CN102399819A - 一种质粒型腺病毒载体pAd-NRIP1及其构建方法 - Google Patents
一种质粒型腺病毒载体pAd-NRIP1及其构建方法 Download PDFInfo
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Abstract
构建含秦川牛NRIP1(nuclear receptor interacting protein 1)基因的重组腺病毒载体，可应用于秦川牛NRIP1基因功能研究、鉴定和对种子细胞进行改造，调控肌肉、脂肪、生殖等组织相关基因的代谢。方法是将秦川牛NRIP1基因插入pAdTrack-CMV腺病毒穿梭质粒的CMV启动子之下，构建重组穿梭质粒pAdTrack-CMV-NRIP1。PmeI线性化后转化含有pAdEasy-1质粒的E.coli BJ5183感受态细胞，利用E.coli BJ5183内Cre重组酶高效的同源重组系统，在细菌中完成pAdTrack-CMV-NRIP1和pAdEasy-1的同源重组。将正确的重组腺病毒质粒命名为pAd-NRIP1。pAd-NRIP1经PacI酶切线性化后，脂质体转染293A细胞产生并获得重组病毒颗粒。
Description
技术领域
本发明涉及一种含有NRIP1基因的质粒型腺病毒载体，该质粒型腺病毒载体腺病毒载体的构建以及在改造种子细胞和牛NRIP1功能鉴定的应用。
背景技术
AdEasyTM系统是由T.C.He等(1998)构建的用来代替传统腺病毒重组系统的一个快捷系统。在这个系统中，只需二步即可产生重组腺病毒：先将表达盒装入转移载体，然后再通过同源重组插入腺病毒基因组。将外源基因插入腺病毒的最有效途径是通过同源重组，原因是：1)腺病毒DNA是大型的线性分子，含有几乎所有的内切酶切位点；2)基因组过大(36kb)，难于操作。
在AdEasyTM载体系统中，含有大部分腺病毒基因组的载体为超螺旋质粒，而不是线性DNA，同源重组 则在大肠杆菌进行。相对于传统系统而言，这两点改进使病毒DNA操作更容易，同时由于利用了大肠杆菌的高效率同源重组特性而使重组载体的筛选更为简单。在本系统中，首先将基因cDNA插入一个转移载体， 将得到的质粒用PmeI线性化，然后在大肠杆菌BJ5183中与病毒DNA质粒pAdEasy-1进行同源重组。pAdEasy-1缺失了E1和E3区，其E1区功能将在293A细胞中得到互补。重组子通过卡那霉素筛选，用内切酶进行鉴定分析。最后将得到的重组腺病毒用PacI线性化，暴露其反向末端重复序列(ITR，Iaverted Termined Repeats)，转染293A细胞后产生重组病毒颗粒。
同源重组在线性化的转移载体和完整的超螺旋腺病毒质粒之间进行。应用完整的腺病毒质粒而不用内切酶进行线性化，对于产生重组腺病毒至关重 要。转移载体中的卡那霉素抗性基因则可用来筛选重组子。由于线性化的转移载体产生卡那霉素抗性克隆的背景较低，因此此同源重组系统有较高的信噪比。大肠杆菌BJ5183有recA活性，但同时缺失介导细菌重组的其它酶，具有高效的转化和重组能力。一旦重组子经确定，则可转入普通的无recA、endA活性的细菌株如DH5α等进行扩增。由于缺乏recA活性，DH5α不能用于腺病毒的同源重组。
与传统系统相比，AdEasyTM系统中筛选和纯化腺病毒所需的时间大大缩短了。克隆和筛选约需1～3周，重组后需验证重组病毒质粒是否含有目的基因。重组子在DH5α中扩增后转入哺乳动物细胞293A，最后将293A细胞中产生的重组病毒颗粒进行纯化和滴定。最终得到的重组腺病毒只能在提供E1区功能的293A细胞中进行增殖。
一般来说，用传统方法产生重组腺病毒并不需要大量工作，每天只需1小时进行细胞传代和观察空斑形成。但由于病毒空斑形成速度慢，整个过程就需要很长时间。而AdEasyTM载体系统用大肠杆菌产生重组病毒，大大缩短了所需时间。
