CN101366949A - 提高动物饲料利用率和/或生长速度和/或减少脂肪积累的方法 - Google Patents
提高动物饲料利用率和/或生长速度和/或减少脂肪积累的方法 Download PDFInfo
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- CN101366949A CN101366949A CNA2008101669012A CN200810166901A CN101366949A CN 101366949 A CN101366949 A CN 101366949A CN A2008101669012 A CNA2008101669012 A CN A2008101669012A CN 200810166901 A CN200810166901 A CN 200810166901A CN 101366949 A CN101366949 A CN 101366949A
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Abstract
本发明公开了一种提高动物饲料利用率和/或生长速度和/或减少脂肪积累的方法。该方法,是对动物施用能够释放生长激素释放因子的表达载体,所述表达载体的核苷酸序列中含有依次串联的启动子序列、GHRF信号肽的编码基因序列和GHRF的编码基因序列。实验证明,本发明的方法能提高饲料效率和生长速度,并减少脂肪积累,产生更好质量的肉。
Description
本申请是申请号为00100010.1、申请日为2000年1月3日、发明创造名称为“提高饲料效率和家畜生长速度的基因处理”的分案申请。
本发明涉及施用内源激素肽,具体地讲是涉及施用含有以基因为基础的物质的内源激素肽,以提高饲料效率和家畜的生长速度。
已有许多刺激家畜生长速度并提高饲料转化效率的方法。这些方法包括使用抗生素、化学物质以及使用像天然或重组生长激素(GH)或生长激素释放因子(GHRF)的生物化合物。这些方法中许多要么有副作用,要么太昂贵以至于不能在现有的农场或牧场上实施。
尽管过去经常用抗生素添加物来提高生长性能和饲料转化,但由于现代化农场管理方面的改进和有向包括人在内的其它动物传播抗生素抗性的潜在危险,抗生素添加物带来的有益效果正逐渐消失。
在生物改进方面最早的尝试是注射脑垂体提取物,脑垂体GH就是后来从中分离并纯化出来的。
GH对身体组织有各种作用,最终导致生长速度和体重的增加。在体内,GH刺激蛋白质的合成,脂肪的裂解,并刺激骺生长。这些作用是由生长调节素例如胰岛素样生长因子I(IGF-I)的增量调节而介导的。
脑垂体GH的产生和释放受两种下丘脑激素肽的控制。促生长素抑制素的抑制作用和GHRF的刺激作用调节胞质GH水平。随动物年龄的增加,GHRF→GH→IGF-I轴发生不利的变化。因此,在较老的动物包括人中,GH的产生速率降低,IGF-I对GH和GHRF的减少作出反应。这会引起骨质疏松、瘦肉减少以及躯干脂肪沉积的增加。因此,已经用从脑垂体提取的GH补充GH的天然减少并促进生长。但是,所述处理还带来了偶发性病原体传播的危险。此外,所述处理的主要缺陷是所涉及的费用太高。由于GH是一种蛋白质,所以,很容易被肠酶降解,因此,必须以注射施用。另外,由于GH的血清半衰期短,因此,得每天施用GH。这进一步增加了处理费用,使其只适用于对人的治疗。
重组DNA技术的发展和使用象大肠杆菌这样的微生物以生产哺乳动物GH,意味着可以大量生产用于治疗的同源GH制品,由此降低成本。另外,通过在一级结构中的工程化取代而在蛋白质稳定化方面取得的最新进展和缓释制剂的发展已经使给药频率减少至每周一次或每两周一次。由于治疗成本的降低,因此,在商业上可用于促进农业生产。例如,目前在美国有三分之一的乳牛场在使用重组牛GH来促进牛奶的生产。
