CN102409064B - 一种提高动物繁殖力的干扰载体 - Google Patents
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Abstract
本发明公开了一种提高动物繁殖力的干扰载体，其序列包含基因序列Sequnence NO.11。本发明针对绵羊干扰素INHA基因的核苷酸序列设计合成多对miRNA单链寡核苷酸，构建了pMD-18T载体，通过功能性实验筛选，得到了抑制效果较强的MR062-3-R(Sequnence NO.6)载体，该载体包含基因序列Sequnence NO.11，进一步将基因序列Sequnence NO.11构建到miRNA慢病毒干扰载体。本发明还公开了上述新型载体的应用。
Description
技术领域
本发明涉及一种新型载体的构建。具体而言，本发明针对绵羊干扰素INHA基因的核苷酸序列设计合成miRNA单链寡核苷酸，构建了miRNA慢病毒干扰载体，得到了具有良好应用前景的新型载体。
背景技术
人类首次从牛的睾丸组织中分离、纯化到了一种糖蛋白激素[McCullagh DR.DUAL ENDOCRINE ACTIVITY OF THE TESTES.Science，1932，76(1957)：19-20.]，根据其具有特异性地抑制垂体细胞分泌及合成促卵泡素(Follicle-Stimulating Hormone，FSH)的生物学活性而将它命名为抑制素(Inhibin，INH)，以后逐渐发现它在多个组织器官中广泛分布。抑制素分子由一个α亚基与一个β亚基通过二硫键连接而成。生物体中只有当α亚基与β亚基处于结合状态时，抑制素表现出生物活性。在生理状况下抑制素能选择性地抑制垂体合成和分泌促卵泡素，从而影响卵巢上卵泡的发育和成熟，是调节家畜繁殖力的重要生殖激素之一。
研究表明抑制素α亚基与动物繁殖性能之间存在密切的关系。采用PCR-SSCP技术检测抑制素α(inhibinα，INHA)基因5′调控区和外显子1在高繁殖力绵羊品种(小尾寒羊)以及低繁殖力绵羊品种(中国美利奴绵羊、考力代绵羊和南非肉用美利奴绵羊)中的单核苷酸多态性[周文然，储明星，孙少华，方丽，叶素成.小尾寒羊高繁殖力候选基因INHA的研究.农业生物技术学报，2007，15(1)：32-36.]，结果发现小尾寒羊、中国美利奴绵羊和考力代绵羊在INHA基因5′调控区发生了1处碱基突变(316C→T)，南非肉用美利奴绵羊没有发生这种突变；小尾寒羊和考力代绵羊在INHA基因外显子1中发生了1处碱基突变(877T→C)，中国美利奴绵羊和南非肉用美利奴绵羊没有发生这种突变；初步表明INHA基因可能是影响小尾寒羊高繁殖力的一个主效基因或是与之存在紧密遗传连锁的一个分子标记。对高繁殖力绵羊品种与低繁殖力绵羊品种之间抑制素α亚基基因多态性的研究发现，高繁殖力绵羊品种与低繁殖力绵羊品种之间基因型分布差异都极显著(P＜0.01)。田秀娥等[田秀娥，孙红霞，王永军.3个绵羊群体INHA基因的遗传多态性及对产羔数的影响.西北农林科技大学学报(自然科学版)，2010，38(1)：23-29.]用PCR-SSCP方法检测和分析3个绵羊品种(滩羊、蒙古羊和小尾寒羊)INHA基因的多态性，研究不同基因型对产羔数的影响，发现滩羊、蒙古羊和小尾寒羊P1(5′调控区282核苷酸处发生的1处A→G突变)位点C、D基因频率分别为0.840和0.160，0.852和0.148及0.162和0.838，均处于中度多态；P2位点E、F基因频率分别为0.784和0.216，0.787和0.213及1.000和0.000，滩羊和蒙古羊在该位点均处于中度多态；引物P3扩增片段中，滩羊和蒙古羊表现为A、B和C三种单倍体基因型，而小尾寒羊仅表现出A和B2种基因型。鉴于INHA对绵羊繁殖力的影响，Li Han等[Li Han，D.G. Mao，D.K.Zhang，A.X.Liang，M.Fang，Muhammad Moaeen-ud-Din，L.G.Yang.Developmentand evaluation of a novel DNA vaccine expressing inhibinα(1-32)fragment forimproving the fertility in rats and sheep.Animal Reproduction Science，2007，109(2008)：251-265.]