CN114957439A - 绵羊pdgfd、编码pdgfd核酸及其重组慢病毒、宿主细胞与应用 - Google Patents
绵羊pdgfd、编码pdgfd核酸及其重组慢病毒、宿主细胞与应用 Download PDFInfo
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- CN114957439A CN114957439A CN202210606481.5A CN202210606481A CN114957439A CN 114957439 A CN114957439 A CN 114957439A CN 202210606481 A CN202210606481 A CN 202210606481A CN 114957439 A CN114957439 A CN 114957439A
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Abstract
本发明提供了一种绵羊PDGFD、编码PDGFD核酸及其重组慢病毒、宿主细胞与应用,涉及分子细胞生物学技术领域,所述血小板源性生长因子PDGFD包括PDGFD‑T1和PDGFD‑T2中的一种或两种;所述PDGFD‑T1的氨基酸序列如SEDIDNO:1所示,所述PDGFD‑T2的氨基酸序列如SEDIDNO:2所示。本发明的PDGFD‑T1和PDGFD‑T2能显著抑制前体脂肪细胞分化成熟,并显著降低成脂分化相关基因CEBPα、PPARγ、FAS、FABP4、LPL的mRNA相对表达量,从而抑制动物脂肪沉积,改善动物肉质,对于在生命科学领域、医学科学领域、畜牧业领域等中都具有重要的指导意义。
Description
技术领域
本发明涉及分子细胞生物学技术领域,具体涉及一种绵羊PDGFD、编码PDGFD核酸及其重组慢病毒、宿主细胞与应用。
背景技术
哺乳动物脂肪组织是一个复杂的器官,在诸多体内因子和体外信号相互影响、协同作用下维持能量平衡。成熟的脂肪组织是由存在于中胚层的干细胞经过脂肪母细胞、前体脂肪细胞、不成熟脂肪细胞逐步分化、发育形成。而动物体内脂肪的沉积过程一方面是成熟脂肪细胞内脂肪的不断合成、蓄积过程;另一方面也是前体脂肪细胞的不断增殖、分化、成熟过程。前体脂肪细胞向脂肪生成的内在程序启动后,会激活一系列转录级联反应,在多种转录因子、脂肪酸合成酶、内环境脂肪因子、各级代谢酶等相关基因及信号通路的协同作用下,促进前体脂肪细胞分化为成熟脂肪细胞。因此,对哺乳动物脂肪沉积的研究从前体脂肪细胞成脂分化的分子调控角度入手尤为重要。
PDGFD基因属于血小板源性生长因子(Platelet-derived Growth Factor,PDGF)家族,该家族共有4个成员,即PDGFA、PDGFB、PDGFC、PDGFD。血小板源性生长因子是成纤维细胞、平滑肌细胞以及其他间充质来源细胞的主要有丝***原和强化学驱动剂,通过与PDGFR受体结合参与调控胚胎发育、细胞增殖、细胞迁移、生存和趋化等。绵羊PDGFD基因位于15号染色体,全长约28.6kb,蛋白质分子量约43kDa,包含CUB和PDGF两种结构域,当PDGFD蛋白被激活时CUB结构域被水解解离,以使PDGF结构域发挥生物活性。迄今为止,还没有关于绵羊PDGFD基因PDGF结构域对前体脂肪细胞分化的功能作用研究。
发明内容
本发明的目的在于提供一种绵羊PDGFD、编码PDGFD核酸及其重组慢病毒、宿主细胞与应用。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种绵羊血小板源性生长因子PDGFD,所述血小板源性生长因子PDGFD包括PDGFD-T1和PDGFD-T2中的一种或两种;
所述PDGFD-T1的氨基酸序列如SED ID NO:1所示,所述PDGFD-T2的氨基酸序列如SED ID NO:2所示。
本发明还提供了一种编码上述血小板源性生长因子PDGFD的核酸,所述的核酸的核苷酸序列如SED ID NO:3~4所示。
本发明还提供了一种含有上述核酸的慢病毒表达载体。
本发明还提供了一种含有上述慢病毒表达载体的重组慢病毒。
本发明还提供了一种宿主细胞,所述宿主细胞含有上述核酸、慢病毒表达载体或重组慢病毒。
本发明还提供了一种抑制动物脂肪沉积的产品,所述产品的有效成分包括上述血小板源性生长因子PDGFD、核酸、慢病毒表达载体、重组慢病毒或宿主细胞。
