CN114214290A - 一种重组腺病毒ADV-lncRNA Gm44275及其构建方法和应用 - Google Patents
一种重组腺病毒ADV-lncRNA Gm44275及其构建方法和应用 Download PDFInfo
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Abstract
一种重组腺病毒ADV‑lncRNA Gm44275及其构建方法和应用，涉及诱导分娩领域。该重组腺病毒ADV‑lncRNA Gm44275是通过在lncRNA Gm44275序列中添加CBH Promoter序列和绿色荧光标签序列构建lncRNA过表达载体，再将lncRNA过表达载体与腺病毒表达载体进行同源重组，经酶切、转染、纯化、浓缩后获得的。该重组腺病毒ADV‑lncRNA Gm44275能够应用在制备具有诱导动物分娩功能药品中。本发明能够促进母畜提前分娩，不会影响母体健康，配方更有普适性，对于各种动物皆使用，不会在动物体内造成激素残留，诱导分娩效果更稳定，受个体差异影响更小。
Description
技术领域
本发明涉及诱导分娩技术领域，具体涉及一种重组腺病毒ADV-lncRNA Gm44275及其构建方法和应用。
背景技术
诱导分娩是指人为干预导致分娩的发生。在动物生产过程中诱导分娩可以提高繁殖效率，可促使母畜集中产仔，集中分娩有利于种群的管理工作，能够使日常管理简单化、集中化、可控化，既可节约人工成本，也可方便对动物进行健康管理，提高自带成活率，进而提高生产效益，具有重大的经济价值。
目前，针对母畜诱导分娩主要有以下几种方式：
(一)母猪诱导分娩
母猪生产过程中常使用前列腺素类似物(如氯前列烯醇、氯前列醇钠等)促进母猪黄体溶解、刺激子宫收缩来诱导母猪分娩。分娩时体内多种激素协同作用共同调节复杂的生理过程，这种激素诱导分娩不当会对母猪和仔猪造成不利影响，若早于妊娠期111d使用前列腺素类似物会增加仔猪的死亡率、降低新生仔猪活力或降低母猪初乳中乳脂含量等。激素类药物的使用同样会根据母猪个体差异如品种、胎次、妊娠期长短等因素影响诱导效果。
(二)母牛诱导分娩
母牛生产过程中通常采用肾上腺皮质醇激素进行处理(如地塞米松、倍他米松等)促进子宫平滑肌收缩诱导母牛分娩，也可采用雌激素或前列腺素的合成制剂诱导分娩，若诱导分娩早于260d会造成犊牛发育不全并影响产奶量。
(三)母羊诱导分娩
妊娠山羊144d肌注射PGF25mg～10mg或地塞米松磷酸钠注射液15mg～20mg，用药后产羔平均时间为32h～120h，相比于不用药197h显著提前了产羔时间。妊娠绵羊则是在144d肌注射地塞米松磷酸钠注射液15mg～20mg，用药后产羔时间为40h～60h。
(四)家兔诱导分娩
家兔习惯于夜间分娩，产仔期相差可达6天左右。颈皮下注射0.02mg氯前列烯醇钠注射液可以诱导母兔同期分娩并在白天产仔。
综上，诱导分娩常用的方法就是依据不同动物和不同妊娠阶段，注射不同药物和不同注射量以达到诱导分娩的效果。但针对不同个体注射量不同，仍会有晚间或延时分娩较多的情况，对于精准分娩调控要求较高，同时存在有较高含量的外源激素会残留在母体和子代中的风险，可能影响肉类食品安全。
发明内容
基于此，本发明提供一种重组腺病毒ADV-lncRNA Gm44275及其构建方法和应用。
本发明为解决技术问题所采用的技术方案如下：
本发明的一种重组腺病毒ADV-lncRNA Gm44275，它是通过在lncRNA Gm44275序列中添加CBH Promoter序列和绿色荧光标签序列构建lncRNA过表达载体，再将lncRNA过表达载体与腺病毒表达载体进行同源重组，经酶切、转染、纯化、浓缩后获得的。
本发明的一种重组腺病毒ADV-lncRNA Gm44275的构建方法，包括以下步骤：
利用体外合成技术合成lncRNA Gm44275，并在其序列中添加CBH Promoter序列和绿色荧光标签序列，得到lncRNA过表达载体，将lncRNA过表达载体与腺病毒表达载体在体外进行同源重组，得到Gm44275-CBH-GFP腺病毒质粒，利用限制性内切酶进行酶切将质粒线性化，将酶切产物转染至HEK293细胞，当细胞出现病态反应并有50％细胞脱落时，裂解细胞收集病毒，经纯化浓缩得到重组腺病毒ADV-lncRNA Gm44275。
作为优选的实施方式，所述lncRNA Gm44275序列见SEQ ID NO.1。
作为优选的实施方式，所述CBH Promoter序列见SEQ ID NO.2。
作为优选的实施方式，所述绿色荧光标签序列见SEQ ID NO.3。
作为优选的实施方式，所述lncRNA过表达载体序列见SEQ ID NO.4。
作为优选的实施方式，所述腺病毒表达载体为pAdeno Vecotr载体。
作为优选的实施方式，所述限制性内切酶为PacI。
作为优选的实施方式，将lncRNA Gm44275替换为lncRNA Gm36582。
本发明的一种重组腺病毒ADV-lncRNA Gm44275在制备具有诱导动物分娩功能药品中的应用。