将抑制素α亚基(1-32)片段插入到HBsAg-S的羧基端，构建出一种抑制素DNA疫苗。用这种疫苗免疫40只绵羊，结果发现经过绵羊的实验组绵羊的双胎率(39.2％)显著高于对照组绵羊的双胎率(10％)(P＜0.05)。结果表明用针对抑制素构建的DNA疫苗进行免疫可诱导产生更多的卵泡，从而增加产仔数。此外许多研究也表明通过抑制素主动免疫，使动物产生抑制素抗体，解除内源性抑制素对垂体FSH合成和释放的抑制作用可以提高动物的繁殖力[Li C，Zhu YL，Xue JH，Zhang SL，Ma Z，Shi ZD.Immunization against inhibinenhances both embryo quantity and quality in Holstein heifers after superovulationand insemination with sex-sorted semen.Theriogenology，2009，71(6)：1011-7.Eamens AL，Waterhouse PM.Vectors and Methods for Hairpin RNA and ArtificialmicroRNA-Mediated Gene Silencing in Plants.Methods Mol Biol，2011，701：179-97.]。
Lee等首先在秀丽新小杆线虫(Caenorhabditis elegans)中发现了第一个可时序调控胚胎后期发育的基因lin-4，时隔七年之后，Reinhart等又在同类线虫中发现第二个异时性开关基因let-7。由于这类基因所编码的能时序调控发育进程、长度约为21个核苷酸的小分子RNA的长度很短而且作用时间也是暂时的，所以开始被命名为stRNA(Small Temporal RNA)。随后多个研究小组分别从线虫、果蝇和人体等多种真核生物细胞中发现了近千个相似的小分子RNA，统称为miRNA。miRNA长21-25nt，由70-90nt的可形成发夹结构的内源性转录前体pri-miRNA加工而成，在进化上具有高度的保守性。miRNA能够通过与靶mRNA特异性的碱基互补配对，引起靶mRNA的降解或者抑制其翻译，在基因调控中扮演重要的角色。研究表明体外构建针对特定基因的miRNA表达载体可有效抑制相应靶基因的表达[Furukawa N，Sakurai F，Katayama K，Seki N，Kawabata K，Mizuguchi H.Optimization of a microRNA expression vector for function analysisof microRNA.J Control Release.2010 Dec 10.Schwab R，Ossowski S，WarthmannN，Weigel D.Directed gene silencing with artificial microRNAs.Methods Mol Biol，2010，592：71-88.]。而慢病毒以其较低的转染条件(可转染分裂和非分裂细胞)和较高的转染效率被广泛应用于基因表达的研究中。曾东风等针对在人肿瘤及肿瘤基质形成过程中发挥重要作用的转录因子HIF-1α构建了miR RNAi慢病毒载体，经测序验证后正式microRNA慢病毒干扰载体构建成功，为进一步探索白血病的治疗创造了新切入点。张红梅等构建了针对基质金属蛋白酶组织抑制因子(TIMPs)的miRNA慢病毒干扰载体，并研究了其对小鼠肝脏星状细胞HSC-T6中TIMP1表达的影响，结果表明构建的慢病毒载体可有效抑制靶基因TIMP1的表达。
抑制素主动免疫这种方法只对亲代动物起作用，其不可能建立一种长效机制，将亲代高繁性状传递给子代动物。为了解决这一问题，本发明从microRNA入手，构建了针对绵羊抑制素α亚基的miRNA慢病毒干扰载体，转入绵羊基因组，抑制绵羊体内抑制素的表达水平，从而可使子代动物获得与亲本相同的繁殖力，为高繁殖力绵羊新品种培育奠定基础。