1995年Cavailles等(Cavailles，Dauvois et al.1995)从雌激素处理的ZR75-1孔腺癌细胞中鉴定出NRIP1，NRIP1成为最早发现的核受体辅助因子之一。1998年Lee等(Lee，Chinpaisal et al.1998)通过酵母双杂交实验分离出小鼠的NRIP1 cDNA。其后，人们鉴定出了大鼠、家兔狗等动物的NRIP1 cDNA。
NRIP1基因在各组织中广泛表达，且在脂肪、肝脏、肌肉等代谢组织中高表达。NRIP1蛋白定位于细胞核内，Gupta等(Gupta，Ho et al.2008)报道NRIP1经PKCε磷酸化修饰后，NRIP1蛋白向细胞质转位。因此，NRIP1蛋 白的亚细胞定位很可能受到多种机制调节。
人NRIP1基因位于第12条染色体上，编码1158个氨基酸，是单一外显子(Cavailles，Dauvois et al.1995)。GenBank显示，牛NRIP1基因位于第1条染色体，编码1156个氨基酸。人和小鼠的氨基酸序列具有83％的相似性，人和牛的氨基酸序列具有88％的相似性。在人上NRIP1存在10个保守氨基酸序列，前9个LXXLL基序(L，赖氨酸；X，任意氨基酸)和第10个LYYML基序(L，酪氨酸；M，甲硫氨酸；Y，任意氨基酸)。NRIP1可被多种核受体所募集，如过氧化物酶体增殖物激活受体(Peroxisome Proliferator-activated Receptors，PPARs)、雌激素相关受体(Estrogen Receptor-related Receptors，ERRs)、甲状腺激素受体(Thyroid Receptors，TRs)等，这几类核受体与LXXLL基序的结合能力不同(Heery，Hoare et al.2001)。
NRIP1包含四个抑制区域(Repression Domain，RD)，通过募集组蛋白去乙酰化酶(Histone Deacetylas，HDAC s)或碳端结合蛋白(C-terminal Binding Protein，CtBP)发挥抑制功能(Lee andWei 1999；Castet，Boulahtouf et al.2004；Augereau，Badia et al.2006)。在人上，RD1抑制区位于27-199为氨基酸残基序列，主要通过募集I、II的HDACs发挥转录抑制作用。RD2位于429-739为氨基酸残基，可与CtBP结合发挥转录抑制功能。RD2也可依次与CtBP、HDAC来发挥转录抑制功能功能，该途径可被曲古抑菌素(TSA)部分阻断。目前，RD3和RD4的功能还未见报道。
研究(Zschiedrih，Hardeland et al.2008)发现，经siRNA沉默的巨噬细胞，炎症反应发生水平显著降低。同时，研究还证明NRIP1对核受体NF-κB的靶基因起转录辅激活作用。
NRIP1活性除受蛋白表达水平影响外，一些翻译后修饰和其他型号途径也能够影响NRIP1的活性。Huq等(Huq，Khan et al.2005)发现了NRIP1氨基酸序列上存在11个磷酸化位点，这些修饰参与了NRIP1生物活性的调节。MAPK介导的Thr202、Thr207磷酸化修饰，能够增强NRIP1的转录辅抑制活性增强，参与RD1募集HDAC的过程(Huq，Khan et al.2005)。利用蛋白质组学(Huq and Wei 2005)的方法检测到NRIP1的8个乙酰化修饰的赖氨酸位点，这些修饰抑制了NRIP1与CtBP的相互作用，降低了NRIP1的辅抑制活性。此外，甲酰化、小泛素化也可调节NRIP1的活性，促进NRIP1向细胞质转位(Rytinki and Palvimo 2008)