本发明还提供了一种上述血小板源性生长因子PDGFD、核酸、慢病毒表达载体、重组慢病毒或宿主细胞在制备抑制动物脂肪沉积的产品中的应用。
优选地,所述产品抑制前体脂肪细胞分化成熟。
本发明还提供了一种上述血小板源性生长因子PDGFD、核酸、慢病毒表达载体、重组慢病毒或宿主细胞在制备CEBPα抑制剂、PPARγ抑制剂、FAS抑制剂、FABP4抑制剂或LPL抑制剂中的应用。
本发明还提供了一种上述血小板源性生长因子PDGFD、核酸、慢病毒表达载体、重组慢病毒或宿主细胞在制备改善动物肉质产品中的应用。
本发明的有益效果如下:
本发明提供了一种绵羊血小板源性生长因子PDGFD:PDGFD-T1和PDGFD-T2,本发明的PDGFD-T1和PDGFD-T2能显著抑制前体脂肪细胞分化成熟,并显著降低成脂分化相关基因CEBPα、PPARγ、FAS、FABP4、LPL的mRNA相对表达量,从而抑制动物脂肪沉积,改善动物肉质,对于在生命科学领域、医学科学领域、畜牧业领域等中都具有重要的指导意义。
附图说明
图1为绵羊PDGFD-T1、PDGFD-T2和PDGFD-T3基因编码序列扩增图示图;
图2为绵羊PDGFD-T1、PDGFD-T2和PDGFD-T3结构域预测结果图;
图3为HA标签抗体检测PDGFD-T1、PDGFD-T2和PDGFD-T3在3T3-L1细胞中的过表达情况图;
图4为油红O染色检测PDGFD-T1、PDGFD-T2和PDGFD-T3组对3T3-L1细胞成脂分化的影响图;
图5为qRT-PCR检测PDGFD-T1和PDGFD-T2对3T3-L1细胞成脂分化相关基因表达的影响图。
具体实施方式
本发明以绵羊PDGFD基因为研究对象,提供了PDGFD基因三种形式的编码序列,其中PDGFD-T1和PDGFD-T2包括CUB和PDGF两种结构域,PDGFD-T3无PDGF结构域,仅保留了CUB结构域。本发明PDGFD-T1、PDGFD-T2和PDGFD-T3对进行功能验证时发现,PDGFD-T1、PDGFD-T2参与抑制前体脂肪细胞3T3-L1分化为成熟脂肪细胞及形成成熟脂滴,而PDGFD-T3在缺失PDGF结构域以后,解除了其对前体脂肪细胞系3T3-L1分化成熟的抑制作用。
基于此,本发明提供了一种绵羊血小板源性生长因子PDGFD,所述血小板源性生长因子PDGFD包括PDGFD-T1和PDGFD-T2中的一种或两种;
所述PDGFD-T1的氨基酸序列如SED ID NO:1所示,所述PDGFD-T2的氨基酸序列如SED ID NO:2所示。
在本发明中,所述PDGFD-T1编码370个氨基酸;所述PDGFD-T2相比于PDGFD-T1的外显子2起始缺失18bp编码,编码364个氨基酸。
本发明还提供了一种编码上述血小板源性生长因子PDGFD的核酸,所述的核酸的核苷酸序列如SED ID NO:3~4所示。
本发明还提供了一种含有上述核酸的慢病毒表达载体。
在本发明中,所述的慢病毒表达载体优选为pLEX-MCS。
本发明还提供了一种含有上述慢病毒表达载体的重组慢病毒。
在本发明中,所述重组慢病毒采用上述的慢病毒表达载体与包装质粒共转染哺乳细胞制备得到。所述的哺乳细胞优选为HEK-293T细胞。所述的慢病毒包装质粒优选为psPAX2和pMD2.G。所述转染的方法优选为磷酸钙转染法。发明的慢病毒表达载体、pSPAX2与pMD2.G的质量比优选为20:15:6。
本发明还提供了一种宿主细胞,所述宿主细胞含有上述核酸、慢病毒表达载体或重组慢病毒。
本发明中,所述的宿主细胞优选为前体脂肪细胞3T3-L1。本发明将PDGFD-T1、PDGFD-T2、PDGFD-T3慢病毒表达载体转染至前体脂肪细胞3T3-L1后,均能成功过表达。
本发明还提供了一种抑制动物脂肪沉积的产品,所述产品的有效成分包括上述血小板源性生长因子PDGFD、核酸、慢病毒表达载体、重组慢病毒或宿主细胞。
本发明还提供了一种上述血小板源性生长因子PDGFD、核酸、慢病毒表达载体、重组慢病毒或宿主细胞在制备抑制动物脂肪沉积的产品中的应用。
在本发明中,所述产品包括试剂或药物。所述产品还包括药学上可接受的载体。作为一优选的实施方式,本发明中,上述血小板源性生长因子PDGFD、核酸、慢病毒表达载体、重组慢病毒或宿主细胞在制备抑制前体脂肪细胞分化成熟的产品中的应用。