发明内容
本发明公布了一种提高动物繁殖力的干扰载体，所述miRNA慢病毒干扰载体包含基因序列Sequnence NO.11。
所述载体可以抑制绵羊抑制素的蛋白表达。由于所述载体在细胞内表达包含基因序列Sequnence NO.11的miRNA，干扰了绵羊抑制素的mRNA水平，使其翻译受限从而抑制了绵羊抑制素的蛋白表达。
将所述miRNA慢病毒干扰表达载体与脂质体和台酚蓝混合，制备成转染液。接着采用微创手术，将上述转染液用分多点注射入绵羊两侧的睾丸中再与雌性交配，得到10只阳性的雌性F1代。进一步将阳性的雌性F1代进行繁育实验，与非转基因绵羊相比其双胎率得到了显著的提高。
所述载体可以应用于培育高繁殖力绵羊新品种。由于所述载体抑制了绵羊抑制素蛋白的表达，减轻内源性抑制素对垂体FSH合成和释放的抑制作用可以提高动物的繁殖力，为高繁殖力绵羊新品种培育奠定基础。
本发明还公开了一种pMD-18T载体，所述pMD-18T载体包含基因序列Sequnence NO.6。
附图说明
图1MR062-1号干扰质粒与INHA高表达质粒共转染HEK293细胞(200×)(A：荧光视野；B：可见光视野)。
图2MR062-2号干扰质粒与INHA高表达质粒共转染HEK293细胞(200×)(A：荧光视野；B：可见光视野)。
图3MR062-3号干扰质粒与INHA高表达质粒共转染HEK293细胞(200×)(A：荧光视野：B：可见光视野)。
图4MR062-4号干扰质粒与INHA高表达质粒共转染HEK293细胞(200×)(A：荧光视野；B：可见光视野)。
图5SR-neg阴性对照质粒与INHA高表达质粒共转染HEK293细胞(200×)(A：荧光视野；B：可见光视野)。
图6空白对照质粒转染HEK293细胞(200×)(A：荧光视野；B：可见光视野)。
图7干扰质粒对INHA基因的沉默效率。
图8MR062-3号慢病毒干扰质粒病毒包装后48h细胞情况(200×)(A：荧光视野；B：可见光视野)。
图9病毒浓缩液稀释103时荧光表达情况(200×)(A：荧光视野；B：可见光视野)。
图10病毒浓缩液稀释104时荧光表达情况(200×)(A：荧光视野；B：可见光视野)。
图11病毒浓缩液稀释105时荧光表达情况(200×)(A：荧光视野；B：可见光视野)。
图12病毒浓缩液稀释106时荧光表达情况(200×)(A：荧光视野；B：可见光视野)。
图13转基因绵羊与非转基因绵羊的双胎率。实验组为用所述载体得到的低表达抑制素的雌性绵羊，对照组为非转基因绵羊。
实施例1慢病毒干扰载体的构建
一.研究方法
1.构建miRNA干扰载体
利用Invitrogen公司miRNA在线设计工具http://rnaidesigner.invitrogen.com/rnaiexpress/针对绵羊INHA基因(L28815)的核苷酸序列设计合成4对miRNA单链寡核苷酸(oligo)(表1)，将4对合成好的oligo用ddH2O溶解成100μM，互补单链各取5μl两两混合，进行退火。然后将4份oligo混合物在95℃加热5分钟，然后放置室温自然冷却20分钟，形成双链oligo。将退火的双链oligo继续稀释成10nM浓度，用载体构建试剂盒BLOCK-iTTM Pol II miR RNAi Expression Vector Kit with EmGFP，将4对双链的miRNA oligo分别插入到miRNA表达载体pcDNATM6.2-GW/EmGFPmiR中，室温连接30分钟，构建4个miRNA表达质粒，并转化至感受态细胞DH5α。，在每个转化平板中分别挑取3个克隆进行测序，验证重组克隆中插入片段序列是否与设计的oligo序列一致。
表1.miRNA oligo序列(注：划线字体为干扰靶序列)
2.INHA高表达载体构建
