CN114214321B - 抑制j亚型禽白血病病毒的长链非编码rna及其载体和应用 - Google Patents
抑制j亚型禽白血病病毒的长链非编码rna及其载体和应用 Download PDFInfo
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Abstract
本发明公开了一种抑制J亚型禽白血病病毒的长链非编码RNA及其载体和应用，所述长链非编码RNA命名为lnc‑LTR5B，其核苷酸序列如SEQ ID NO.1所示。本发明提供的长链非编码RNA高表达后，可以有效抑制ALV‑J病毒感染和复制能力，可以用于制备预防或治疗J亚型禽白血病疫苗或药物，为研究抗ALV‑J病毒药物提供新的靶点，进一步的相关研究也可以作为新的防治策略与手段。
Description
技术领域
本发明属于畜禽医药研究领域，具体涉及抑制J亚型禽白血病病毒的长链非编码RNA及其载体和应用。
背景技术
禽白血病是严重危害我国养禽业的重要疫病，其致病病毒可分为ALV-A、B、C、D、E、J和K等亚群。其中J亚群禽白血病病毒对家禽养殖业仍是一个重大威胁。ALV-J病毒感染可引起鸡造血细胞恶性增生以及免疫抑制，导致蛋鸡产蛋量下降，肉鸡生长缓慢，死淘率增加。ALV-J爆发会给养禽业带来了巨大的损失。现有技术中对ALV-J的致病机理知之甚少，且尚无可利用的疫苗和有效抗病毒药物，对ALV-J的防控主要采取严格的净化和控制程序。因此，针对ALV-J防治，需要发现新的抗病毒免疫或治疗策略，以解决目前ALV-J防控中存在的困难。
长非编码RNA(Long noncoding RNA,lncRNAs)是一类长度大于200nt的非编码RNA。研究发现，lncRNA作为一种新型的调控因子，通过影响基因转录，蛋白质稳定性、细胞定位和其他过程来影响细胞的生理功能。近年来，研究发现lncRNA在调控病毒复制和抗病毒免疫方面也起着关键作用，某些lncRNA已被作为病毒防治的靶点或抗病毒治疗的载体来重点研究。有鉴于此，对ALV-J感染相关的lncRNA的鉴定和功能研究，将有可能发现防治ALV-J的新靶点或抗病毒药物，相关的发明研究或可以作为其新的防治策略与手段。
发明内容
发明目的：针对现有技术中存在的不足，本发明提供一种全新的抑制J亚型禽白血病病毒的长链非编码RNA，通过高表达该长链非编码RNA，可以有效抑制ALV-J病毒感染和复制能力，从而达到防治J亚型禽白血病的目的。
本发明还提供所述的抑制J亚型禽白血病病毒的长链非编码RNA表达载体和应用。
技术方案：为了实现上述目的，本发明所述一种抑制ALV-J病毒的长链非编码RNA，所述长链非编码RNA命名为lnc-LTR5B，其核苷酸序列如SEQ ID NO.1所示。
其中，检测所述lnc-LTR5B表达水平的引物对为q-lnc-LTR5B-F和q-lnc-LTR5B-R，其核苷酸序列分别如SEQ ID NO.2-3所示。
其中，用于扩增所述抑制ALV-J病毒的长链非编码RNA的5′-末端和3′-末端基因特异性引物的核苷酸序列分别如SEQ ID NO.4-5所示。
其中，用于扩增所述抑制ALV-J病毒的长链非编码RNA的全长序列的引物对lnc-LTR5B-F和lnc-LTR5B-R，其核苷酸序列分别如SEQ ID NO.6-7所示。
含有本发明所述的抑制ALV-J病毒的长链非编码RNA的表达载体。
本发明所述的表达载体的构建方法，包括如下步骤：
(1)从鸡胚成纤维细胞CEF提取总RNA，将其逆转录成cDNA；
(2)以步骤(1)所获得的cDNA产物为模板，设计引物，扩增带同源臂的lnc-LTR5B全长序列；
(3)对PCR扩增产物切胶回收产物与pcDNA3.1线性质粒载体进行无缝连接，然后连接产物直接转化大肠杆菌感受态细胞，经阳性克隆筛选，DNA测序鉴定，测序结果正确的质粒，命名为pcDNA3.1-lnc-LTR5B。
其中，步骤(2)中用于扩增带同源臂lnc-LTR5B全长序列所用引物RT-lnc-LTR5B-F和RT-lnc-LTR5B-R的核苷酸序列分别如SEQ ID NO.8-9所示。步骤(2)的引物中设计了与载体一致的同源臂，通过同源重组法将lncRNA全长和载体连接在一起。
本发明所述的长链非编码RNA或者所述的表达载体在抑制ALV-J病毒中的应用。
本发明所述的长链非编码RNA或者所述的表达载体在制备抗ALV-J病毒药物中的应用。
其中，所述的长链非编码RNA或者表达载体通过抑制ALV-J病毒感染和复制能力在制备抗ALV-J病毒基因药物中的应用。
本发明在DF-1细胞过表达长链非编码lncRNA-LTR5B时，lnc-LTR5B可以显著降低ALV-J病毒的感染及胞内复制能力，可以用于制备预防或治疗J亚型禽白血病药物。
有益效果：与现有技术相比，本发明具有以下优点：
本发明首次克隆到一种全新序列的长链非编码RNA，该长链非编码RNA(lnc-LTR5B)可以有效抑制ALV-J病毒复制，与对照组相比抑制效率可达50％以上，可以用于制备预防或治疗J亚型禽白血病药物。
本发明通过高表达lnc-LTR5B，可以有效抑制ALV-J病毒感染和复制能力，为研究抗ALV-J病毒复制的药物提供新的靶点，进一步的相关研究也可以作为禽白血病新的防治策略与手段。
附图说明
图1为ALV-J感染导致lnc-LTR5B转录水平下降。A：qRT-PCR分析ALV-J感染对CEF细胞中lnc-LTR5B表达的影响；B：qRT-PCR分析ALV-J感染对DF-1细胞中lnc-LTR5B表达的影响。
图2为本发明中利用RACE技术鉴定lnc-LTR5B全长序列电泳结果。
图3为本发明中过表达lnc-LTR5B对ALV-J病毒复制的影响。A为qRT-PCR检测过表达lnc-LTR5B的表达情况，以GAPDH基因作为内参；B为qRT-PCR检测ALV-J病毒RNA的相对表达水平，以GAPDH基因作为内参。B：Western Blotting检测ALV-J病毒囊膜蛋白Env的表达情况，和经GAPDH内参校正后的相对表达量。