NRIP1参与对脂肪细胞的成熟(Sawada，Miyoshi et al.2010)的调控，但是并不影响脂肪组织的发生。基因敲除NRIP1(Powelka Seth et al.2006)后，三羧酸循环、糖酵解、脂肪酸氧化、线粒体氧化磷酸化的相关基因表达明显增加，说明NRIP1调控的靶基因与代谢密切相关。而且，白色脂肪组织(White Adipose Tissue，WAT)中的NRIP1较褐色脂肪组织(Brown AdiPose Tissue，BAT)表达水平更高(Powelka，Seth et al.2006；Seale 2010)。NRIP1敲除小鼠WAT线粒体耗氧量明显增加，可能与UCP1和CPT1b基因表达增加导致线粒体能量无效释放有关(Leonardsson，Steel et al.2004)。研究表明，肥胖不仅是采食和能量消耗的结果，而且也是棕色脂肪组织和储存能量的白色脂肪组织平衡的结果(Gesta，Tseng et al.2007)。NRIP1敲除小鼠能够抑制肥胖发生。在高脂饲喂时，NRIP1敲除小鼠糖耐受能力明显改善(Powelka，Seth et al.2006)。所以，降低NRIP1的表达能够增加胰岛素敏感性、促进糖摄取，NRIP1有望成为治疗肥胖、II型糖尿病的有效靶点。
骨骼肌纤维被分为3种类型：I型，IIa型和IIb型，慢肌I型和快肌IIa型含有较多的线粒体具有相对较高的氧化代谢率。与之相反，IIb型纤维含有较少的线粒体，并进行糖酵解代谢。有报道发现糖酵解型代谢的指伸肌和腓肠肌NRIP1的mRNA水平高于氧化型代谢的比目鱼肌。NRIP1敲除小鼠(Fritah，Christian et al.2010)的氧化型肌纤维比例升高，肌肉组织中的线粒体活性增加。免疫荧光共沉淀发现NRIP1能够作用于FABP3和MACD的启动子序列，抑制脂肪酸摄取、线粒体β氧化等代谢过程，说明了NRIP1能够对骨骼肌的氧化代谢进行负向调控。
小鼠肝脏甘油三酯(Triglycerides，TG)代谢紊乱时，NRIP1的表达上调。特异性沉默肝脏NRIP1后，小鼠肝脏TG分解增加。说明通过抑制肝脏NRIP1基因表达可以治疗肝脏变性等肝脏疾病。研究报道，NRIP1与肝X受体(LiverX Receptor，LXRs)(Herzog，Hallberg et al.2007)结合调节肝脏脂质与糖代谢。Wang等(Wang and Collins 2010)报道肝X受体能够在脂肪组织中诱导NRIP1结合并抑制UCP1基因的转录调控。
因此对NRIP1的表达和功能研究对于了解、掌握和调控细胞代谢和增殖具有重要的意义。此外，今后对于研制以NRIP1为靶位的药物，治疗肥胖症、糖尿病、不育症、乳腺癌(Caplan，Mamillapalli et al.2008；Docquier，Harmand et al.2010)等疾病具有重要的应用和借鉴意义。
NRIP1作为重要的调节代谢因子，能够显著的调控糖酵解、甘油三酯合成、三羧酸循环、脂肪酸β氧化、氧化磷酸化等能量代谢过程相关基因的表达。通过秦川牛NRIP1基因腺病毒载体的构建能够为黄牛肉质性能、生长性能的标记辅助选择和新的优良品种的育种提供重要的研究资料，最终为转基 因动物的研究提供理论依据。
发明内容
本发明构建了能够表达秦川牛NRIP1基因的重组腺病毒载体，本发明所构建的载体，转染原代培养的秦川牛肌肉细胞后，可获得NRIP1 mRNA的高效表达，为NRIP1基因功能鉴定和改造细胞，调控代谢和生殖奠定了基础。
本发明是通过以下技术方案来实现：
PCR扩增NRIP1后与pGM-T载体连接，转化E.coli DH5α感受态细胞。试剂盒抽提pGM-T-NRIP1重组质粒，限制性内切酶BglII和NotI酶切，酶切产物通过1.0％琼脂糖凝胶电泳鉴定，1.0％琼脂糖凝胶电泳结果与理论上图谱吻合。将质粒送交上海金斯瑞公司进行测序，NRIP1基因测序结果与已报告的基因序列吻合。