对INHA基因的编码区进行全序列分析，检查基因内部有无特别复杂二级结构和重复序列；根据基因序列分析的结果，进行单链oligo的设计及合成，并在序列的两端制造粘性末端。利用PCR将合成的oligo拼接成完整的基因；将合成好的序列装入pMD-18T载体并转化至感受态细胞DH5α；测序验证重组克隆中基因序列是否与要求相符；通过重叠PCR将基因序列中突变点修复；将突变后的全长片段用XhoI和EcoRI酶切后连接至目的载体pIRES2-eGFP，并转化到感受态细胞DH5α中；测序验证重组克隆中目的基因片段的序列信息，获得正确的INHA高效表达载体。
3.干扰载体的筛选
1)细胞转染
将生长状态良好的HEK293细胞消化计数后，按照每孔5×105个细胞接种于六孔板中。待细胞生长状态稳定，融合率在80％左右时，进行转染实验；用无血清Opti-MEM轻洗孔底细胞一次，加入1.5ml Opti-MEM；分别用250ul Opti-MEM稀释3ug干扰质粒MR062-1、MR062-2、MR062-3、MR062-4、阴性对照质粒SR-neg以及1ug高表达质粒，轻轻混匀；用250ul Opti-MEM稀释10ul Lipofectamine2000试剂，轻轻混匀，室温孵育5min；轻轻混合已稀释质粒和Lipofectamine2000稀释液，室温放置20分钟；将每管500ul脂质体质粒复合物慢慢加入到各细胞孔中，轻轻混匀；在37℃，5％的CO2的培养箱中培养4-6小时后，换上完全培养液(不含抗生素)，放置在37℃，5％的CO2的培养箱中继续培养过夜；第二天观察是否有荧光表达。
2)qPCR检测
Trizol法提取转染成功的HEK293细胞中的总RNA，反转录得到cDNA，然后用得到的cDNA进行q-PCR检测，50ul反应体系，反应条件为：95℃，2分钟；95℃，10秒钟，60℃，20秒钟，72℃，30秒钟以上三步进行40个循环；72℃，10分钟；然后60℃开始，每升高0.5℃，保持温度30秒钟，70个循环后升至95℃。最后计算出基因的表达量和目标基因的沉默效率，计算方法：
ΔΔct＝(待测样品的目的基因的ct的平均-待测样本的看家基因的ct的平均)-(对照样品的目的基因的ct的平均-对照样本的看家基因的ct的平均)
基因的表达量F＝2-ΔΔct目标基因的沉默效率为1-2-ΔΔct
4.慢病毒干扰载体构建
使用Invitrogen公司的BP重组系统：2ul pcDNATM6.2-GW/EmGFPmi-SR(60-150ng)、1ul pDONR221载体、2ul BP克隆酶II、5ul超纯水组成的BP重组反应体系，25℃反应1小时；然后加入1ul的蛋白酶K，37℃反应10分钟；取5ul重组反应液转化100ul的DH5α感受态细胞，选阳性克隆并测序验证，保留测序验证正确的BP重组质粒。从而将miRNA表达序列重组到载体pDONR221上，使用Invitrogen公司的LR重组系统：1ul BP重组产生的BP重组质粒、1ulpLenti6.3/V5-DEST、2ul LR克隆酶II 6ul超纯水组成的LR重组反应体系，25℃反应1小时；然后加入1ul的蛋白酶K，37℃反应10分钟；取5ul重组反应液转化100ul的Stbl3感受态细胞，选阳性克隆并测序验证，保留测序验证正确的BP重组质粒。从而将miRNA表达序列重组到慢病毒载体pLenti6.3/V5-DEST上。
5.慢病毒制备
取细胞状态良好，处于对数生长期的293T细胞，细胞消化计数后，每个10cm的细胞培养皿接种3×106个细胞，37℃，5％的CO2培养箱中培养过夜。9ugPackaging Mix和3ug重组慢病毒质粒加入至1.5ml Opti-MEM中，36ulLipofectamine2000加入至1.5ml Opti-MEM中，室温放置5min；混合质粒和Lipofectamine 2000稀释液，轻轻混匀，室温孵育20min；细胞培养皿中细胞，用Opti-MEM小心的洗两遍后，加入5ml Opti-MEM。将3ml质粒脂质体复合物小心地加入到细胞培养皿中，轻轻混匀，37℃，5％的CO2培养箱中孵育4-6个小时后，更换完全培养基。48h后收集细胞培养上清，3000rpm离心15min，去除细胞残余，上清用0.45um的滤器过滤分装，每管1.0ml。将病毒液放置于-80℃保存。
6.病毒滴度的测定