本发明还提供了一种上述血小板源性生长因子PDGFD、核酸、慢病毒表达载体、重组慢病毒或宿主细胞在制备CEBPα抑制剂、PPARγ抑制剂、FAS抑制剂、FABP4抑制剂或LPL抑制剂中的应用。
在本发明中,本发明的PDGFD-T1和PDGFD-T2均能显著降低成脂分化相关基因CEBPα、PPARγ、FAS、FABP4、LPL的mRNA相对表达量。其中,与PDGFD-T1相比,PDGFD-T2更加显著降低成脂分化相关基因相对表达量。
本发明还提供了一种上述血小板源性生长因子PDGFD、核酸、慢病毒表达载体、重组慢病毒或宿主细胞在制备改善动物肉质产品中的应用。
在本发明中,本发明的PDGFD-T1和PDGFD-T2能显著抑制前体脂肪细胞分化成熟,并显著降低成脂分化相关基因CEBPα、PPARγ、FAS、FABP4、LPL的mRNA相对表达量,从而抑制动物脂肪沉积,改善动物肉质,提高瘦肉率。
在本发明中,若无特殊说明,所有的原料组分均为本领域技术人员熟知的市售商品。
下面将结合本发明中的实施例,对本发明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1
1.绵羊PDGFD慢病毒表达载体的构建
1.1PDGFD编码区序列扩增
以NCBI中绵羊PDGFD基因转录本XM_004015965.5序列信息为参考,使用BioEdit软件分析PDGFD编码区序列酶切位点分布情况,并参照pLEX MCS载体图谱,选择BamH I和XhoI作为构建表达载体的酶切位点。利用Primerpremier 5.0软件设计扩增PDGFD基因编码区序列的引物,并在引物的5’端加入保护碱基、酶切位点、KOZAK序列、HA标签序列(表1),以绵羊脂肪cDNA为模板PCR扩增(表2)PDGFD基因编码区序列。
表1 PDGFD基因编码区序列扩增引物
注:小写加粗字母为保护碱基,大写加粗字母为酶切位点(F:BamH I、R:Xho I),斜体字母为KOZAK序列,下划线字母为HA标签序列。
表2 PCR反应体系
反应程序为:98℃10s,55.5℃5s,72℃5s/kb;共33个循环。
1.2重组真核表达质粒PLEX-PDGFD的构建
(1)将目的片段PCR产物和pLEX-MCS载体经限制性内切酶BamH I-HF(购自NEB#R3136)和Xho I(购自NEB#R0146)双酶切处理。反应体系如表3所示。
表3目的片段和pLEX-MCS载体双酶切反应体系
(2)酶切产物回收纯化后,参照T4 DNA ligase试剂盒(NEB#M0202S)说明书,将酶切后的目的片段连接至同样被酶切后的pLEX-MCS载体。反应体系如表4所示。
表4 PDGFD基因表达载体连接体系
(3)目的片段与载体连接后,经转化、单克隆筛选,对于测序鉴定正确的、无碱基突变的重组质粒,按照QIAGEN Midi kit说明书提取质粒,使用Nanodrop One核酸定量仪测定所提取质粒的浓度及纯度,于-20℃保存备用。
2.绵羊PDGFD基因编码区扩增序列结构域预测分析
(1)将上述获得的重组质粒测序,结果显示,重组质粒PDGFD有三种形式的编码序列(见图1),分别为①PCR扩增全长1113bp,编码370个氨基酸,后续命名为PDGFD-T1;②PCR扩增全长1095bp,编码364个氨基酸,与①相比外显子2起始缺失18bp,后续命名为PDGFD-T2;③PCR扩增全长1088bp,与①相比外显子5起始缺失25bp,导致终止密码子TAA提前出现,共编码191个氨基酸,后续命名为PDGFD-T3,其中绵羊PDGFD基因编码序列扩增图示见图1。
具体的,PDGFD-T1氨基酸序列如下(如SED ID NO:1所示):
MHRLILVYTLVCANFCSYRDTSATPQSASIKALRNANLRRDESNHLTDLYRRDETIQVTGHGHVQSPRFPNSYPRNLLLTWRLHSQEKTRIQLAFDNQFGLEEAENDICRYDFVEVEDISETSTVIRGRWCGHKEVPPRITSRTNQIKITFKSDDYFVAKPGFKIYYSFVEDFQPAAASETNWESVTSSISGVSYHSPSVTDPTLTADALDKTIAEFDTVEELLKHFNPESWQDDLENLYLDTPHHRGRSYHDRKSKVDLDRLNDDVKRYSCTPRNYSVNLREELKLTNVVFFPRCLLVQRCGGNCGCGTVNWKSCACNSGKTVKKYHEVLKFEPGHFKRRGRAKHMALVDIQLDHHERCDCICSSRPPR。