用猪GH处理猪可提高生长率,增加屠体蛋白质,同时减少脂肪的比例。已由Palmer J.Holden(Porcine somatotropin(pST),BiotechnologyInformation Series(Bio-4),Iowa State University,1993)就猪GH对猪生长及有关安全和经济方面的益处作了综述。将二十年来的研究数据做一总结,说明注射了GH的猪生长速度提高15%,而消耗的饲料减少21%,同时,使肌肉蛋白增加并且背部脂肪减少。但是,在生产率方面的这些提高要付出相当的代价。首先,由于GH的血清半衰期短并且被迅速从血液中清除,所以必须每天注射GH才有效。因此,在为期一至两个月的修整期内,必须每天给猪施用高达4mg的猪GH蛋白以维持血浆GH水平并促进生长。这就使得每头猪最终总共获得了高达400mg的GH。其次,高剂量可能是有毒的并会引起某些副作用,例如呼吸困难,参见美国专利5134120。此外,至今在文献中所记载的处理方案过于繁琐并且对动物来讲是创伤性的。尽管美国专利5015626和5134120分别记载了使用猪GH来改善肉的质量和促进体重增加,但是目前在以GH肽为基础的生长促进剂方面的技术还未达到足以用于商品化农业的水平。
生长激素释放因子(GHRF)的发现开拓了一种更好地促进生长的方法。GHRF是一种由下丘脑分泌的肽激素,它特异性地刺激垂体前叶促生长激素细胞合成和释放GH。通过刺激内源性GH的生产,使所需的有效剂量远远低于GH的剂量,从而提供了成本更低、更符合生理模式的处理方法。然而,据我们所知,仍没有令人满意并且经济的用于家畜的处理方法。例如,在EP0289186中记载了每天施用两次人GHRF蛋白注射液来刺激绵羊生长的方法。但是,长期注射人GHRF会引起绵羊的免疫应答,对人蛋白产生中和抗体。这会降低这种处理方法的效率,并且人力和物力的浪费使该方法变得不经济。GHRF肽注射液曾被用于持续地刺激猪GH的产生。尽管证实某些试验在引起体重增加和降低屠体脂肪含量上是成功的,但是快速清除的问题意味着需要频繁地给药。因此,需要新的用于促进生长的补充GH的方法。
自从第一次证实了肌肉内注射的质粒DNA载体可被骨骼肌细胞吸收并在体内表达数月,基于骨骼肌的基因治疗方法在治疗蛋白的全身传递方面的前景一直很好。这种可被分泌到循环系统到达远端靶位点由骨骼肌生产的重组蛋白非常适用于治疗因血清蛋白不足而引起的疾病。尽管质粒载体在蛋白表达方面的效率远远低于病毒载体,但其优点是免疫原性较低,并且不会整合到宿主基因组内。因此,它们被认为更安全一些。从而,质粒载体特别适于治疗蛋白在低浓度下有效的情况。因此,注射表达GHRF基因的质粒载体是用于长期补充足以促进动物生长之有效剂量的GHRF的一种理想方法。此外,持续的GHRF产生可大大减少处理频率,由此使成本降低至经济上适用的水平。
最近表明,将含有受鸡最小骨骼α-肌动蛋白启动子控制的人GHRF(hGHRF)的质粒注射到小鼠肌肉内后可引起血清GH水平的升高(Draghia-Akli等,自然生物技术(Nature Biotechnology),15,p1285-1289)。此外,据报道在注射3周后,生长增加了约15%。但是,该方法引起了免疫应答,表现在注射hGHRF基因21和28天后,抗hGHRF的中和抗体的血清水平增加。已证实注射质粒中的基因的原位表达水平的限制因素是肌肉纤维摄入质粒DNA的程度极低。为了提高hGHRF表达的水平,Draghia-Akli及其同事在将质粒DNA注射到再生的肌肉内之前,首先注射丁哌卡因(一种已知的肌毒素物质)。尽管已经证实了这可以通过增加肌肉纤维内摄入的质粒来提高表达水平,但是这种技术既不适宜也不宜用于供人消费的饲养动物。
