐-聚-L-赖氨酸-藻酸盐(APA)膜。APA半渗透膜的制备见Bas ic et al.,1996,该文献全文引入作为参考。
本发明第六方面提供向宿主给予多肽的方法,包括向宿主给予本发明第一方面的表达盒。
本发明第七方面提供向宿主给予多肽的方法,包括在宿主中植入本发明第五方面的胶囊。
宿主可以是任何动物或人。在优选实施方案中,宿主是家畜。在进一步优选的实施方案中,宿主选自放牧牛、圈养牛、奶牛、猪和家禽。
本领域技术人员会认识到,本发明提供了传递遗传物质到宿主中的改进***。胰岛素分泌信号与生物活性多肽的连接导致从重组细胞分泌的多肽增加。分泌后,分泌信号可以切除,留下生物活性多肽。重组细胞包封在半渗透膜中时,可以分泌相当数量的生物活性多肽,借助半渗透膜可以通过外来方式控制基因表达。植入包封的重组细胞的优势在于植入需要的手术最轻。此外,半渗透膜减少了免疫监视和细胞排斥,这意味着非宿主细胞可以用在胶囊中。
在优选实施方案中,半渗透膜是持久耐用的,从而具有限制细胞生长并阻止在宿主受体中的不受控制增殖的优势。胶囊的另一个优势在于它们可以被取出并再次使用。
为了更清楚地理解本发明的特点,以下参照非限定性实施例和附图叙述本发明的优选方式。
附图简述:
图1:胰岛素分泌信号-pST基因构建体。
图2:胰岛素分泌信号-pST肽序列。
图3:对照和pST-L6IXS处理猪的增重率(从0天起)。
图4:对照和pST-L6IXS处理猪的增重百分比。
图5:对照和pST-L6IXS处理动物的血浆pST水平。
图6:Plate 1-pST-L6IXS胶囊给药部位评估。
Plate 2-pST-L6IXS胶囊置于培养基中进行来自体内的评估。
图7:从宿主动物取出后24小时从胶囊分泌pST的来自体内的评估。
图8:在给予pST胶囊之前(白柱体)和一周后(黑柱体)平均血浆pST(3小时@30分钟间隔)(*显著性)
图9:分别植入分泌25ng/ml和500ng/ml的胶囊的两只猪206和228的每日血浆pST浓度。
图10:实施例4中的猪的增重率(ROG,公斤/日,黑方块)和P2背脂肪测定。
图11:植入pST分泌或对照免疫中性基因治疗(IGT)胶囊后公猪增重率(ROG)(±SEM)。
图12:植入pST分泌或对照免疫中性基因治疗(IGT)胶囊后公猪背脂肪(P2)(±SEM)。
图13:植入pST分泌或对照免疫中性基因治疗(IGT)胶囊后公猪腰(眼)肌肉面积(±SEM)。
发明详述
实施例1:ISS-pST构建体的克隆
pST基因获自Southern Cross Biotechnology Pty Ltd的大肠杆菌。含有pST基因的质粒pMG939使用标准质粒制备技术从细菌中分离。设计用来扩增pST基因的PCR引物,在5’末端加入Xho I位点,3’末端加入XbaI位点,以进行连接。
修饰pST基因序列然后连接至来自前原胰岛素cDNA的分泌信号序列(ISS)。Nhe I(GCTAGC)和Xba I(TCTAGA)限制位点分别构建在ISS起始密码子之前pST 3’末端密码子之后,以便整合进pCI-neo质粒(Promega)。然后分离pST融合构建体并测序,以验证编码区(图1)。
大鼠成肌细胞(L6)用胰蛋白酶处理后2小时,用LipoTAXI(Stratagene)进行转染(pST基因整合细胞)。PST转染L6细胞克隆在培养物中维持,用G418选择,直至产生>107细胞。培养物上清的等分试样(2ml)在-20℃保存,直至用悉尼大学(Camden)的Dr P.Wynn首创的pST放射免疫试验(RIA)测定pST浓度。RIA敏感性被认为大于0.4ng/ml,CV为12.4%。多克隆抗血清用pST肽抗原在豚鼠中产生。RIA结果(表1)表明pST基因构建体产生一种蛋白(图2),它被由猪脑垂体纯化的天然形式pST产生的多克隆抗血清识别。L6克隆pCI/pst-1.5如下所述由修饰转染技术产生。
修饰转染法
一般说来,L6细胞粘附在培养板上,需要用胰蛋白酶解吸附才能传代;转染一般在24小时后进行。该方法产生分泌6-18ng/ml pST的L6细胞克隆(n=10)。胰蛋白酶处理2小时后,加入LipoTAXI(Prome ga)和ISS/pST质粒至L6细胞增加pST分泌速率10到20倍(>180ng/ml,n=5克隆)。该较高的pST分泌速率降低了促进生长所需的细胞数(胶囊数)。
表1:用ISS-pST转染的各克隆的浓度(ng/ml)
L6克隆 | pST(ng/ml) |
pCI/pst-1* | 182 |
pCI/pst-2* | 188 |
pCI/pst-3* | 188 |
pCI/pst-4* | 140 |
pCI/pst-5* | 200 |
pCI/pst-6 | 17 |
pCI/pst-7 | 12 |
pCI/pst-8 | 8 |
pCI/pst-9 | 9 |
pCI/pst-10 | 7 |
pCI/pst-11 | 7 |
pCI/pst-12 | 10 |
pCI/pst-13 | 8 |