本发明的有益效果是：
本发明通过在lncRNA Gm44275序列中添加CBH Promoter序列和绿色荧光标签序列构建lncRNA过表达载体，再将lncRNA过表达载体与腺病毒表达载体进行同源重组，经酶切、转染、纯化、浓缩后获得一种重组腺病毒ADV-lncRNA Gm44275，将所得重组腺病毒ADV-lncRNA Gm44275直接注射到母畜子宫，通过lncRNA在母畜子宫的表达，进而诱导母畜提前分娩，达到进一步精确控制母畜分娩的目的。针对不同动物，只需在注射量上有差异，皆能成功诱导分娩，而灭活的ADV在动物体内仅有几天时间的表达，外源激素不会残留在母体和子代中，满足肉类食品安全需求。
本发明利用腺病毒表达载体包装lncRNA过表达载体，获得的重组腺病毒ADV-lncRNA Gm44275能够促进母畜提前分娩，不会影响母体健康，配方更有普适性，对于各种动物皆使用，不会在动物体内造成激素残留，诱导分娩效果更稳定，受个体差异影响更小。
附图说明
图1为pAdeno Vecotr载体结构模式图。
图2为小鼠a和小鼠d子宫注射ADV 39小时子宫照片。其中，图A为子宫注射重组腺病毒ADV-lncRNA Gm44275 39小时分娩完成小鼠a子宫照片，图B为子宫注射重组腺病毒ADV-lncRNA Gm44275 39小时小鼠a照片，图C为子宫注射ADV-NC 39小时小鼠d子宫照片，D为子宫注射ADV-NC 39小时小鼠d照片。
图3为小鼠a和小鼠d子宫注射ADV 39小时切片图。其中，图A为小鼠d子宫注射ADV-NC 39小时切片白光，图B为小鼠d子宫注射ADV-NC 39小时切片荧光，图C为小鼠a子宫注射重组腺病毒ADV-lncRNA Gm44275 39小时切片白光，图D为小鼠a子宫注射重组腺病毒ADV-lncRNA Gm44275 39小时切片荧光。
图4为手术剖开暴露小鼠子宫。
图5为小鼠b子宫注射重组腺病毒ADV-lncRNA Gm44275后32-37小时分娩完成的小鼠b照片和小鼠b子宫照片。
图6为小鼠c子宫注射重组腺病毒ADV-lncRNA Gm44275后32-39小时分娩完成的小鼠c照片和小鼠c子宫照片。
图7为小鼠e子宫注射ADV-NC后48小时分娩完成的小鼠e子宫照片和小鼠e照片。
图8为小鼠f子宫注射ADV-NC后39小时分娩完成的小鼠f子宫照片和小鼠f照片。
图9为小鼠c和小鼠f子宫注射ADV 39小时切片图。其中，图A为小鼠f子宫注射ADV-NC 39小时切片白光，图B为小鼠f子宫注射ADV-NC 39小时切片荧光，图C为小鼠e子宫注射重组腺病毒ADV-lncRNA Gm44275 39小时切片白光，图D为小鼠e子宫注射重组腺病毒ADV-lncRNA Gm44275 39小时切片荧光。
具体实施方式
本发明的一种重组腺病毒ADV-lncRNA Gm44275，它是通过在lncRNA Gm44275序列中添加CBH Promoter序列和绿色荧光标签序列构建lncRNA过表达载体，再将lncRNA过表达载体与腺病毒表达载体进行同源重组，经酶切、转染、纯化、浓缩后获得的。
本发明的一种重组腺病毒ADV-lncRNA Gm44275的构建方法，具体包括以下步骤：
利用体外合成技术合成lncRNA Gm44275(SEQ ID NO.1)，并在其序列中添加CBHPromoter序列(SEQ ID NO.2)和绿色荧光标签序列(SEQ ID NO.3)，得到lncRNA过表达载体(SEQ ID NO.4)，将lncRNA过表达载体与腺病毒表达载体(优选的，pAdeno Vecotr载体)在体外进行同源重组，得到Gm44275-CBH-GFP腺病毒质粒，利用限制性内切酶(优选的，PacI)进行酶切将质粒线性化，将酶切产物转染至HEK293细胞，当细胞出现病态反应并有50％细胞脱落时，裂解细胞收集病毒，经纯化浓缩得到重组腺病毒ADV-lncRNA Gm44275。
其中，可将lncRNA Gm44275替换为lncRNA Gm36582，lncRNA Gm44275与lncRNAGm36582的序列相同。
本发明的一种重组腺病毒ADV-lncRNA Gm44275在制备具有诱导动物分娩功能药品中的应用。
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1lncRNA过表达载体的构建
长链非编码RNA(long noncoding RNA，lncRNA)是指长度大于200个核苷酸的非编码RNA。该种非编码RNA在表观遗传调控、细胞周期调控、细胞分化等生命活动中发挥着重要作用，某些lncRNA具有组织和时空特异性。本发明首先利用体外合成技术合成lncRNAGm44275(lncRNA Gm36582)，其序列见SEQ ID NO.1；然后在lncRNA Gm44275序列中添加CBHPromoter序列(其序列见SEQ ID NO.2)以及绿色荧光标签序列(其序列见SEQ ID NO.3)，得到lncRNA过表达载体，其序列见SEQ ID NO.4。