因此,本发明的目的是提供了一种通过施用内源激素肽GHRF来刺激动物,特别是家畜的生长速度并提高饲料转化效率的经济可行的方法。最起码,本发明的目的是给公众提供一种有用的选择。
因此,提供了一种通过提高饲料效率和/或动物生长速度和/或减少脂肪积累的方法,该方法包括施用由外源基因序列编码的内源激素肽GHRF以刺激GHRF的产生,所述外源基因序列含有编码用于基因表达的DNA序列、编码GHRF信号肽的互补DNA(cDNA)序列和编码GHRF的cDNA序列。
本发明特别优选的实施方案是肌动蛋白启动子的各种启动子变体。根据本发明具体优选的实施方案,肌动蛋白启动子是骨骼肌动蛋白启动子。编码骨骼肌动蛋白启动子的DNA序列最好含有骨骼肌动蛋白启动子的全部DNA序列。
根据本发明的另一优选的实施方案,所述外源基因序列还包括编码所述启动子或GHRF 3′非翻译、含多聚腺苷酸信号区的DNA序列。
根据本发明另一优选的实施方案,所述外源基因序列还包括编码抗生素抗性基因的DNA序列。
由所述cDNA序列编码的GHRF和其信号肽可以是天然的或重组的或合成的GHRF和其信号肽,或者是它们的生物活性片段或具有相似活性的类似物。本发明特别优选的实施方案是启动子的DNA序列、编码GHRF信号肽的cDNA序列和编码GHRF的cDNA序列都是种特异性的。
刺激GHRF产生的、作为DNA序列的外源手段优选与可皮下施用的适宜载体混合。根据本发明具体优选的实施方案,外源DNA序列经肌肉内施用。
给每只动物施用的刺激GHRF产生的、作为DNA序列的外源手段的量优选是每kg动物体重1-100μg。
本发明的另一方面是提供刺激GHRF内源产生的外源基因序列,以提高饲料效率和/或动物的生长速度和/或减少动物的脂肪积累,其中,所述外源基因序列含有编码用于基因表达的启动子的DNA序列、编码GHRF信号肽的cDNA序列和编码GHRF的互补DNA(cDNA)序列。
本发明的再一方面是提供制备提高饲料效率和/或动物生长速度和/或减少动物脂肪积累的药物的方法,该方法包括提供编码用于基因表达的启动子的DNA序列,连接编码GHRF信号肽的cDNA序列,再连接编码GHRF的cDNA序列,以形成一基因序列,然后将所述肽与适宜的载体混合。
从下列描述中,本发明的其它目的、特征、优点和方面对于本领域技术人员来说将是显而易见的。但是应理解,下列描述和具体实施例,尽管是表明木发明的优选实施方案,但是它们仅仅是说明性的。在阅读下列描述和本发明公开的其它部分后,在公开的本发明精神和范围内的各种改变和修饰对于本领域技术人员来说将是显而易见的。
现在利用实施例并参考附图来解释本发明的优选实施方案,其中:
图1是pUPAGRF、pGHRF表达盒的图示,其中包括启动子、pGHRF编码区和多聚腺苷酸化信号的排列,同时,通过缺失pGHRF编码区得到对照质粒pUPAX。
图2表示构建pUPAGRF质粒时的主要步骤,并给出了所用的关键酶。
图3是表示从pUPAGRF构建pUPAX的图示。
图4说明在4周的时间内,pGHRF基因注射液4个处理组对雄性C57BL/6J小鼠平均体重的影响,pUPAGRF质粒的剂量为30、100和200μl,pUPAX对照为100μl,在下午2:00和4:00进行注射和称重。
图5表示在用pGHRF基因注射的小鼠中,体重增加百分比的差异。
图6表示pGHRF基因的剂量对雄性Landrace/Yorkshire/Duroc(LYD)猪的体重增加的影响。
图7表示在pGHRF基因注射后,Landrace/Yorkshire猪中平均背部脂肪厚度的变化。图7a为GHRF基因处理对雄性LY猪的平均背部脂肪厚度的影响;图7b为GHRF基因对雌性LY猪的平均背部脂肪厚度的影响。