pCI/pst-14 | 6 |
pCI/pst-15 | 18 |
实施例2:猪促生长素-大鼠成肌细胞(L6)免疫中性表达***(pST-L6IXS)的制备
按照Basic et al,1996所述进行包封步骤,包括如下改进。
在室温下包封细胞,利用氯化钙(或乳酸钙)(100mM)使藻酸盐(1.5%w/v)液滴形成凝胶,然后立即用盐水(0.9%NaCl)洗涤,再重悬于聚-L-赖氨酸(0.05%)中5分钟。37℃氯化钙交联10分钟产生与细胞活力更相容的藻酸盐基质。
聚-L-赖氨酸包被和盐水洗涤之后,加入另一层藻酸盐。柠檬酸钠(55mM)室温处理4分钟软化胶囊至一定坚度,增加进一步操作的难度。柠檬酸钠处理4分钟显著降低细胞活力至<35%。柠檬酸钠处理之前将胶囊置于细胞滤过器使得37℃下处理1分钟提高细胞活力达>98%。
对包封方法的步骤和设备改进提高了包封的效率(时间和资源),细胞活力一般增加64%。
实施例3:pST-L6IXS植入猪的初步试验(1)
将pST-L6IXS给予生长中的小鼠获得的初步结果表明了较好的生长特性。在用公猪进行的初步试验(n=9,平均活体重61公斤)中,在不同部位给予不等量的pST-L6IXS(0天,每只动物:颈部肌肉,3个胶囊,i.m.;颈部,3个胶囊,s.c.;耳根部,10个胶囊,s.c.;颈部,20个胶囊,i.m.;颈部,29个胶囊,i.m.)。颈静脉穿刺采集血样(10ml),给药后-14、0、7、14、21、28和36天记录P2超声(us)测定结果。试验中全程监测pST-L6IXS给药部位的组织反应事件。36天将动物无痛处死,对躯体各部分进行分析(背部脂肪厚度,BF(mm);眼肌面积,EMA(cm);前臂骨长,BONE(cm);心脏重量,HEART(gm);脾脏重量,SPLEEN(gm);肝脏重量,LIVER(gm))并记录(见表2),回收pST-L6IXS。图3代表对照(con,平均值±SE,n=4)和各只pST-L6IXS处理猪的增重率(从0天起)。对照(con)(平均值±SE)和各只pST-L6IXS处理动物相对于pST-L6IXS处理的增重百分比在图4中示出。对照(平均值±SE)(con)和各只pST-L6IXS处理动物的血浆pST(ng/ml)通过放射免疫试验(RIA)确定并在图5中示出。处死时评价pST-L6IXS给药部位(图6,Plate 1,箭头),然后取出并置于培养基中进行来自体内的24小时pST分泌评估(图6,Plate 2)(图6)。在36天i.m.或s.c.给药均未观察到明显的组织损害或免疫反应。但是,置于耳部(s.c.)的胶囊似乎高度血管化,而且100%可回收。置于颈部的胶囊可回收的小于10%。
pST-L6IXS在体内36日仍有效,并且似乎在胶囊中增殖(Plate2),胶囊可以取出,从而打消对食用转基因材料的担忧。而且,如果胶囊受损(即由于严重的组织创伤),正常的宿主-移植物排斥会摧毁L6细胞,防止转染材料的增殖。小鼠和猪中进行的试验已经证实pST-L6IXS可有效改变血清pST,提高生长特性,还可能提高动物的免疫能力。
表2:pST-L6IXS初步试验:
公猪由Westmil lpiggery(Young,NSW)
在EMAI的高度安全猪舍进行试验
ACEC号:98/20
实施例4:pST-L6IXS植入猪的初步试验(2)
为了优化通过胶囊的pST-L6IXS传递,从而获得类似于每日注射pST的能量再分配的生长应答,进行了第二次初步试验。
如实施例1所述,制备了多种分泌速率(6-200ng/ml)的pST分泌细胞。对pST分泌细胞外加胁迫(即细菌污染)后,2-25ng/ml的pST分泌速率似乎是最稳定的(数据未示出)。因此,选择分泌约5ng/ml(克隆pCI/pst-14)和约10ng/ml(pCI/pst-12)的克隆进行该初步试验。在公猪(n=10,平均活体重78.1kg)耳根部以s.c.途径给予不同数量的胶囊(按照实施例2所述步骤制备)(表3)。
猪 | 胶囊数量 | 克隆 |
204 | 1 | a |
216 | 1 | b |
230 | 3 | a |
202 | 3 | b |
226 | 5 | a |
206 | 5 | b |
208 | 10 | a |
224 | 10 | b |
222 | 100 | a |
228 | 100 | b |
a=克隆pCI/pst-14(5ng/ml)
b=克隆pCI/pst-12(10ng/ml)
试验开始和结束时记录体重。动物分别圈养于围栏(2m2),并在控制的环境条件下(22℃)稳定一周。动物自由饮水,定量给予标准颗粒状食料(3 kg/日@0:900hrs),记录每日残留。插管置于耳静脉(evc),24小时后开始取样。对照猪(即不给予pST胶囊)血浆(10毫升)每30分钟收集一次,共3小时。系列取样后,耳后部给予pST胶囊。通过evc收集血样(10毫升)(每日@11:00hrs),插管仍留置。处理(给予pST胶囊后7日)血浆(10ml)每30分钟收集一次,共3小时。处死和躯体分析在21日后活体重为约100kg时进行。然后由耳部回收pST胶囊,置于体外培养(进行pST分析)。评估胶囊植入部位的免疫应答(如淋巴细胞浸润)。