PDGFD-T1编码区核苷酸序列如下(如SED ID NO:3所示):
ATGCACCGGCTCATCCTTGTCTACACGCTAGTCTGCGCAAACTTTTGCAGCTACCGGGACACCTCTGCCACCCCGCAGAGCGCATCTATCAAAGCTTTGCGTAACGCCAACCTCAGGCGAGATGAGAGCAATCACCTCACAGACTTGTACCGAAGAGACGAGACCATCCAGGTGACAGGACACGGCCACGTGCAGAGTCCCCGCTTCCCAAACAGCTACCCTCGCAACCTGCTTCTGACCTGGCGGCTCCACTCCCAGGAGAAAACAAGGATACAGCTAGCCTTTGACAATCAGTTTGGATTAGAGGAAGCGGAAAATGATATCTGTAGGTATGATTTTGTAGAAGTTGAAGACATATCTGAAACCAGTACTGTTATTAGAGGACGATGGTGTGGACACAAGGAAGTTCCTCCAAGGATAACATCAAGAACAAACCAGATTAAAATAACGTTCAAGTCTGATGACTACTTTGTGGCTAAACCTGGATTCAAGATTTATTATTCTTTTGTGGAAGATTTCCAACCTGCAGCAGCCTCAGAGACCAACTGGGAGTCAGTCACAAGCTCTATCTCAGGGGTATCCTATCACTCTCCATCAGTAACGGACCCCACTCTCACTGCGGATGCTCTGGACAAAACGATTGCAGAATTTGATACTGTGGAAGAGCTGCTCAAGCACTTCAATCCCGAATCATGGCAAGACGATCTTGAGAATCTGTATTTGGATACCCCTCATCATCGAGGCAGATCGTATCATGACAGGAAGTCAAAAGTTGACCTGGACAGGCTCAACGATGATGTCAAGCGTTACAGTTGCACTCCCAGGAATTACTCCGTCAACTTGAGAGAAGAGCTGAAGCTTACCAATGTGGTCTTCTTTCCACGCTGCCTCCTTGTGCAGCGCTGCGGAGGAAACTGTGGCTGTGGAACTGTCAACTGGAAGTCCTGTGCGTGCAATTCAGGGAAAACTGTGAAAAAGTATCACGAGGTGTTAAAGTTTGAACCTGGCCATTTCAAGAGGAGGGGCAGAGCGAAGCACATGGCTCTCGTTGACATCCAGTTGGATCATCATGAGCGGTGCGACTGTATCTGCAGCTCAAGACCACCTCGATAA。
PDGFD-T2氨基酸序列如下(如SED ID NO:2所示):
MHRLILVYTLVCANFCSYRDTSATPQSASIKALRNANLRRDDLYRRDETIQVTGHGHVQSPRFPNSYPRNLLLTWRLHSQEKTRIQLAFDNQFGLEEAENDICRYDFVEVEDISETSTVIRGRWCGHKEVPPRITSRTNQIKITFKSDDYFVAKPGFKIYYSFVEDFQPAAASETNWESVTSSISGVSYHSPSVTDPTLTADALDKTIAEFDTVEELLKHFNPESWQDDLENLYLDTPHHRGRSYHDRKSKVDLDRLNDDVKRYSCTPRNYSVNLREELKLTNVVFFPRCLLVQRCGGNCGCGTVNWKSCACNSGKTVKKYHEVLKFEPGHFKRRGRAKHMALVDIQLDHHERCDCICSSRPPR。
PDGFD-T2编码区核苷酸序列如下(如SED ID NO:4所示):