优选用一质粒载体构建肌肉特异性GHRF表达系统。从公开的肽一级结构设计编码GHRF信号肽和GHRF本身的互补DNA(cDNA)序列并优化成用于哺乳动物的密码子。用常规寡核苷酸合成化学法合成该cDNA。由cDNA序列编码的GHRF和其信号肽可以是天然的或重组的或合成的GHRF和其信号肽,或者是生物活性片段或具有相似活性的类似物。GHRF前导序列在新的GHRF肽中编码信号肽,所述信号肽指导所述GHRF肽从肌肉细胞中转运出来并以活性形式分泌到总循环中。
通过聚合酶链反应分别克隆启动子/增强子元件的DNA序列和启动子/GHRF基因的3′非翻译的、含多聚腺苷酸化信号的区。例如,使用骨骼α-肌动蛋白(skαa)启动子/增强子,但不应认为这是对启动子/增强子选择的限制。使用全套的肌动蛋白启动子,而不是肌动蛋白的最小亚单位如Draghia-Akli等人描述。原因是,利用直接基因注射的体内研究已表明,与其它截短形式相比,完整骨骼α-肌动蛋白启动子在小鼠肌肉中可以得到最高水平的基因表达(Reecy et al.,AnimalBiotechnology 2:101-120,1998)。
作为本发明的一个优选的实施方案,肌动蛋白启动子的DNA序列、编码GHRF信号肽的cDNA序列和编码GHRF的cDNA序列是种特异性的。
将这些DNA片段以下列顺序插入到pUC质粒中:肌动蛋白启动子;人GHRF信号肽cDNA;猪GHRF(1-44)cDNA;肌动蛋白3′非翻译区。所述pUC质粒还含有抗生素例如氨苄青霉素抗性基因,所述抗性基因可以在质粒转化到大肠杆菌细菌中后进行选择,以及选择产生高拷贝数质粒的复制原点ColEI。
然后,将用GHRF表达质粒转化的大肠杆菌在氨苄青霉素选择下使之于发酵罐中生长。收集细胞并用碱溶解释放质粒后,用柱色谱将质粒DNA与RNA、细菌DNA与其它细胞污染物分开。通过限制酶消化/琼脂糖凝胶电泳和DNA测序确定纯化质粒DNA的完整程度。通过在OD260/280测量吸收值和琼脂糖凝胶电泳来确定纯度。
将分析过的质粒DNA溶解在生理学可接受的载体中,检测热原水平,调整剂量,然后经肌肉内注射施用给动物。作为本发明优选的实施方案,每只动物应施用的所述质粒DNA的总量应为每kg家畜体重1-100μg。
在注射到动物肌肉内后,质粒表达载体作为DNA被摄入到肌肉细胞内。然后质粒表达载体指导细胞产生GHRF肽。所述肽被释放到循环系统中,然后在到达脑垂体后刺激GH的产生。这样引起循环系统中总GH水平的升高,从而促进生长,使生长速度加快,增加瘦肉量并使食物转化更好。此外,产生的肉更瘦,脂肪更少,因此具有较高的市场价值。
此外,由于GHRF释放受组成性肌肉特异性启动子的控制,因此,促生长素抑制素对GH的正常负反馈控制被绕过了。这样可以连续产生GH。
提供下列实施例进一步说明,而不是限制本发明的实施方案。
实施例1 构建pGHRF表达载体
设计pGHRF表达构建体以便在转染到哺乳动物肌肉细胞中后,得到pGHRF肽的最佳生产和分泌(图1)。
骨骼α-肌动蛋白(skαA)启动子是熟知的启动子,已表明在分化后的肌肉纤维细胞中产生高度特异性的基因表达(Reecy et al.,Gene180:23-28,1996)。此外,它与人、牛和鸡skaα启动子有着高度的序列同一性,并享有许多转录因子共同序列结合位点。这表明该启动子在哺乳动物和鸟之间是高度保守的。这一点得到体外研究的支持,体外研究表明了猪skαa启动子在小鼠、大鼠和猪细胞中赋予肌肉特异性基因表达的能力。因此,选用该2kb启动子驱动pGHRF cDNA的肌肉特异性表达。
在含有编码人GHRF信号肽和猪GHRF成熟肽的DNA序列的开放读码框前剪接所述猪skαa启动子。