实施例2ADV包装lncRNA过表达载体
腺病毒(Adenovirus，ADV)是指一种大分子(36kb)双链无包膜DNA病毒。通过受体介导的内吞作用进入细胞内，将腺病毒基因组转移至细胞核内。灭活后的腺病毒可作为载体将目的基因片段整合，注射到人或动物体内，促进该基因的表达。
pAdeno Vecotr载体是一种腺病毒表达载体，载体结构模式如图1所示。本发明将上述lncRNA过表达载体与pAdeno Vecotr载体在体外进行同源重组，将其中的lncRNAGm44275(lncRNA Gm36582)序列转移至pAdeno Vecotr载体上，得到Gm44275-CBH-GFP腺病毒质粒。
通过PacI酶切将质粒线性化，将酶切产物转染至HEK293细胞中，当细胞出现病态反应并有50％左右细胞脱落时，裂解细胞收集病毒，纯化浓缩，得到重组腺病毒ADV-lncRNAGm44275。
试验例1诱导小鼠分娩试验
选择孕期相同的母鼠六只，每只鼠腹腔注射0.4mL三嗅乙醇麻醉，碘伏清洁腹部皮肤，剖开腹腔，暴露子宫(图4)，将重组腺病毒ADV-lncRNA Gm44275直接注射到其中三只母鼠子宫(小鼠a、b、c)，同时将ADV-NC(pAdeno Vecotr空载体)以同等实验量直接注射到另三只母鼠子宫(小鼠d、e、f)，手术线缝合皮肤，碘伏清洁缝合部分皮肤，等待小鼠苏醒及分娩，结果图2至图9所示。
如图2所示，为小鼠a和小鼠d子宫注射ADV 39小时子宫照片。其中，图A为子宫注射重组腺病毒ADV-lncRNA Gm44275 39小时分娩完成小鼠a子宫照片，图B为子宫注射重组腺病毒ADV-lncRNA Gm4427539小时小鼠a照片，小鼠a子宫注射重组腺病毒ADV-lncRNAGm4427539小时后完成分娩；图C为子宫注射ADV-NC 39小时小鼠d子宫照片，D为子宫注射ADV-NC 39小时小鼠d照片，小鼠d子宫注射ADV-NC 39小时后，小鼠d子宫无变化，小鼠d状态无变化。
如图3所示，为小鼠a和小鼠d子宫注射ADV 39小时切片图。其中，图A为小鼠d子宫注射ADV-NC 39小时切片白光，小鼠d子宫冰冻切片质量较好，轮廓清晰；图B为小鼠d子宫注射ADV-NC 39小时切片荧光，携带绿色荧光的ADV-NC载体成功感染小鼠d子宫；图C为小鼠a子宫注射重组腺病毒ADV-lncRNA Gm44275 39小时切片白光，小鼠a子宫冰冻切片质量一般；图D为小鼠a子宫注射重组腺病毒ADV-lncRNA Gm44275 39小时切片荧光，携带绿色荧光的重组腺病毒ADV-lncRNA Gm44275成功感染小鼠a子宫。
如图5所示，为小鼠b子宫注射重组腺病毒ADV-lncRNA Gm44275后32-37小时分娩完成的小鼠b照片和小鼠b子宫照片。小鼠b子宫注射重组腺病毒ADV-lncRNA Gm44275 32-37小时后完成分娩。
如图6所示，为小鼠c子宫注射重组腺病毒ADV-lncRNA Gm44275后32-39小时分娩完成的小鼠c照片和小鼠c子宫照片。小鼠c子宫注射重组腺病毒ADV-lncRNA Gm44275 32-39小时后完成分娩。
如图7所示，为小鼠e子宫注射ADV-NC后48小时分娩完成的小鼠e子宫照片和小鼠e照片，小鼠e子宫注射ADV-NC 39小时后，小鼠e子宫无变化，小鼠e状态无变化。
如图8所示，为小鼠f子宫注射ADV-NC后39小时分娩完成的小鼠f子宫照片和小鼠f照片。小鼠f子宫注射ADV-NC 39小时后，小鼠f子宫无变化，小鼠f状态无变化。
如图9所示，为小鼠c和小鼠f子宫注射ADV 39小时切片图。其中，图A为小鼠f子宫注射ADV-NC 39小时切片白光，小鼠f子宫冰冻切片质量较好，轮廓清晰；图B为小鼠f子宫注射ADV-NC 39小时切片荧光，携带绿色荧光的ADV-NC载体成功感染小鼠f子宫；图C为小鼠c子宫注射重组腺病毒ADV-lncRNA Gm44275 39小时切片白光，小鼠c子宫冰冻切片质量一般；图D为小鼠c子宫注射重组腺病毒ADV-lncRNA Gm44275 39小时切片荧光，携带绿色荧光的重组腺病毒ADV-lncRNA Gm44275成功感染小鼠c子宫。
上述试验结果表明：E15.5注射浓度为10^10的重组腺病毒ADV-lncRNA Gm44275100μL可在32-39小时成功诱导小鼠分娩。
本发明公开了一种重组腺病毒ADV-lncRNA Gm44275及其构建方法和应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的产品已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的产品进行改动或适当变更与组合，来实现和应用本发明技术。
序列表
<110> 吉林大学
<120> 一种重组腺病毒ADV-lncRNA Gm44275及其构建方法和应用