pGM-T-NRIP1重组质粒和pAdTrack-CMV穿梭质粒经BglII和NotI双酶切，凝胶回收试剂盒回收NRIP1基因片段和pAdTrack-CMV载体片段，回收的载体片段和目的基因片段T4DNA Ligase进行酶连反应。酶连产物转化E.coli DH5α感受态细胞，纯化试剂盒抽提pAdTrack-CMV-NRIP1重组穿梭质粒，BglII和NotI双酶切鉴定证明获得正确的pAdTrackCMV-NRIP1重组质粒。
限制性内切酶PmeI线性化pAdTrack-CMV-NRIP1重组穿梭质粒，凝胶回收试剂盒回收线性化pAdTrack-CMV-NRIP1重组穿梭质粒，转化含有pAdEasy-1质粒E.coli BJ5183感受态细胞，利用E.coli BJ5183内Cre重组酶高效的同源重组系统，在细菌中pAdTrack-CMV-NRIP1和pAdEasy-1同源重组获得携带NRIP1基因的重组腺病毒质粒，将同源重组成功的重组腺病毒质粒命名为pAd-NRIP1。试剂盒抽提同源重组质粒，0.7％琼脂糖凝胶电泳，选择比pAdEasy-1大的重组腺病毒质粒，PacI酶切。酶切产物通过0.7％琼脂糖凝胶电泳鉴定，琼脂糖凝胶电泳结果与理论上同源重组质粒多克隆位点图 谱吻合。pAd-NRIP1重组腺病毒质粒转化E.coli DH5α感受态细胞，大量抽提质粒备用。pAd-NRIP1重组腺病毒质粒用PacI线性化暴露反向的末端重复序列，采用脂质体转染包装细胞(293A cells)进行病毒包装。转染24h后荧光显微镜直接观察，可见穿梭质粒带有的绿色荧光蛋白(GFP)基因表达。重组病毒上清感染293A细胞扩增重组腺病毒，荧光显微镜观察绿色荧光蛋白基因的表达。
附图说明
图1为PCR扩增的秦川牛NRIP1基因：其中1为D15000Marker，条带分别为1000，2500，5000，7500，10000，15000bp；2～6为NRIP1基因片段，大小为3517bp。
图2为秦川牛NRIP1基因pGM-T-NRIP1重组质粒酶切鉴定：其中1，2为BglII和NotI双酶切；3为EcoRI酶切；4为D15000Marker，条带分别为1000，2500，5000，7500，10000，15000bp。
图3为pAdTrack-CMV-NRIP 1重组质粒双酶切鉴定：1为D15000Marker，条带分别为250，1000，2500，5000，7500，10000，15000bp；2～3为BglII和NotI双酶切。
图4为pAdTrack-CMV-NRIP1重组质粒PmeI线性化：1，2为线性化的质粒；3为D15000Marker，条带分别为250，1000，2500，5000，7500，10000，15000bp。
图5为重组子质粒图谱：2，3，7，8为重组成功质粒；13为空pAdEasy-1。
图6为pAd-NRIP1重组腺病毒质粒PacI酶切鉴定：1、PacI酶切产生一个4.5kb的小片度；2、为D15000Marker，条带分别为250，1000，2500，5000，7500，10000，15000bp。
图7为阴性对照质粒转染96h后293A图。
图8为pAd-NRIP1重组腺病毒质粒转染96h后293A图。
以下通过具体实施方式来描述本发明，应该指出的是，所描述的具体实施方式不应理解为对本发明的限制。
具体实施方式
本发明利用质粒的体外酶切及连接技术，应用pAdEasy-1系统，将线性化的穿梭质粒转化含有pAdEasy-1质粒的E.coli BJ5183感受态细胞进行同源重组，所得重组腺病毒质粒可以通过卡那霉素筛选出，并通过质粒大小和限制性内切酶分析鉴定。最后，PacI酶切后的重组腺病毒质粒转染293A细胞包装产生重组腺病毒。
实施例1pAd-NRIP1重组腺病毒质粒的构建
1、材料和方法
1.1仪器