由于得不到编码猪GHRF的DNA序列,我们通过逆翻译已知的肽结构来设计该DNA序列,并根据哺乳动物密码子偏好来进行优化。本发明的进一步修饰包括在编码区的下游加入猪骨骼α-肌动蛋白基因的3′非翻译区。这样通过稳定mRNA转录本并延长其半衰期可提高pGHRF产物的表达。在下文详细描述构建pGHRF表达载体的方法并总结于图2中。
PolyA区:从猪肾中纯化基因组DNA并用于扩增猪基因序列。用PCR扩增750bp的skαa基因的3′末端。该区为已公开序列的核苷酸数2464-3204(Genbank Accession No.U16368)并含有3′非翻译区和推测的多聚腺苷酸化信号。然后用限制酶Xba I和Sac II切割所得产物(利用与PCR引物一起导入的切割位点),克隆到质粒载体pBluescript KS+中,得到pSαApA。
GHRF编码区:GHRF编码区是一个开放读码框(ORF),它含有编码人GHRF信号肽的DNA“前导序列”,其后接有一框架内猪GHRF(1-44)cDNA。从人基因组DNA PCR扩增所述前导序列,该前导序列含人GHRF基因的所有外显子2和部分外显子3(GenbankAccession No.L10034-5)。用下列两个序列将猪GHRF cDNA构建为两个寡核苷酸:
LGPGRF1
5′-TACGCCGACG CCATCTTCAC CAACAGCTAC AGGAAGGTGC
TGGGCCAGCT GAGCGCCAGG AAGCTGCTGC AGGACATCAT G-3′
LGPGRF2
5′-CGTCTAGATCACAGCCTCACCCTGGCGCCCTGCTCCTGGTTCC
TCTCGCCCTGCTGCCTGCTCATGATGTCCTGCAGCAGC-3′
这些寡核苷酸含有一个重叠,用所述重叠使其彼此退火,然后用Vent聚合酶填平。然后用Vent聚合酶经PCR扩增退火的产物。将前导序列和猪GHRF cDNA产物均用T4多核苷酸激酶激酶化,然后用聚丙烯酰胺凝胶电泳纯化。然后用T4DNA连接酶将产物连在一起并将265bp的连接产物再用聚丙烯酰胺凝胶进一步纯化。然后用含Xma I和Xba I限制酶位点的引物,经PCR再扩增该ORF产物。然后用XmaI和Xba I酶切割ORF,经纯化得到245bp片段。然后将该片段克隆到pSαApA中,得到pPGRFpA。
启动子区:从猪基因组DNA经PCR扩增全长2kb猪骨骼α肌动蛋白(skαa)启动子/增强子。在第一PCR反应的1/50后,进行嵌套聚合酶链反应(PCR)以得到跨猪skαa基因的-1909至+76bp(Reecy et al,Gene180:23-28,1996)的DNA产物。用T4DNA聚合酶将该产物制成平整末端,然后用Eco RI切割。将凝胶纯化过的启动子片段克隆到pPGRFpA的Eco RI和Eco RV位点以得到中间构建体pSαAPGRFpA。
为了得到最终的构建体,用侧翼Kpn I和Sac I限制酶切割该表达盒,然后与质粒pUC19相连,命名为pUPAGRF。用Xho I进行部分消化,从质粒pUPAGRF中除去全部的pGHRF编码区,得到第二构建体(图3)。用该第二质粒pUPAX作为动物试验中的对照。
将所述质粒分别转化到大肠杆菌K-12(菌株DH5α)中,然后使之在用150μg/ml氨苄青霉素补充的培养基中于发酵罐内生长。当培养达到稳定期后,收集细菌细胞并用经典碱溶解法溶解。离心回收质粒,然后通过阴离子交换色谱将其与污染的细胞DNA、RNA和蛋白质分开。通过在260和280nm测量吸收值来检测纯度。通过琼脂糖凝胶电泳检测纯化质粒DNA的完整性。最后,通过限制酶消化和DNA测序确定同一性。