第一天，细胞胰酶消化计数细胞后，接种24孔板，37℃培养过夜，感染时细胞长至30～50％的融合密度；第二天，转染时，将保存于-80℃冰箱中病毒液融解，用含有2％FBS的细胞培养基进行10倍梯度稀释，从10-1稀释到10-6(每个浓度梯度可以做二个重复)。注：病毒梯度稀释时，轻轻地颠倒混匀稀释液，不能漩涡混匀；去掉24孔板中的培养基，轻轻颠倒每管慢病毒稀释液，各取1ml加入每孔细胞中，加入聚酰胺(Polybrene)终浓度至8ug/ml，轻摇使混匀，37度过夜培养；第三天，去除含慢病毒的培养基，加入2ml完全培养基；第四/五天，在荧光显微镜下观察各孔中GFP表达量，计算病毒滴度(TU)/ml：
二.结果
经测序验证，miRNA表达载体构建成功，INHA高效表达载体构建成功，且q-PCR法检测INHA的表达情况，结果表明，转染高表达质粒pIRES2-EGFP-INHA的细胞中，目的基因INHA的表达量与细胞本底相比升高1010倍(表2)。将构建好的miRNA表达载体与INHA高效表达载体转入HEK293细胞进行筛选，其在荧光显微镜下的结果见图1、图2、图3、图4、图5、图6。通过q-PCR的检测发现MR062-3号干扰质粒干扰效率最高结果见表3，目标基因被沉默的效率将近85％。转染阴性对照质粒SR-neg和干扰质粒MR062-1、MR062-2、MR062-3、MR062-4对INHA基因的沉默效率见图7。用MR062-3号干扰质粒构建慢病毒干扰质粒，测序结果表明慢病毒质粒构建成功。对构建成功的慢病毒质粒进行慢病毒包装，获得滴度为3.18×107TU/ml的慢病毒液。慢病毒液滴度测定时荧光显微镜下的结果见图8、图9、图10、图11、图12。
表2高表达质粒的qPCR检测结果
检测结果显示，转染高表达质粒pIRES2-EGFP-INHA的细胞中，目的基因INHA的表达量与细胞本底相比升高1010倍。
表3干扰质粒与高表达质粒共转染HEK293细胞qPCR检测结果
MR062-3号干扰质粒干扰效果最好，达到84.82％。MR062-3-R(SequnenceNO.6)的序列为CCTGAGAGTAA CCTCTCCGAG GTGTCAGTCA GTGGCCAAAA CACCTCGGATGGAGGTTACTCTC；其中，该干扰质粒的发挥作用的核心序列为ACCTCGGATGGAGGTTACTCT(Sequnence NO.11)。本发明构建的针对绵羊抑制素α亚基的miRNA慢病毒干扰载体MR062-3号干扰质粒，明显的抑制了抑制素的表达水平，为高繁殖力绵羊新品种培育奠定了基础。
实施例2慢病毒干扰载体在绵羊育种中的应用
挑选28只雄性绵羊，将INHA慢病毒干扰表达载体与脂质体和台酚蓝混合，制备成转染液。接着采用微创手术，将上述转染液用分多点注射入绵羊两侧的睾丸中再与雌性交配。最后通过PCR技术，检测F1代绵羊INHA基因的表达。通过Southern印迹检测排除假阳性F1代绵羊，得到11只阳性的雄性F1代，10只阳性的雌性F1代。
将得到的阳性的雌性F1代进行繁育实验。实验分为两组，其中实验组为10只阳性的雌性F1代绵羊与非转基因的雄性绵羊交配，对照组为20对非转基因绵羊交配做对照。比较两组的双胎率(图13)，其中实验组的双胎率为60％(6/10)，对照组双胎率为15％(3/20)。本发明载体转基因动物实验证明，INHA慢病毒干扰表达载体可以明显提高动物的繁殖力。
Claims (4)
1.一种提高绵羊繁殖力的干扰载体，其特征在于，所述干扰载体为miRNA慢病毒干扰载体，通过以下方法制备：
1)将序列为SEQ ID NO.5和SEQ ID NO.6的一对合成好的oligo用ddH2O溶解成100μM，互补单链各取5μl混合，进行退火，然后将oligo混合物在95℃加热5分钟，然后放置室温自然冷却20分钟，形成双链oligo，将退火的双链oligo继续稀释成10nM浓度，用载体构建试剂盒BLOCK-iTTM Pol II miR RNAi Expression Vector Kit with EmGFP，将上述得到的双链oligo插入到miRNA表达载体pcDNATM6.2-GW/EmGFPmiR中，室温连接30分钟，并转化至感受态细胞DH5α，在每个转化平板中分别挑取3个克隆进行测序，验证重组克隆中插入片段序列是否与设计的oligo序列一致；