ATGCACCGGCTCATCCTTGTCTACACGCTAGTCTGCGCAAACTTTTGCAGCTACCGGGACACCTCTGCCACCCCGCAGAGCGCATCTATCAAAGCTTTGCGTAACGCCAACCTCAGGCGAGATGACTTGTACCGAAGAGACGAGACCATCCAGGTGACAGGACACGGCCACGTGCAGAGTCCCCGCTTCCCAAACAGCTACCCTCGCAACCTGCTTCTGACCTGGCGGCTCCACTCCCAGGAGAAAACAAGGATACAGCTAGCCTTTGACAATCAGTTTGGATTAGAGGAAGCGGAAAATGATATCTGTAGGTATGATTTTGTAGAAGTTGAAGACATATCTGAAACCAGTACTGTTATTAGAGGACGATGGTGTGGACACAAGGAAGTTCCTCCAAGGATAACATCAAGAACAAACCAGATTAAAATAACGTTCAAGTCTGATGACTACTTTGTGGCTAAACCTGGATTCAAGATTTATTATTCTTTTGTGGAAGATTTCCAACCTGCAGCAGCCTCAGAGACCAACTGGGAGTCAGTCACAAGCTCTATCTCAGGGGTATCCTATCACTCTCCATCAGTAACGGACCCCACTCTCACTGCGGATGCTCTGGACAAAACGATTGCAGAATTTGATACTGTGGAAGAGCTGCTCAAGCACTTCAATCCCGAATCATGGCAAGACGATCTTGAGAATCTGTATTTGGATACCCCTCATCATCGAGGCAGATCGTATCATGACAGGAAGTCAAAAGTTGACCTGGACAGGCTCAACGATGATGTCAAGCGTTACAGTTGCACTCCCAGGAATTACTCCGTCAACTTGAGAGAAGAGCTGAAGCTTACCAATGTGGTCTTCTTTCCACGCTGCCTCCTTGTGCAGCGCTGCGGAGGAAACTGTGGCTGTGGAACTGTCAACTGGAAGTCCTGTGCGTGCAATTCAGGGAAAACTGTGAAAAAGTATCACGAGGTGTTAAAGTTTGAACCTGGCCATTTCAAGAGGAGGGGCAGAGCGAAGCACATGGCTCTCGTTGACATCCAGTTGGATCATCATGAGCGGTGCGACTGTATCTGCAGCTCAAGACCACCTCGATAA。
PDGFD-T3氨基酸序列如下(如SED ID NO:7所示):
MHRLILVYTLVCANFCSYRDTSATPQSASIKALRNANLRRDESNHLTDLYRRDETIQVTGHGHVQSPRFPNSYPRNLLLTWRLHSQEKTRIQLAFDNQFGLEEAENDICRYDFVEVEDISETSTVIRGRWCGHKEVPPRITSRTNQIKITFKSDDYFVAKPGFKIYYSFVEDFQPAAASETNWESVTSSIS。
PDGFD-T3编码区核苷酸序列如下(如SED ID NO:8所示):
ATGCACCGGCTCATCCTTGTCTACACGCTAGTCTGCGCAAACTTTTGCAGCTACCGGGACACCTCTGCCACCCCGCAGAGCGCATCTATCAAAGCTTTGCGTAACGCCAACCTCAGGCGAGATGAGAGCAATCACCTCACAGACTTGTACCGAAGAGACGAGACCATCCAGGTGACAGGACACGGCCACGTGCAGAGTCCCCGCTTCCCAAACAGCTACCCTCGCAACCTGCTTCTGACCTGGCGGCTCCACTCCCAGGAGAAAACAAGGATACAGCTAGCCTTTGACAATCAGTTTGGATTAGAGGAAGCGGAAAATGATATCTGTAGGTATGATTTTGTAGAAGTTGAAGACATATCTGAAACCAGTACTGTTATTAGAGGACGATGGTGTGGACACAAGGAAGTTCCTCCAAGGATAACATCAAGAACAAACCAGATTAAAATAACGTTCAAGTCTGATGACTACTTTGTGGCTAAACCTGGATTCAAGATTTATTATTCTTTTGTGGAAGATTTCCAACCTGCAGCAGCCTCAGAGACCAACTGGGAGTCAGTCACAAGCTCTATCTCATAACGGACCCCACTCTCACTGCGGATGCTCTGGACAAAACGATTGCAGAATTTGATACTGTGGAAGAGCTGCTCAAGCACTTCAATCCCGAATCATGGCAAGACGATCTTGAGAATCTGTATTTGGATACCCCTCATCATCGAGGCAGATCGTATCATGACAGGAAGTCAAAAGTTGACCTGGACAGGCTCAACGATGATGTCAAGCGTTACAGTTGCACTCCCAGGAATTACTCCGTCAACTTGAGAGAAGAGCTGAAGCTTACCAATGTGGTCTTCTTTCCACGCTGCCTCCTTGTGCAGCGCTGCGGAGGAAACTGTGGCTGTGGAACTGTCAACTGGAAGTCCTGTGCGTGCAATTCAGGGAAAACTGTGAAAAAGTATCACGAGGTGTTAAAGTTTGAACCTGGCCATTTCAAGAGGAGGGGCAGAGCGAAGCACATGGCTCTCGTTGACATCCAGTTGGATCATCATGAGCGGTGCGACTGTATCTGCAGCTCAAGACCACCTCGATAA。
(2)利用在线软件SMART(http://smart.embl-heidelberg.de/)对PDGFD三种编码序列进行结构域预测。
结果显示,绵羊PDGFD-T1和T2包括CUB和PDGF两种结构域,CUB结构域由PDGFD基因外显子2和3编码,PDGF结构域由外显子6和7编码;但PDGFD-T3中PDGF结构域丢失,仅保留了CUB结构域(见图2)。
3.PLEX-PDGFD重组慢病毒的包装
按照PLEX-MCS慢病毒包装说明书,将重组慢病毒质粒PDGFD-T1、PDGFD-T2、PDGFD-T3分别与包装质粒(psPAX2和pMD2.G)通过磷酸钙转染法共转染至293T细胞内进行慢病毒包装,具体步骤如下:
(1)每10cm细胞培养板中接种2~2.5×106个293T细胞,待细胞贴壁并达到70~80%的生长汇合度时准备转染慢病毒质粒;
(2)将重组慢病毒质粒与包装质粒(psPAX2和pMD2.G)通过磷酸钙转染法共转染至293T细胞内进行慢病毒包装。转染体系(1mL/10cm plate):重组慢病毒质粒20μg;包装质粒(psPAX2)15μg;包膜质粒(pMD2.G)6μg;用无菌H2O稀释至500μL;加入2×HBS(Hepes缓冲盐水)500μL;通过快速涡旋将混合物完全混合。
(3)在涡旋的同时,将50μL 2.5M CaCl2逐滴缓慢加入上述混合物中。室温孵育20分钟后将混合物加至细胞培养板中。
(4)12~14h后更换新鲜培养基,10h后将细胞转移至32℃培养箱内。
(5)14~16h后收集细胞上清,并用0.45μm过滤器过滤上清液(即为慢病毒感染液),过滤后的上清液可直接用于感染靶细胞。
4.PLEX-PDGFD重组慢病毒感染靶细胞
(2)将含有慢病毒包装质粒的细胞上清液和新鲜培养基(v/v,1:1)与10μg/mL的聚凝胺加入到靶细胞中。然后,将细胞置于32℃的培养箱中。
(3)14~16h后将细胞转移至37℃细胞培养箱,10h后更换新鲜培养基继续培养。
(4)48h后将细胞传至10cm细胞培养板,同时加入含1.5μg/mL的嘌呤霉素细胞培养液进行抗性细胞筛选培养,期间每3~4d更换一次含有1.5μg/mL的嘌呤霉素细胞培养液。
5.3T3-L1细胞中PDGFD过表达检测
将上述PDGFD-T1、PDGFD-T2、PDGFD-T3慢病毒表达载体转染至前体脂肪细胞3T3-L1后,利用HA标签抗体(购自Sigma#H3663)按常规Western Blot实验方法检测3T3-L1细胞中PDGFD-T1、PDGFD-T2、PDGFD-T3过表达情况。
结果显示,PDGFD-T1、PDGFD-T2、PDGFD-T3均在3T3-L1细胞中过表达成功(见图3),后续可继续使用过表达PDGFD的3T3-L1细胞进行相关实验。
6.PDGFD基因PDGF结构域对前体脂肪细胞3T3-L1成脂分化的功能影响
(1)前体脂肪细胞诱导分化及油红O染色检测
将过表达PDGFD-T1、PDGFD-T2、PDGFD-T3的3T3-L1细胞和对照组按照该细胞系培养说明书(CL-173TM)进行诱导分化处理,其中所述对照组为未作任何处理的前体脂肪细胞系3T3-L1,待诱导分化处理10天后,参照油红O染色液使用说明书(购自Solarbio,#G1260)对诱导后的细胞进行染色,以检测PDGFD对3T3-L1细胞分化成熟形成脂滴能力的影响。