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agatatacga gagaacagcc agaagcatcg gaaagagtcc agagcagaga gagaaagaag 1020
tagactgaac atagccagaa aactggacca gggcacgaga gaaacaggag cacagcagag 1080
agagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 1140
agagaaacag agacacagag agagacacag agagactatc acagaaatta gcagagtttt 1200
aaagagtagc tgaggggagc cagtgagctg gaaaactttg ggtaggagag gaggccagaa 1260
gagctaggat gctagcatgg accttctaag gagtaacagt acttgtgata ctgaaggatc 1320
ctggatgact ctgtgagagc tctgagtgaa taaaatagta aatatgaaca tgtcacatct 1380
cgctttgcac aatctaggtc aacattaaaa tggatattga atatttacct agcaggttaa 1440
catttaccaa gtgtcttata tttaatgccc cagttcataa tgaaagcttc ctgcaggcta 1500
gtgctagcca ggctctaact ggttccaaag gattggcatc cttggtattg ttgcccttgg 1560
ttagccactc cacactgtag ggctgacctg tgtaaccaat agactctgca agaatgaggg 1620
gtacagtccc caggattaga ctgtcatgaa cattgcaatt ctcacctcat tctgtcttgg 1680
gtgactccct ctgagcaaga tagctgatgt ctgtgagtac ttcaagctcc ctcggagaag 1740
cccacatcgt gataacaggc aagaatagct agaaagaaga gcatccagtc ccccagggag 1800
tctttggata cctgcactca ctgccaatcc catttgggtt ttttgcttgt tttatataat 1860
gtctttattg aaatataatt cacatgccat ataattcagc cacttaaaag gcacagttga 1920
tggcatttta gaacagttac agatacacgt aagccatcaa cacctgtgcc ctttagctgt 1980
cagctcctca tttccccggc ctgcgtcaca aagcaaccac cgaacaactt ctagattcat 2040
ctctgtcgat ttgccactta gatatatttg aagaagtgga ttcctttaga ctaggttttt 2100
tttcttttca gtttatttat tattgtatgt ttatgtgcat gattcaagga ggcagagact 2160
tacatgccac ggtgtgattg ggagtcagag gacagcttta tcagccttca ctctcccact 2220
ctaggtgggt tctgggaatg aaacctgggt cactaggctt gcctgggaaa caccttcacc 2280
cactgagtct ttcccctggg ccagggtgta gtagtttctg actgacgggt atttaccgtt 2340
atgtttgcaa agtcagtctg ggttatagca tgtgtcagaa cctcgtttcc tttcatggct 2400
gaatagaatt ctgttcccgt tgtttatacg tgtattttgc ttgcccattc atggacattg 2460
ggttgcttcc aactttgggc tattgtgaac actgttacta taaacactga tatacaggtg 2520
tcgactataa ggatgatgac gacaaatgag ctagcacata acttacggta aatggcccgc 2580
ctggctgacc gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt 2640
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 2700
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 2760
attgtgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 2820
tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc 2880
ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg 2940
gggcgggggg ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg 3000
gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccaaaa gtttcctttt 3060
atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc 3120
gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg 3180
ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc 3240
tgtaattagc ttcgaacgcc accatggtga gcaagggcga ggagctgttc accggggtgg 3300
tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg 3360
agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 3420
agctgcccgt gccctggccc accctcgtga ccaccttgac ctacggcgtg cagtgcttcg 3480
cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 3540
acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg 3600
tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 3660
aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aaggtctata 3720
tcaccgccga caagcagaag aacggcatca aggtgaactt caagacccgc cacaacatcg 3780
aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 3840
ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca 3900
acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg 3960
gcatggacga gctgtacaag taa 3983
Claims (10)
1.一种重组腺病毒ADV-lncRNA Gm44275，其特征在于，它是通过在lncRNA Gm44275序列中添加CBH Promoter序列和绿色荧光标签序列构建lncRNA过表达载体，再将lncRNA过表达载体与腺病毒表达载体进行同源重组，经酶切、转染、纯化、浓缩后获得的。
2.如权利要求1所述的一种重组腺病毒ADV-lncRNA Gm44275的构建方法，其特征在于，包括以下步骤：
利用体外合成技术合成lncRNA Gm44275，并在其序列中添加CBH Promoter序列和绿色荧光标签序列，得到lncRNA过表达载体，将lncRNA过表达载体与腺病毒表达载体在体外进行同源重组，得到Gm44275-CBH-GFP腺病毒质粒，利用限制性内切酶进行酶切将质粒线性化，将酶切产物转染至HEK293细胞，当细胞出现病态反应并有50％细胞脱落时，裂解细胞收集病毒，经纯化浓缩得到重组腺病毒ADV-lncRNAGm44275。
3.根据权利要求2所述的构建方法，其特征在于，所述lncRNA Gm44275序列见SEQ IDNO.1。
4.根据权利要求2所述的构建方法，其特征在于，所述CBH Promoter序列见SEQ IDNO.2。
5.根据权利要求2所述的构建方法，其特征在于，所述绿色荧光标签序列见SEQ IDNO.3。
6.根据权利要求2所述的构建方法，其特征在于，所述lncRNA过表达载体序列见SEQ IDNO.4。
7.根据权利要求2所述的构建方法，其特征在于，所述腺病毒表达载体为pAdenoVecotr载体。
8.根据权利要求2所述的构建方法，其特征在于，所述限制性内切酶为PacI。
9.根据权利要求2所述的构建方法，其特征在于，将lncRNA Gm44275替换为lncRNAGm36582。
10.如权利要求1所述的一种重组腺病毒ADV-lncRNA Gm44275在制备具有诱导动物分娩功能药品中的应用。
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