接受pST胶囊(分泌5到1000ng/ml)之前和之后7天,猪平均(3小时、30分钟间隔)血浆pST浓度测定结果示于图8。由图8可见,免疫中性pST(5-100ng/ml)分泌胶囊处理后1周,猪血浆pST明显减少。
个体间和个体中血浆pST浓度的变化在对照系列取样期间似乎更明显。这种现象反映在平均测定浓度的标准误中。此外,稳定基线和pST脉冲间隔(一般3-4小时)不能被设计来鉴别激素脉冲的计算机程序识别。但是,稳定基线和明显的pST脉冲在pST胶囊给药后一周的动物中可以观察到(图9)。
动物的增重率(ROG)似乎以剂量依赖方式对pST胶囊分泌有反应(图10)。30ng/ml的分泌率(即3个胶囊各分泌10ng/ml)似乎是观察到生长速率增加所需的最小剂量。大多数evc在21天保持有效,此时动物用巴比妥盐无痛处死以进行躯体分析。躯体背部脂肪分析(不计皮肤的P2)测定值进一步表明30ng/ml是pST胶囊给药21天内观察到能量再分配所需的最小剂量(图10)。
包括接受100个胶囊的动物在内,所有动物在试验过程中未观察到副反应,即增重下降或不利免疫应答。
实施例5:pST-L6IXS植入猪的初步试验(3)
实施例4后,进行试验以评估在处死前不同时间(即处死前2、4和6周)向猪给予最佳pST分泌速率/胶囊数对背部脂肪的影响。每个处理使用8只动物,8只动物周作对照(即无pST胶囊)。
增重率测定结果在图11中给出。
在全躯体冷冻(24小时@4℃)后进行背部脂肪测定(图12)。对距脊柱中心65毫米的第12肋骨记录P2测定值。观察到给予分泌pST的胶囊2、4和6周的猪背部脂肪较少约46%。给予pST IGT胶囊4周的动物背部脂肪反应差异更大,这可能与从多只这样的动物中不能回收胶囊有关。
分泌胶囊处理的猪腰部肌肉面积只有在pST IGT胶囊处理6周后才显著增加(22%)(图13)。
本领域技术人员会认识到,对本发明的具体技术方案可以进行多种改进和/或修饰,而不会偏离本发明的精神和范围。因此,本文所述的各种实施方案仅是示例性的而非限制性的。
参考文献
Basic et al.,遗传工程细胞的微包封和植入:体细胞基因治疗的新途径。Art.Cells,Blood subs and Immob.Biotech24(3):219-255。
Magari et al.,(1997)植入裸鼠的人源化基因治疗***的药物动力学控制。J.Clin.Invest.100:2865-2872。
Schrader et al.,(1996)核受体R2R/ROR的天然单体应答元件的鉴定。它们也结合COUP-TF同型二聚体。J.Biol.Chem.271:19732-19736。
序列表
申请人:Commonwealth Scientific and Industrial ResearchOrganisation
University of Western Sydney(Nepean)
Pig Research and Development Corporation
发明名称:猪促生长素传递***
在先申请号:PP 6556
在先申请日:1998-10-16
序列数:4
软件:PatentIn Ver.2.1
SEQ ID NO:1
长度: 24
类型: PRT
有机体:人
序列: 1
Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu AlaLeu Leu Ala Leu
1 5 10 15
Trp Gly Pro Asp Pro Ala Ala Ala
20
SEQ ID NO: 2
长度:72
类型:DNA
有机体:人
序列:2
atggccctgt ggatgcgcct cctgcccctg ctggcgctgc tggccctctg gggacctgac 60
ccagccgcag cc
SEQ ID NO:3
长度:666
类型:DNA
有机体:人工序列
特征:
其它信息:人工序列描述ISS-pST基因构建体
序列:3
gctagcatgg ccctgtggat gcgcctcctg cccctgctgg cgctgctggc cctctgggga 60
cctgacccag ccgcagccct cgagatgttt ccagctatgc cactttcttc tctgttcgct 120
aacgctgttc ttcgggccca gcacctgcac caactggctg ccgacaccta caaggagttt 180
gagcgcgcct acatcccgga gggacagagg tactccatcc agaacgccca ggctgccttc 240
tgcttctcgg agaccatccc ggcccccacg ggcaaggacg aggcccagca gagatcggac 300