如图4可知:PDGFD-T1组“戒环样”脂滴数量明显低于CK组(对照组),PDGFD-T2组无法观察到“戒环样”脂滴,缺少PDFD结构域的PDGFD-T3组与CK组相比,油红O染色结果无明显差异。
(2)3T3-L1细胞成脂分化相关基因的表达检测
为进一步验证PDGFD对前体脂肪细胞分化成熟的影响,从分子水平对3T3-L1细胞成脂分化相关基因的表达进行检测。取诱导分化10天的3T3-L1细胞抽提RNA并反转录为cDNA,以cDNA为模板,进行qRT-PCR检测(qRT-PCR扩增引物见表5)。结果显示(图5),在诱导分化10天后,PDGFD-T1、PDGFD-T2组成脂分化相关基因CEBPα、PPARγ、FAS、FABP4、LPL的mRNA相对表达量均显著低于对照组CK(P<0.05);缺少PDFD结构域的PDGFD-T3组成脂分化相关基因CEBPα、PPARγ、FAS、FABP4、LPL的mRNA相对表达量均与对照组CK无显著差异。
表5成脂分化相关基因qRT-PCR扩增引物
综合以上实验结果,本发明PDGFD-T1、PDGFD-T2显著抑制前体脂肪细胞3T3-L1分化为成熟脂肪细胞及形成成熟脂滴;PDGFD-T3在缺失PDGF结构域以后,解除了其对前体脂肪细胞3T3-L1分化成熟的抑制作用,表明PDGF结构域是PDGFD发挥抑制前体脂肪细胞3T3-L1分化成熟功能的重要组成部分。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 新疆畜牧科学院生物技术研究所(新疆畜牧科学院中国-澳大利亚绵羊育种研究中心)
<120> 绵羊PDGFD、编码PDGFD核酸及其重组慢病毒、宿主细胞与应用
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 370
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met His Arg Leu Ile Leu Val Tyr Thr Leu Val Cys Ala Asn Phe Cys
1 5 10 15
Ser Tyr Arg Asp Thr Ser Ala Thr Pro Gln Ser Ala Ser Ile Lys Ala
20 25 30
Leu Arg Asn Ala Asn Leu Arg Arg Asp Glu Ser Asn His Leu Thr Asp
35 40 45
Leu Tyr Arg Arg Asp Glu Thr Ile Gln Val Thr Gly His Gly His Val
50 55 60
Gln Ser Pro Arg Phe Pro Asn Ser Tyr Pro Arg Asn Leu Leu Leu Thr
65 70 75 80
Trp Arg Leu His Ser Gln Glu Lys Thr Arg Ile Gln Leu Ala Phe Asp
85 90 95
Asn Gln Phe Gly Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Tyr Asp
100 105 110
Phe Val Glu Val Glu Asp Ile Ser Glu Thr Ser Thr Val Ile Arg Gly
115 120 125
Arg Trp Cys Gly His Lys Glu Val Pro Pro Arg Ile Thr Ser Arg Thr
130 135 140
Asn Gln Ile Lys Ile Thr Phe Lys Ser Asp Asp Tyr Phe Val Ala Lys
145 150 155 160
Pro Gly Phe Lys Ile Tyr Tyr Ser Phe Val Glu Asp Phe Gln Pro Ala
165 170 175
Ala Ala Ser Glu Thr Asn Trp Glu Ser Val Thr Ser Ser Ile Ser Gly
180 185 190
Val Ser Tyr His Ser Pro Ser Val Thr Asp Pro Thr Leu Thr Ala Asp
195 200 205