超净工作台、生化培养箱、基因扩增仪、PTC-200单槽梯度基因扩增仪、Heraeus冷冻高速离心机(德国)、Bio-Rad凝胶成像分析仪(USA)、CO2培养箱、HZS-H水浴振荡器(哈尔滨)、Eppendorf移液器、DYY-III型稳压稳流电泳仪(北京六一)、DYY-III31A和DYY-III28D电泳槽(北京六一)、制冰仪、MDF-382E超低温冰箱(日本三洋)、Eppendorf台式高速离心机、Sartorious电子天平(德国)、常规冰箱等。
1.2主要生化试剂和试剂盒
Long Taq聚合酶、PrimeSTAR DNA聚合酶、DNA限制性内切酶(NotI、PacI、PmeI、BglII等)、胶原蛋白、胰蛋白酶、DNA Marker、DNA连接酶、Trizol、反转录试剂盒、克隆载体(pGM-T)、表达载体(pAdTrack-CMV、pAdEasy-1)、质粒提取试剂盒、DNA凝胶回收试剂盒、胎牛血清、细胞培养基等。
1.3培养基和抗性
LB培养基：胰蛋白胨，酵母粉，NaCl，琼脂粉；
抗性：氨苄青霉素，卡纳霉素等。
1.4普通试剂
肝素钠，Tris，EDTA，NaCl，NaOH，无水乙醇，醋酸钠，十二烷基磺酸钠(SDS)，抗生素，溴化乙锭(EB)，溴酚蓝，二甲基苯菁FF，乙酸，蔗糖，去离子甲酰胺，硝酸，盐酸，硝酸银，无水碳酸钠，硫代硫酸钠，甲醛，硼酸，琼脂糖，KCl，Na2HPO4，KH2PO4，Tris饱和酚(pH＝8.0)，氯仿，异戊醇，甘油，石蜡油。
1.5秦川牛NRIP1基因PCR引物的设计
本研究参照GenBank公布的NRIP1mRNA序列(XM_603300.3)，设计引物：
上游引物：
5’＞GGAAGATCTGCCCACCATGACTCATGGAGAAGAGCT＜3’；
下游引物：
5’＞ATTTGCGGCCGCTTAATGATGATGATGATGATGGTTCTGATTCCTTTTTTATTG＜3’，其中BglII，NotI分别为上下游的酶切位点。
1.6PCR扩增秦川牛的NRIP1基因片段
(1)PCR总反应体系为50ul，见表1：
表1本发明的PCR反应体系
(2)PCR反应程序，见表2；
表2本发明的PCR反应程序
1.7携带NRIP1基因的pGM-T-NRIP1重组质粒的构建
将PCR扩增的NRIP1基因片段经加A反应后，与pGM-T载体经T4Ligase16℃过夜连接，得到pGM-T-NRIP1重组质粒，后者转化E.coli DH5α感受态细胞，挑选菌落扩增，质粒回收后BglII和NotI双酶切鉴定。
BglII和NotI酶切反应体系20ul，见表3：
表3本发明的BglII和NotI酶切反应体系
注：酶切消化条件为37℃，3～10h
1.8携带NRIP1基因的pAd-TrackCMV-NRIP1重组质粒的构建
BglII和NotI双酶切携带NRIP1基因的pGM-T-NRIP1重组质粒和pAdTrack-CMV质粒，胶回收后T4 Ligase 16℃过夜连接，得到 pAdTrack-CMV-NRIP1重组质粒。后者转化E.coli DH5α感受态细胞，挑选菌落扩增，质粒回收后BglII和NotI双酶切鉴定。
连接体系25μL，见表4：
表4本发明的连接反应体系
注：条件为16℃，10～12h。
1.9携带NRIP1基因的pAd-NRIP1重组腺病毒质粒的构建
限制性内切酶PmeI线性化pAdTrack-CMV-NRIP1重组穿梭质粒，凝胶回收试剂盒回收线性化pAdTrack-CMV-NRIP1重组穿梭质粒，转化包含有pAdEasy-1质粒的E.coli BJ5183感受态细胞，利用E.coli BJ5183内Cre重组酶高效的同源重组系统，在细菌中完成pAdTrack-CMV-NRIP1和pAdEasy-1同源重组，试剂盒抽提同源重组质粒，0.7％琼脂糖凝胶电泳，选择比pAdEasy-1大的重组腺病毒质粒，PacI酶切。酶切产物通过0.7％琼脂糖凝胶电泳鉴定，琼脂糖凝胶电泳结果与理论上同源重组质粒多克隆位点图谱吻合。具体步骤如下：