gtggagctgc tgcgcttctc gctgctgctc atccagtcgt ggctcgggcc cgtgcagttc 360
ctcagcaggg tcttcaccaa cagcctggtg tttggcacct cagaccgcgt ctacgagaag 420
ctgaaggacc tggaggaggg catccaggcc ctgatgcggg agctggagga tggcagcccc 480
cgggcaggac agatcctcaa gcaaacctac gacaaatttg acacaaactt gcgcagtgat 540
gacgcgctgc ttaagaacta cgggctgctc tcctgcttca agaaggacct gcacaaggct 600
gagacatacc tgcgggtcat gaagtgtcgc cgcttcgtgg agagcagctg tgccttctag 660
tctaga 666
SEQ ID NO:4
长度:217
类型:PRT
有机体:人工序列
特征:
其它信息:人工序列描述:ISS-pST肽序列
序列:4
Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu
1 5 10 15
Trp Gly Pro Asp Pro Ala Ala Ala Leu Glu Met Phe Pro Ala Met PrO
20 25 30
Leu Ser Ser Leu Phe Ala Asn Ala Val Leu Arg Ala Gln His Leu His
35 40 45
Gln Leu Ala Ala Asp Thr Tyr Lys Glu Phe Glu Arg Ala Tyr Ile Pro
50 55 60
Glu Gly Gln Arg Tyr Ser Ile Gln Asn Ala Gln Ala Ala Phe Cys Phe
65 70 75 80
Ser Glu Thr Ile Pro Ala Pro Thr Gly Lys Asp Glu Ala Gln Gln Arg
85 90 95
Ser Asp Val Glu Leu Leu Arg Phe Ser Leu Leu Leu Ile Gln Ser Trp
100 105 110
Leu Gly Pro Val Gln Phe Leu Ser Arg Val Phe Thr Asn Ser Leu Val
115 120 125
Phe Gly Thr Ser Asp Arg Val Tyr Glu Lys Leu Lys Asp Leu Glu Glu
130 135 140
Gly Ile Gln Ala Leu Met Arg Glu Leu Glu Asp Gly Ser Pro Arg Ala
145 150 155 160
Gly Gln Ile Leu Lys Gln Thr Tyr Asp Lys Phe Asp Thr Asn Leu Arg
165 170 175
Ser Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu Ser Cys Phe Lys
180 185 190
Lys Asp Leu His Lys Ala Glu Thr Tyr Leu Arg Val Met Lys Cys Arg
195 200 205
Arg Phe Val Glu Ser Ser Cys Ala Phe
210 215
Claims (17)
1.一种表达盒,包括编码胰岛素分泌信号的序列,该序列与一种异源序列有效连接,所述异源序列编码成熟猪促生长素,其中所述胰岛素分泌信号具有SEQ ID NO:1所示氨基酸序列,或具有修饰的氨基酸序列,所述修饰通过对SEQ ID NO:1所示氨基酸序列进行一个或几个氨基酸***、缺失和/或取代实现,并且修饰的氨基酸序列具有与SEQ ID NO:1所示氨基酸序列相同的总体生物活性。
2.权利要求1的表达盒,其中胰岛素分泌信号具有SEQ ID NO:1所示氨基酸序列。
3.权利要求1或2的表达盒,还包括使异源序列脉冲表达的一种或多种调控序列。
4.包括权利要求1到3任一项的表达盒的载体。
5.包括权利要求1到3任一项的表达盒的重组细胞。
6.权利要求5的重组细胞,其中细胞是细菌、酵母、昆虫或哺乳动物细胞。
7.权利要求6的重组细胞,其中细胞是哺乳动物细胞。
8.权利要求7的重组细胞,其中细胞是大鼠成肌细胞(L6)。
9.制备猪促生长素的方法,包括在可以表达和分泌猪促生长素的条件下培养权利要求5到8任一项的重组细胞。
10.权利要求9的方法,其中所述方法进一步包括分离所表达和分泌的猪促生长素。
11.用于植入宿主的胶囊,所述胶囊包括包封权利要求5到8任一项的重组细胞的半渗透膜。
12.权利要求11的胶囊,其中半渗透膜是藻酸盐-聚-L-赖氨酸-藻酸盐(APA)膜。