图4为本发明中pcDNA3.1-lnc-LTR5B质粒图谱。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
鸡原代成纤维细胞(CEF细胞)来源：由江苏立华牧业有限公司提供的受精蛋在孵化箱中孵育至11日龄，无菌条件下取出鸡胚，放入灭菌1xPBS中漂洗血污，去除四肢和头部后，取躯干部份再次漂洗血污，用剪刀将躯干部分组织剪碎成约1mm2大小的碎块，静置后弃去上清，沉淀用0.25％胰酶消化5min，待组织呈粘稠絮状时，加入3倍体积的含10％FBS完全培养基终止消化，室温800rpm离心10min，弃上清，再用含10％FBS完全培养基重悬细胞沉淀，悬液经四层灭菌纱布过滤后，将滤液转移至细胞培养瓶中，37℃，5％CO2培养，12h后贴壁细胞即为CEF细胞。
DF-1细胞源于美国ATCC(CRL-12203)，由本实验室传代保种。
ALV-J病毒(JS09GY3株)由扬州大学提供。
实施例1
ALV-J感染对lnc-LTR5B表达水平的影响
ALV-J感染CEF和DF-1细胞，使用荧光定量PCR测定不同感染时间点lnc-LTR5B表达量的变化。具体步骤如下：
将CEF和DF-1细胞分别铺于12孔板中，待细胞融合度达到70％时，将培养基换为无血清DMEM，加入MOI 0.1ALV-J病毒(JS09GY3株)MOI 0.1孵育，并设置未感染对照组。感染2h后换为含2％胎牛血清的维持培养基(含1％的青/链霉素)。
在分别感染12、24、36和48h后，提取CEF细胞和DF-1细胞总RNA，逆转录为cDNA后通过荧光定量PCR检测lnc-LTR5B表达水平，引物序列如下：
上游引物q-lnc-LTR5B-F：
5′-AATCCCTCCTCTTCCTTCTT-3′(SEQ ID NO.2)
下游引物q-lnc-LTR5B-R：
5′-GATAACTTGGCTGCTGGTA-3′(SEQ ID NO.3)
结果如图1所示，ALV-J病毒分别感染CEF和DF-1细胞后，lnc-LTR5B的表达量均显著下调，表明lnc-LTR5B表达与ALV-J的复制相关。
实施例2
利用RACE方法扩增lnc-LTR5B全长核苷酸序列
(1)RACE技术鉴定lnc-LTR5B的5′和3′末端序列
由RACE 5’/3’Kit试剂盒(Takara,#634859)来完成。首先，用/>Reagent提取鸡胚成纤维细胞CEF总RNA，然后用RNase-free DNase I去除基因组。在SMARTScribeReverse Transcriptase(由RACE试剂盒提供)的作用下分别将1μg去除基因组的RNA逆转录成5′-或3′-RACE产物。
然后，按照RACE 5’/3’Kit试剂盒的操作说明，利用通用引物UPM分别与5′-末端或3′-末端基因特异性引物(gene-specific primer，GSP)进行PCR扩增(RACE琼脂糖凝胶电泳图见图2)，克隆测序获得lnc-LTR5B的5′末端和3′末端序列，用于后期鉴定lnc-LTR5B的末端序列。其中，所用5′-末端或3′-末端基因特异性引物的核苷酸序列如下：
5’-RACE引物：
5′-CCAGTGGCAGGGAGGCAGAAAATGACCT-3′(SEQ ID NO.4),
3’-RACE引物：
5′-TGGTTGGGATCAAGCAGCAAGTCTATCC-3′(SEQ ID NO.5)。
(2)PCR扩增lnc-LTR5B全长。其中，所用引物核苷酸序列如下：
lnc-LTR5B-F：
5′-CTCTTGCTGGCTGCACAG-3′(SEQ ID NO.6)
lnc-LTR5B-R：
5′-TTTCTTTGAGTTGCAGGTTA-3′(SEQ ID NO.7)
其反应体系包括：100ng鸡胚成纤维细胞cDNA产物作为模板，1μL(10μM)上游引物lnc-LTR5B-F与1μL(10μM)lnc-LTR5B-R分别作为扩增引物，1μL DNA Polymerase，10μL 5xSF Buffer，1μL(10μM)dNTP Mix和32μL ddH2O。设置反应条件为：95℃3min；95℃15s，58℃30s，72℃1min，35x循环；72℃10min；4℃维持。
(3)以步骤(2)中PCR扩增产物进行琼脂糖凝胶电泳，然后切胶回收产物，TA克隆并测序，获得lnc-LTR5B的全长cDNA序列。测序结果表明，lnc-LTR5B全长为590nt，具体核苷酸全长序列如SEQ ID NO.1。
实施例3
lnc-LTR5B过表达载体构建
本实施例中，使用从实施例2中所获得的lnc-LTR5B全长序列，设计全长扩增引物，构建lnc-LTR5B过表达质粒。具体包括如下步骤：
(1)使用TRIzol法从鸡胚成纤维细胞CEF提取总RNA，用RNase-free DNase I去除基因组后，使用PrimeScript RT reagent Kit试剂盒(Takara,#RR047A)将其逆转录成cDNA。
(2)以步骤(1)所获得的cDNA产物为模板，使用高保真酶扩增带同源臂的lnc-LTR5B全长序列，其中，所用引物RT-lnc-LTR5B-F和RT-lnc-LTR5B-R的核苷酸序列如下：
RT-lnc-LTR5B-F:
5′-ACCCAAGCTGGCTAGCGTTTCTCTTGCTGGCTGCACAG-3′(SEQ ID NO.8)
RT-lnc-LTR5B-R:
5′-GGCTGATCAGCGGGTTTTTTCTTTGAGTTGCAGGTTA-3′(SEQ ID NO.9)
其反应体系包括：100ng步骤(1)所获得的cDNA产物作为模板，1μL(10μM)上游引物RT-lnc-LTR5B-F与1μL(10μM)RT-lnc-LTR5B-R分别作为扩增引物，1μL DNA Polymerase，10μL 5x SF Buffer，1μL(10μM)dNTP Mix和32μL ddH2O。
其反应条件为：95℃3min；95℃15s，58℃30s，72℃1min，35x循环；72℃10min；4℃维持。