2)使用Invitrogen公司的BP重组系统：2μl第1)步得到的含有SEQ ID NO.5和SEQ ID NO.6的miRNA表达载体pcDNATM6.2-GW/EmGFPmiR、1μlpDONR221载体、2μlBP克隆酶II、5μl超纯水组成的BP重组反应体系，25℃反应1小时；然后加入1μl的蛋白酶K，37℃反应10分钟；取5μl重组反应液转化100μl的DH5α感受态细胞，选阳性克隆并测序验证，保留测序验证正确的BP重组质粒，从而将miRNA表达序列重组到载体pDONR221上，使用Invitrogen公司的LR重组系统：1μl BP重组产生的BP重组质粒、1μlpLenti6.3/V5-DEST、2μl LR克隆酶II、6μl超纯水组成的LR重组反应体系，25℃反应1小时；然后加入1μl的蛋白酶K，37℃反应10分钟；取5μl重组反应液转化100μl的Stb13感受态细胞，选阳性克隆并测序验证，保留测序验证正确的BP重组质粒，从而得到含有SEQ ID NO.5和SEQ ID NO.6的单链寡核苷酸对的慢病毒载体pLenti6.3/V5-DEST。
2.根据权利要求1所述的干扰载体，其特征在于，所述载体可以抑制绵羊抑制素的蛋白表达。
3.根据权利要求1所述的干扰载体，其特征在于，所述载体可以应用于培养高繁殖力绵羊品种。
4.一种pMD-18T载体，其特征在于，所述pMD-18T载体包含基因序列SEQ ID NO.6。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000025831A1 (en) * | 1998-11-03 | 2000-05-11 | Federman & Sons (Holdings) Ltd. | A new composition and method for increasing production yield of agriculturally useful species |
| JP2005116204A (ja) * | 2003-10-03 | 2005-04-28 | Daikin Ind Ltd | 放電装置および空気浄化装置 |
| CN101029340A (zh) * | 2007-04-11 | 2007-09-05 | 中国农业科学院北京畜牧兽医研究所 | 一种筛选高繁殖力山羊的方法及其专用引物 |
| CN101338340A (zh) * | 2008-08-29 | 2009-01-07 | 中国农业科学院北京畜牧兽医研究所 | 一种检测山羊繁殖力的方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000025831A1 (en) * | 1998-11-03 | 2000-05-11 | Federman & Sons (Holdings) Ltd. | A new composition and method for increasing production yield of agriculturally useful species |
| JP2005116204A (ja) * | 2003-10-03 | 2005-04-28 | Daikin Ind Ltd | 放電装置および空気浄化装置 |
| CN101029340A (zh) * | 2007-04-11 | 2007-09-05 | 中国农业科学院北京畜牧兽医研究所 | 一种筛选高繁殖力山羊的方法及其专用引物 |
| CN101338340A (zh) * | 2008-08-29 | 2009-01-07 | 中国农业科学院北京畜牧兽医研究所 | 一种检测山羊繁殖力的方法 |
Non-Patent Citations (1)
| Title |
|---|
| 小尾寒羊高繁殖力候选基因INHA的研究;周文然等;《农业生物技术学报》;20071231;32-36 * |
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