13.权利要求1的表达盒在制备用于向宿主给予猪促生长素的药物中的用途。
14.权利要求11的胶囊在制备用于向宿主给予猪促生长素的药物中的用途。
15.权利要求13或14的用途,其中宿主是动物或人。
16.权利要求15的用途,其中宿主是家畜。
17.权利要求16的用途,其中家畜是猪。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AUPP6556 | 1998-10-16 | ||
AUPP6556A AUPP655698A0 (en) | 1998-10-16 | 1998-10-16 | Delivery system for porcine somatotropin |
Publications (2)
Publication Number | Publication Date |
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CN1323348A CN1323348A (zh) | 2001-11-21 |
CN1325650C true CN1325650C (zh) | 2007-07-11 |
Family
ID=3810777
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNB998122564A Expired - Fee Related CN1325650C (zh) | 1998-10-16 | 1999-10-18 | 猪促生长素传递*** |
Country Status (14)
Country | Link |
---|---|
US (2) | US6967089B1 (zh) |
EP (1) | EP1121449A4 (zh) |
JP (1) | JP2002527112A (zh) |
KR (1) | KR20010099687A (zh) |
CN (1) | CN1325650C (zh) |
AU (1) | AUPP655698A0 (zh) |
BR (1) | BR9914547A (zh) |
CA (1) | CA2346799A1 (zh) |
ID (1) | ID30149A (zh) |
MX (1) | MXPA01003810A (zh) |
NZ (1) | NZ510943A (zh) |
RU (1) | RU2268940C2 (zh) |
WO (1) | WO2000023601A1 (zh) |
ZA (1) | ZA200102965B (zh) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AUPP655698A0 (en) * | 1998-10-16 | 1998-11-05 | Commonwealth Scientific And Industrial Research Organisation | Delivery system for porcine somatotropin |
EP3291843B1 (en) * | 2015-05-07 | 2023-03-22 | University of South Florida | Modified ube3a gene for a gene therapy approach for angelman syndrome |
US20200354745A1 (en) * | 2018-01-23 | 2020-11-12 | Paul B. Fisher | Mda-7/il secretory variants and methods of use |
CN109929849B (zh) * | 2019-03-18 | 2021-05-04 | 华南农业大学 | 一种优化的pGH基因、蛋白及在提高毕赤酵母分泌表达pGH产量和活性中的应用 |
Citations (3)
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WO1987003300A1 (en) * | 1985-11-25 | 1987-06-04 | Biogen N.V. | Enhanced secretion of heterologous proteins by hosts using substituted promoters |
US4992367A (en) * | 1986-05-12 | 1991-02-12 | Hoffmann-La Roche Inc. | Enhanced expression of human interleukin-2 in mammalian cells |
WO1997035959A1 (en) * | 1996-03-27 | 1997-10-02 | Cytel Corporation | Production of galactosyltransferase |