(1)、将2管含有pAdEasy-1的BJ5183感受态细胞置于冰上，1管加入6ulPmeI线性化的pAdTrack-CMV-NRIP1轻弹混匀，另一管做空对照，冰浴30min；
(2)、42℃水浴90秒，迅速置于冰中静置3min；
(3)、每管加入37℃预热的LB培养基1000ul，37℃震荡培养60min；
(4)、将转化物铺3～5块LB/Kan(50ug/ml)培养板，37℃培养24h。分别铺100、300和600ul，以提高得到可分离菌落的机率，约得到40～100个菌落；
(5)、挑出24个最好的克隆，转入2ml LB/Kan(50ug/ml)培养液中培养10～15h。因可能产生非目的重组子，应尽快抽提重组质粒；
(6)、用传统碱裂解法小量制备质粒，取一半进行0.7％琼脂糖凝胶电泳检测质粒的大小。重组质粒大小约40kb，由于它是低拷贝质粒，所以小量制备时应相应调整具体操作；
(7)、20％以上的菌落含有大质粒(约40kb)，这些是侯选重组子。与对照培养板生长的菌落进行对照即可估计线性化穿梭质粒再连接所致的背景强弱；
(8)、重组质粒的筛选和扩增
将侯选重组子进行酶切分析，验证其结构。PacI酶切后通常得到约30kb的片段和一个3.0或4.5kb的小片段，小片段的大小因重组位置在左臂还是复制子而不同。
挑选最佳阳性重组子转入DH5α中进行扩增，这一步对于扩增时保持重组质粒的结构是必需的。因为重组质粒在BJ5183中是不稳定的，而在DH5α中能很容易地获得大量重组质粒。
1.10携带NRIP 1基因的pAd-NRIP1重组质粒脂质体LIPOFECTAMINE 2000转染293A细胞
(1)、转染前一天，以合适的细胞密度接种到6孔培养板上。转染时，细胞要 达到90～95％的融合；
(2)、溶液1：240ul无血清培养基+10ul LIPOFECTAMINE 2000(温育5min)；
(3)、溶液2：Xul无血清培养基+4ug质粒(总体积250ul)；
(4)、将溶液1与溶液2混合，室温下置20min；
(5)、与此同时，将6孔板中的细胞用无血清培养基冲洗细胞两遍后，加入2ml无血清培养基；
(6)、将溶液1与溶液2的混合液逐滴加入孔中，摇动培养板，轻轻混匀。在37℃，5％的CO2中保温5～6h；
(7)、6h后，更换含有血清的全培养基，在37℃，5％的CO2中24～72h检测转染水平。
1.11携带秦川牛NRIP1基因的重组腺病毒的鉴定
取重组腺病毒上清500ul，加入至90％融合的293A细胞中，37℃，5％的CO2中培养。镜下观察到95％～100％细胞出现病变后，离心收集上清。并将细胞于-80℃和37℃之间反复冻融3次，以3000r/min离心收集上清，对腺病毒上清进行离心纯化。取5ul病毒上清加入10ul蛋白酶K，55℃孵育1h，再煮沸5min，离心后取2ul做PCR。
1.12携带秦川牛NRIP1基因的重组腺病毒的滴度测定
(1)、细胞准备：96孔板中接种100ul 293A细胞，每孔细胞数约104个，以2％DMEM培养；
(2)、稀释病毒液准备：以2％DMEM将病毒液稀释成8个较高浓度(如10-3～10-10)，每个浓度重复10个，每孔加入病毒稀释液100ul。另留两排不加病毒液作为阴性对照。37℃下，孵箱培养10天；
(3)、10d后观察细胞，记数每排出现Cpe的孔数，计算细胞病变率(如某一浓度各孔细胞全部病变，比率为1，如无细胞病变，则比率为0)；
(4)、计算T＝10×101+d(S-0.5)/ml
d＝Log10稀释度(如为10倍稀释度，d＝1)
S＝各浓度细胞病变比率之和。
2结果
2.1NRIP1基因PCR扩增结果
以秦川牛基因组为模板，利用PrimeSTAR DNA聚合酶，成功扩增出NRIP1。见图1。
2.2携带NRIP1基因的pGM-T-NRIP1重组质粒的酶切鉴定结果
为了鉴定秦川牛NRIP1基因的pGM-T-NRIP1阳性重组质粒，以BglII和NotI双酶切，得到了3517bp的NRIP基因片段。见图2。