将质粒DNA沉淀并再溶于无热原的磷酸盐缓冲盐水(PBS)中,通过测量在260nm的吸收值来计算浓度。完成热原检测后,此时的DNA可用于动物注射。
实施例2
为了检测pUPAGRF构建体促进动物生长的能力,我们将该质粒注射到小鼠的四头肌中。
从C57BL/6J小鼠的育种群体中,挑选48只体重14.5-18g的雄性小鼠,随机分成12只一组的4个组。给各组的12只小鼠分别注射一次100μl含100μg对照质粒或者30、100或200μg GHRF表达质粒(pUPAGRF)的PBS。将小鼠称重,作趾标记用于鉴别,然后在左四头肌中部进行一次注射。在控制气候、最少疾病环境和确定的标准大鼠食物和水供应的情况下将小鼠保持12小时的白昼循环。每周记录两次体重。用氯胺酮/赛拉嗪(0.15ml/100g体重)麻醉小鼠以得到血液和组织样品。通过心脏针刺,将血液收集到1ml胰岛素注射管中,注射管中含30μl 0.5M EDTA作为抗凝剂。将收集的血液保持在冰中,直到通过以6000g离心10分钟沉淀细胞而得到血浆后。将包含注射位点的完整四头肌切下,然后迅速冷冻于液氮中。还切下肝的右叶并立即冷冻。将所有样品储存于-80℃直到进一步分析。
结果:
在注射后的4周内,所有4个处理组中的小鼠均有迅速生长和体重增加。在注射后(P.I)21天,所有组之间的体重值没有显著差异。尽管在注射后25天,施用30μg pUPAGRF的小鼠体重增加最多,而最高剂量(200μg)组滞后(图4),但在较高剂量和对照之间有显著差异。注射后25天,200、100和30μg组的平均体重分别为26.3、26.5和24.7g,而对照组的体重为24.5g(p<0.032,单一因素ANOVA)。200和100μg处理组小鼠在注射后28天的平均体重分别为26.8和26.6g,而30μg和对照处理组的分别为24.5和25.0g。另外,与对照处理小鼠的体重相比亦有显著差异(p<0.0045)。
在图5中示出了pUPAGRF注射液对体重增加百分比的影响。注射质粒4周后,对照处理小鼠体重增加的百分比为48.7%,30和100μgpUPAGRF的值为56.6%,而200μg组的值为68.4%。这些结果表明注射一次pGHRF产生质粒能够使年轻小鼠的生长速度有显著提高。在剂量为100μg时,有约8%的提高,而200μg的剂量能够使体重增加百分比达到19%。这些结果与Draghia-Akli用再生小鼠肌肉得到的观察结果相同,尽管其注射方法需要先注射肌毒素丁哌卡因以提高质粒的摄入和表达。
实施例3
然后,我们检测了pGHRF基因处理提高饲养猪的生长的能力。
在本研究中使用Landrace/Yorkshire/Duroc(LYD)三重杂交的杂交猪。将育种场上10周龄的年轻雄性猪随机分到3个相邻的栏中。用1.5英寸16号针头在臀肌中给猪注射一次质粒制品。给A和B栏中的猪分别注射1mg和4mg的pUPAGRF,而给C栏中的猪注射4mg对照质粒pUPAX。不限制供给猪的饲料和水,然后每周称重。
结果:
在6周的试验期内,给猪注射一次1mg pUPAGRF可使体重从62.53增加至87.17kg(增加37.8%)。给猪注射4mg,使其平均体重从60.20kg增加至98.28kg(增加44.1%)。在相同的时间内,对照组中的猪从58.59增加至89.33kg(增加34.4%)。因此,6周后,注射pGHRF表达质粒的猪比注射对照质粒的猪的体重增加百分比要高。此外,由于与1mg剂量组相比,4mg剂量组使体重增加量超过了1mg剂量组的2倍,因此,似乎存在着剂量依赖反应的趋势。就饲料转化效率而言,1mg剂量组尽管生长速度中等,但消耗的饲料少了10%(表1)。此外,在对照和高剂量组之间,消耗的饲料量没有差异,因此,1mg和4mg剂量的pUPAGRF均能使饲料转化提高。