Family Cites Families (14)
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GR70279B (zh) * | 1979-09-12 | 1982-09-03 | Univ California | |
US4923808A (en) | 1985-03-12 | 1990-05-08 | Genentech, Inc. | Method for identifying mutants secreting high levels of heterologous proteins |
US5200509A (en) | 1987-04-06 | 1993-04-06 | Celtrix Pharmaceuticals, Inc. | Human somatomedin carrier protein subunits and process for producing them; recombinant DNA molecules, hosts, processes and human somatomedin carrier protein-like polypeptides |
US5681562A (en) | 1991-06-25 | 1997-10-28 | Sidney Kimmel Cancer Center | Lymphokine gene therapy of cancer |
US5932211A (en) | 1991-11-12 | 1999-08-03 | Women's And Children's Hospital | Glycosylation variants of iduronate 2-sulfatase |
US5545423A (en) | 1991-11-25 | 1996-08-13 | Vivorx, Inc. | Cytoprotective, biocompatible, retrievable macrocapsule containment systems for biologically active materials |
US5858751A (en) * | 1992-03-09 | 1999-01-12 | The Regents Of The University Of California | Compositions and methods for producing sialyltransferases |
US5641665A (en) | 1994-11-28 | 1997-06-24 | Vical Incorporated | Plasmids suitable for IL-2 expression |
GB9526733D0 (en) * | 1995-12-30 | 1996-02-28 | Delta Biotechnology Ltd | Fusion proteins |
EP1109898A1 (en) * | 1998-09-04 | 2001-06-27 | ZK Pharmaceuticals Inc. | Method for selecting peptides inhibiting viral surface protein binding to cell surface receptor |
AUPP655698A0 (en) * | 1998-10-16 | 1998-11-05 | Commonwealth Scientific And Industrial Research Organisation | Delivery system for porcine somatotropin |
WO2002024908A1 (fr) * | 2000-09-22 | 2002-03-28 | Takeda Chemical Industries, Ltd. | Nouveau polypeptide secretoire specifique d'un tissu et adn correspondant |
WO2002066064A1 (fr) * | 2001-01-19 | 2002-08-29 | Takeda Chemical Industries, Ltd. | Peptide de type galanine |
US6811785B2 (en) * | 2001-05-07 | 2004-11-02 | Mount Sinai School Of Medicine Of New York University | Multivalent MHC class II—peptide chimeras |
-
1998
- 1998-10-16 AU AUPP6556A patent/AUPP655698A0/en not_active Abandoned
-
1999