(3)以步骤(2)中PCR扩增产物进行琼脂糖凝胶电泳，然后切胶回收产物与pcDNA3.1线性质粒载体(淼灵生物，P0157)进行无缝连接。无缝连接按照MultiF SeamlessAssembly Mix(ABclonal)试剂盒说明书进行，然后连接产物直接转化大肠杆菌感受态细胞，经阳性克隆筛选，提取质粒，DNA测序鉴定等步骤，测序结果正确的质粒，命名为pcDNA3.1-lnc-LTR5B，质粒图谱如图4，质粒全长序列如SEQ ID NO.10。
实施例4
lnc-LTR5B对ALV-J病毒复制的影响
本实施例中，使用从实施例3中所获得的过表达质粒pcDNA3.1-lnc-LTR5B转染DF-1细胞，再感染ALV-J病毒，通过荧光定量PCR和Western-blot方法评估了lnc-LTR5B表达对J亚群禽白血病病毒复制水平的影响。具体包括如下步骤：
(1)待DF-1细胞融合度为50％-60％时，使用QuickShuttle-basic转染试剂(博奥龙，KX0110041)分别转染0.5μg和1.0μg pcDNA3.1-lnc-LTR5B质粒，将相应体积的质粒加入到100μL opti-MEM培养基中，再按质粒：转染试剂＝1：2的比例加入转染试剂，同时转染pcDNA3.1空质粒作为对照。18h后换为无血清DMEM，再用MOI为0.1的ALV-J病毒接种细胞，2h后换为含2％FBS的培养基，持续感染48h后收集细胞。
(2)提取步骤(1)中细胞总RNA，逆转录为cDNA后通过荧光定量PCR方法检测病毒Env基因RNA表达水平。Env基因荧光定量PCR上下游引物为：
上游引物q-JY03-F:TTGGTTCGGTGTGCTATG
下游引物q-JY03-R:GTCTCGTTGCTGGTGAAT
(3)提取步骤(2)中细胞总蛋白，进行SDS-PAGE凝胶电泳，结束后使用湿转法转印至NC膜上，5％脱脂乳封闭后，4℃过夜孵育ALV-J Env蛋白抗体(JE9 mAb)，并以GAPDH为内参蛋白；然后再用HRP标记的山羊抗鼠IgG室温孵育1h，于蛋白成像仪中观察结果。
实验结果如图3所示，与对照组相比，当DF-1细胞过表达lnc-LTR5B时(图3A)，ALV-J病毒RNA显著受到抑制(图3B)，且病毒蛋白Env的相对表达水平也显著受到抑制，抑制率可达到50％以上(图3C)。由此表明，lnc-LTR5B可以显著降低ALV-J病毒的感染及胞内复制能力。因此，本发明克隆的长链非编码RNA lnc-LTR5B能作为防治J亚型禽白血病的潜在核酸药物。
序列表
<110> 扬州大学
<120> 抑制J亚型禽白血病病毒的长链非编码RNA及其载体和应用
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aaaagcatag cgtcatacca ggcactatga agaaaatcgt ctctgtttca gctgtaacca 180
ggaaagagga acagctggtt gggatcaagc agcaagtcta tccaggcttg tctgaatccc 240
tcctcttcct tcttcagatg gtttgagcaa agaacacatc aaagatgttg acgtgtctct 300
catcaactaa ataccagcag ccaagttatc taaggtcatt ttctgcctcc ctgccactgg 360
gaagaaagtg ccttcagggg ttctcattgt gtaccccaga agtacacagc ataacaactc 420
ctgagtaaac acaggcacag tcagcatccc ccatcctcgc ctctgaaaca gtttgtattc 480
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gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttctcttg ctggctgcac agtgtgagaa gctgaaaaac tgcaacgtct ttggctctgt 960
acagtgctgc tcagcaaaaa actaaaacat cagtttgttc ttagcaatgt ttttctcctt 1020
aggcaaaagc atagcgtcat accaggcact atgaagaaaa tcgtctctgt ttcagctgta 1080
accaggaaag aggaacagct ggttgggatc aagcagcaag tctatccagg cttgtctgaa 1140
tccctcctct tccttcttca gatggtttga gcaaagaaca catcaaagat gttgacgtgt 1200
ctctcatcaa ctaaatacca gcagccaagt tatctaaggt cattttctgc ctccctgcca 1260
ctgggaagaa agtgccttca ggggttctca ttgtgtaccc cagaagtaca cagcataaca 1320
actcctgagt aaacacaggc acagtcagca tcccccatcc tcgcctctga aacagtttgt 1380
attcacaata aaacccctgc tttgatctct tgccaagaat aacacagaca gtctattcaa 1440
acatctttaa taacatgata taattaaaaa tatctaacct gcaactcaaa gaaaaaaccc 1500
gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg 1560
tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa 1620
ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca 1680
gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg 1740
cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg 1800
gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 1860
ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 1920
cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 1980
tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga 2040
cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 2100
ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga 2160
tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct 2220
gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 2280
gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc 2340
aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac 2400
tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact 2460
aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta 2520
gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc 2580
cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg 2640
attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca 2700
acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt 2760
tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg 2820
gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga 2880
agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca 2940
ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct 3000
tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac 3060
tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc 3120
gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt 3180
gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt 3240
catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg 3300
tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat 3360
cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc 3420
gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc 3480
gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc 3540
tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt 3600
attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 3660
tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 3720
tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg 3780
tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 3840
gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 3900
ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 3960
ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 4020
gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 4080
tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 4140
aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 4200
aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 4260
ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 4320
tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 4380
agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 4440
gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 4500
tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 4560
acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc 4620
tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 4680
caaaccaccg ctggtagcgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 4740
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 4800
tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 4860
aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 4920
taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 4980
gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 5040
agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 5100
cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 5160
tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 5220
gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 5280
agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 5340
gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 5400
atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 5460
gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 5520
tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 5580
atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 5640
agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 5700
gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 5760
cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 5820
tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 5880
ccgcgcacat ttccccga 5898
Claims (5)
1.一种抑制ALV-J病毒的长链非编码RNA，其特征在于，所述长链非编码RNA命名为lnc-LTR5B，其核苷酸序列如SEQ ID NO.1所示。
2.一种含有权利要求1所述的抑制ALV-J病毒的长链非编码RNA的表达载体。
3.一种权利要求2所述的表达载体的构建方法，其特征在于，包括如下步骤：
（1）从鸡胚成纤维细胞CEF提取总RNA，将其逆转录成cDNA；
（2）以步骤（1）所获得的cDNA产物为模板，设计引物，扩增带同源臂的lnc-LTR5B全长序列，
（3）对PCR扩增产物切胶回收产物与pcDNA3.1线性质粒载体进行无缝连接，然后连接产物直接转化大肠杆菌感受态细胞，经阳性克隆筛选，DNA测序鉴定，测序结果正确的质粒，命名为pcDNA3.1-lnc-LTR5B。
4.根据权利要求3所述的构建方法，其特征在于，步骤（2）中用于扩增带同源臂lnc-LTR5B全长序列所用引物RT-lnc-LTR5B-F和RT-lnc-LTR5B-R的核苷酸序列分别如SEQ IDNO.8-9所示。
5.一种权利要求1所述的长链非编码RNA或者权利要求2所述的表达载体在制备抗ALV-J病毒药物中的应用。
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| CN111575288A (zh) * | 2020-05-16 | 2020-08-25 | 扬州大学 | 抑制鸡c-myc基因表达和J亚群禽白血病病毒增殖的ch-MYC-AS1及其应用 |
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| CN107502611A (zh) * | 2017-09-26 | 2017-12-22 | 扬州大学 | 一种抗j亚群禽白血病病毒增殖的内源性反转录病毒rna及其制备方法和应用 |
| CN111575288A (zh) * | 2020-05-16 | 2020-08-25 | 扬州大学 | 抑制鸡c-myc基因表达和J亚群禽白血病病毒增殖的ch-MYC-AS1及其应用 |
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