2.3pAdTrack-CMV-NRIP1重组质粒酶切鉴定结果和PmeI线性化结果
经过连接和扩繁，以BglII和NotI双酶切pAdTrack-CMV-NRIP1重组质粒，琼脂糖检测有3517bp片段，证明成功获得pAdTrack-CMV-NRIP1阳性重组质粒。图3。后经PmeI酶切，琼脂糖检测线性化完全。见图4。
2.4携带NRIP1基因的pAd-NRIP1质粒的构建结果
如图5所示，携带NRIP1基因的pAd-NRIP1重组质粒构建成功。后经PacI酶切鉴定，进一步证实携带NRIP1基因的pAd-NRIP1重组质粒构建成功。见图6。
2.5携带NRIP1基因的pAd-NRIP1重组质粒脂质体LIPOFECTAMINE 2000转染293A细胞效果
24h转染效果见图7和图8，证明转染成功。
2.6携带秦川牛NRIP1基因的重组腺病毒的滴度测定
重组腺病毒经繁殖、扩增后，计算出病毒滴度为1.96×1010pfu/ml，说明病毒滴度较高，能够满足转染细胞及其他应用。
实施例2携带秦川牛NRIP1基因的重组腺病毒转染原代培养的秦川牛肌肉 细胞
1秦川牛肌肉细胞的原代培养
(1)、将新鲜组织置于培养皿中，于无菌操作台中用Hank’s液洗涤三次，并剔除脂肪、血液等杂物；
(2)、用手术剪将肌内剪成小块(1mm2)，再用Hank’s液洗三次，转移至小青霉素瓶中；
(3)、视组织块量加入5～6倍的0.25％胰酶液，37℃中消化20～40min，每隔5min振荡一次，或用吸管吹打一次，使细胞分离；
(4)、加入3～5ml培养液以终止胰酶消化作用(或加入胰酶抑制剂)；
(5)、静置5～10mi n，使未分散的组织块下沉，取悬液加入到离心管中；
(6)、1000rpm，离心10min，弃上清液；
(7)、加入Hank’s液5ml，冲散细胞，再离心一次，弃上清液；
(8)、加入培养液1～2ml(视细胞量)，血球计数板计数；
(9)、将细胞调整到5×105/ml左右，转移至75ml2方瓶中37℃培养。2NRIP1重组腺病毒转染原代细胞后的RT-PCR检测
至原代细胞密度达到60％，加入适量病毒上清感染细胞。细胞感染病毒(MOI＝200)72h时，RT-PCR检测证明，与阴性对照相比(阴性对照为空pAdTrack-CMV与pAdEasy-1重组产生的重组腺病毒质粒，没有携带NRIP1基因)，NRIP1基因mRNA的表达量增加了25％。同时90％的肌肉细胞表达绿色荧光，且肌肉细胞形态良好。
实施例3感受态细胞的制备
1步骤
(1)、在15ml离心管中普能4ml LB培养液摇菌过夜；
(2)、次日1∶100接种扩大培养于200ml Rich LB(见表5)培养液中(37℃) 至OD600＝0.6-0.7；
(3)、置于冰上10min，摇动使其冷却，分装到50ml离心管，每管42ml；
(4)、4℃，2000rpm，15min，收集细胞(去尽上清)；
(5)、1/6体积的CCMB 80buffer(见表6)重悬，置冰上20min；
(6)、4℃，2000rpm，15min，收集细胞(去尽上清)；
(7)、用1/24体积CCMB80buffer重悬，每100ul于一个离心管，-80℃冻存。
表5Rich LB的组分
表6CCMB80 buffer的配方
CCMB80中各种试剂的应称量精确，先用一定量的ddH2O溶解各化合物，再加入甘油，搅拌均匀，最后用ddH2O定容到所需体积。CCMB80不可高压灭菌，将pH调至6.4后，先用滤纸过滤，然后用0.22um滤膜过滤，保存于4℃。
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Claims (10)
1.一种质粒型腺病毒载体pAd-NRIP1，其特征在于所述载体含有SEQ IDNO：1所示核苷酸序列的秦川牛NRIP1基因。