- 1999-10-18 EP EP99970688A patent/EP1121449A4/en not_active Withdrawn
- 1999-10-18 RU RU2001113279/13A patent/RU2268940C2/ru not_active IP Right Cessation
- 1999-10-18 CN CNB998122564A patent/CN1325650C/zh not_active Expired - Fee Related
- 1999-10-18 NZ NZ510943A patent/NZ510943A/en unknown
- 1999-10-18 CA CA002346799A patent/CA2346799A1/en not_active Abandoned
- 1999-10-18 US US09/807,519 patent/US6967089B1/en not_active Expired - Fee Related
- 1999-10-18 JP JP2000577308A patent/JP2002527112A/ja not_active Withdrawn
- 1999-10-18 MX MXPA01003810A patent/MXPA01003810A/es unknown
- 1999-10-18 KR KR1020017004624A patent/KR20010099687A/ko not_active Application Discontinuation
- 1999-10-18 BR BR9914547-2A patent/BR9914547A/pt not_active Application Discontinuation
- 1999-10-18 ID IDW00200101076A patent/ID30149A/id unknown
- 1999-10-18 WO PCT/AU1999/000896 patent/WO2000023601A1/en not_active Application Discontinuation
-
2001
- 2001-04-10 ZA ZA200102965A patent/ZA200102965B/en unknown
-
2005
- 2005-06-27 US US11/166,340 patent/US20060062772A1/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1987003300A1 (en) * | 1985-11-25 | 1987-06-04 | Biogen N.V. | Enhanced secretion of heterologous proteins by hosts using substituted promoters |
US4992367A (en) * | 1986-05-12 | 1991-02-12 | Hoffmann-La Roche Inc. | Enhanced expression of human interleukin-2 in mammalian cells |
WO1997035959A1 (en) * | 1996-03-27 | 1997-10-02 | Cytel Corporation | Production of galactosyltransferase |
Also Published As
Publication number | Publication date |
---|---|
MXPA01003810A (es) | 2003-10-14 |
US20060062772A1 (en) | 2006-03-23 |
RU2268940C2 (ru) | 2006-01-27 |
WO2000023601A1 (en) | 2000-04-27 |
BR9914547A (pt) | 2001-06-26 |
CN1323348A (zh) | 2001-11-21 |
KR20010099687A (ko) | 2001-11-09 |
ID30149A (id) | 2001-11-08 |
JP2002527112A (ja) | 2002-08-27 |
EP1121449A1 (en) | 2001-08-08 |
NZ510943A (en) | 2004-10-29 |
EP1121449A4 (en) | 2004-12-01 |
US6967089B1 (en) | 2005-11-22 |
CA2346799A1 (en) | 2000-04-27 |
ZA200102965B (en) | 2001-10-12 |
AUPP655698A0 (en) | 1998-11-05 |
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