表1 注射质粒后LYD猪的平均每天饲料消耗
对照 | 1mg | 4mg | |
每头猪的平均每天饲料消耗(kg) | 3.960 | 3.563 | 3.963 |
对照的% | 100.00 | 89.98 | 100.08 |
相对于对照的% | 0.00 | -10.02 | 0.08 |
实施例4
本研究是确定给Landrace/Yorkshire(LY)猪注射2mg pUPAGRF质粒对背部脂肪积累的影响。20头约32周龄、平均体重55kg的LY猪被分成两组,10头一组,每组5头雄性,5头雌性。一组通过肌肉内注射施用2毫克pUPAGRF质粒。第二组用质粒pUPAX同样处理,作为对照。用Renco超声测量仪每周测量背侧至最后胸椎的背部脂肪厚度。分别在距最后肋椎骨侧至脊柱中心45mm、65mm和80mm的位置PI、PII和PIII取3个测量值,平均后得到平均背部脂肪厚度。
结果
结果总结于图7中。在雄性猪中,尽管施用pGHRF表达质粒的猪的背部脂肪增加的较少,但是经pGHRF处理和对照猪的背部脂肪厚度均随动物年龄的增加而逐渐增加。雌性对照猪的背部脂肪也逐渐增加,但该增加受pGHRF处理的抑制。表2列出了在试验期内背部脂肪厚度的相对增加值。在注射后60天,在雄性和雌性LY猪中,pGHRF处理减少了背部脂肪积累的量。
表2 注射质粒后在LY猪中背部脂肪厚度的相对增加
这些实施例的结果表明用pGHRF基因处理能够对小鼠和猪的生长均产生益处。生长速度的增加可与进行GH处理的方案相媲美,但本发明的成本更加经济和有效。每个动物所用的DNA量比文献中报道的生长激素注射液的量至少少100倍。此外,猪的数据表明所需的饲料量减少,而且背部脂肪厚度减少。因此,本发明通过提高饲料效率和家畜的生长速度而给家畜工业带来益处。
尽管已通过实施例详细描述了本发明的优选实施方案,但是本领域技术人员可对本发明进行的修饰和改变是显而易见的。但是应理解,所述修饰和改变在本发明范围内,正如下列权利要求的范围内。另外,本发明的实施方案不应被解释为仅受实施例的限制。
Claims (10)
1.提高动物饲料利用率和/或生长速度和/或减少脂肪积累的方法,是对动物施用能够释放生长激素释放因子的表达载体,所述表达载体的核苷酸序列中含有依次串联的启动子序列、GHRF信号肽的编码基因序列和GHRF的编码基因序列。
2.根据权利要求1所述的方法,其特征在于:所述肌动蛋白启动子为骨骼激动蛋白启动子。
3.根据权利要求2所述的方法,其特征在于:所述骨骼激动蛋白启动子为猪骨骼激动蛋白启动子。
4.根据权利要求3所述的方法,其特征在于:所述猪骨骼激动蛋白启动子为猪骨骼α肌动蛋白启动子。
5.根据权利要求2-4中任意一项所述的方法,其特征在于:所述GHRF的编码基因序列的3′端还连接有骨骼肌动蛋白基因的3′端非翻译区序列。
6.根据权利要求5所述的方法,其特征在于:所述骨骼肌动蛋白基因的3′端非翻译区序列为猪骨骼肌动蛋白基因的3′端非翻译区序列。
7.根据权利要求6所述的方法,其特征在于:所述骨骼肌动蛋白基因的3′端非翻译区序列为猪骨骼α肌动蛋白基因的3′端非翻译区序列。
8.根据权利要求1-7中任意一项所述的方法,其特征在于:所述GHRF信号肽的编码基因序列为人GHRF信号肽的编码基因序列;所述GHRF的编码基因序列为猪GHRF的编码基因序列。
9.根据权利要求8所述的方法,其特征在于:所述载体的核苷酸序列中还含有抗生素抗性筛选基因。
10.根据权利要求9所述的方法,其特征在于:所述表达载体的用量为每kg动物体重1-100μg表达载体;所述表达载体与可药用载体在施用前混合。
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