2.如权利要求1所述的质粒型腺病毒载体，其特征在于是由AdEasyTM系统构建完成。
3.如权利要求1所述的质粒型腺病毒载体，其特征在于NRIP1基因连接于穿梭质粒CMV启动子之下。
4.如权利要求1所述的质粒型腺病毒载体，其特征在于NRIP1基因起始密码子前含有Kozak序列(GCCCACC)。
5.如权利要求1所述的质粒型腺病毒载体，其特征在于NRIP1基因终止密码子前含有6个His标签序列。
6.如权利要求1所述的质粒型腺病毒载体，其特征在于NRIP1基因CDS区1301位碱基序列为C。
7.一种构建权利要求1至6中任意一权利要求所述质粒型腺病毒载体的方法，所述方法包括如下步骤：
PCR扩增NRIP1后与pGM-T载体连接，转化E.coli DH5α感受态细胞。试剂盒抽提pGM-T-NRIP1重组质粒；
pGM-T-NRIP1重组质粒和pAdTrack-CMV穿梭质粒经BglII和NotI双酶切，凝胶回收试剂盒回收NRIP1基因片段和pAdTrack-CMV载体片段，回收的载体片段和目的基因片段T4DNA Ligase进行酶连反应，酶连产物转化E.coli DH5α感受态细菌，纯化试剂盒抽提pAdTrack-CMV-NRIP1重组穿梭质粒，BglII和NatI双酶切鉴定证明获得正确的pAdTrackCMV-NRIP1重组质粒；
限制性内切酶PmeI线性化pAdTrack-CMV-NRIP1重组穿梭质粒，凝胶回收试剂盒回收线性化pAdTrack-CMV-NRIP1重组穿梭质粒，转化含有pAdEasy-1质粒E.coli BJ5183感受态细菌，利用E.coli BJ5183内Cre重组酶高效的同源重组系统，在细菌中pAdTrack-CMV-NRIP1和pAdEasy-1同源重组获得携带NRIP1基因的重组腺病毒质粒，将同源重组成功的重组腺病毒质粒命名为pAd-NRIP1。
8.权利要求1至6中任意一权利要求所述的质粒型腺病毒载体，作为牛NRIP1基因功能鉴定的应用。
9.如权利要求1至6中任意一权利要求所述的质粒型腺病毒载体，作为细胞改造的应用。
10.如权利要求1至6中任意一权利要求所述的质粒型腺病毒载体，作为代谢和生殖调控的应用。
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| 刘栋等: "秦川牛NRIP1基因重组腺病毒载体的构建", 《中国畜牧兽医学会养牛学分会2011年学术研讨会论文集》 * |
| 陈艳芳等: "转录辅抑制因子RIP140在代谢组织中的作用及机制", 《中国药理学通报》 * |
| 马云等: "秦川牛分子标记研究进展", 《中国牛业科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102766653A (zh) * | 2012-06-25 | 2012-11-07 | 西北农林科技大学 | 一种质粒型腺病毒载体pAd-PPARGC1A及其构建方法 |
| CN103205462A (zh) * | 2012-12-09 | 2013-07-17 | 西北农林科技大学 | 一种质粒型腺病毒载体pAd-IGF2及其构建方法和应用 |
| CN103031336A (zh) * | 2012-12-10 | 2013-04-10 | 西北农林科技大学 | 牛质粒型腺病毒载体pAd-ZBED6及其构建方法和用途 |
| CN104988181A (zh) * | 2015-07-23 | 2015-10-21 | 温州医科大学附属第二医院 | S100a9重组腺病毒、其构建及应用 |
| CN104988181B (zh) * | 2015-07-23 | 2018-03-27 | 温州医科大学附属第二医院 | S100a9重组腺病毒、其构建及应用 |
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