Ala Leu Asp Lys Thr Ile Ala Glu Phe Asp Thr Val Glu Glu Leu Leu
210 215 220
Lys His Phe Asn Pro Glu Ser Trp Gln Asp Asp Leu Glu Asn Leu Tyr
225 230 235 240
Leu Asp Thr Pro His His Arg Gly Arg Ser Tyr His Asp Arg Lys Ser
245 250 255
Lys Val Asp Leu Asp Arg Leu Asn Asp Asp Val Lys Arg Tyr Ser Cys
260 265 270
Thr Pro Arg Asn Tyr Ser Val Asn Leu Arg Glu Glu Leu Lys Leu Thr
275 280 285
Asn Val Val Phe Phe Pro Arg Cys Leu Leu Val Gln Arg Cys Gly Gly
290 295 300
Asn Cys Gly Cys Gly Thr Val Asn Trp Lys Ser Cys Ala Cys Asn Ser
305 310 315 320
Gly Lys Thr Val Lys Lys Tyr His Glu Val Leu Lys Phe Glu Pro Gly
325 330 335
His Phe Lys Arg Arg Gly Arg Ala Lys His Met Ala Leu Val Asp Ile
340 345 350
Gln Leu Asp His His Glu Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro
355 360 365
Pro Arg
370
<210> 2
<211> 364
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met His Arg Leu Ile Leu Val Tyr Thr Leu Val Cys Ala Asn Phe Cys
1 5 10 15
Ser Tyr Arg Asp Thr Ser Ala Thr Pro Gln Ser Ala Ser Ile Lys Ala
20 25 30
Leu Arg Asn Ala Asn Leu Arg Arg Asp Asp Leu Tyr Arg Arg Asp Glu
35 40 45
Thr Ile Gln Val Thr Gly His Gly His Val Gln Ser Pro Arg Phe Pro
50 55 60
Asn Ser Tyr Pro Arg Asn Leu Leu Leu Thr Trp Arg Leu His Ser Gln
65 70 75 80
Glu Lys Thr Arg Ile Gln Leu Ala Phe Asp Asn Gln Phe Gly Leu Glu
85 90 95
Glu Ala Glu Asn Asp Ile Cys Arg Tyr Asp Phe Val Glu Val Glu Asp
100 105 110
Ile Ser Glu Thr Ser Thr Val Ile Arg Gly Arg Trp Cys Gly His Lys
115 120 125
Glu Val Pro Pro Arg Ile Thr Ser Arg Thr Asn Gln Ile Lys Ile Thr
130 135 140
Phe Lys Ser Asp Asp Tyr Phe Val Ala Lys Pro Gly Phe Lys Ile Tyr
145 150 155 160
Tyr Ser Phe Val Glu Asp Phe Gln Pro Ala Ala Ala Ser Glu Thr Asn
165 170 175
Trp Glu Ser Val Thr Ser Ser Ile Ser Gly Val Ser Tyr His Ser Pro
180 185 190
Ser Val Thr Asp Pro Thr Leu Thr Ala Asp Ala Leu Asp Lys Thr Ile
195 200 205
Ala Glu Phe Asp Thr Val Glu Glu Leu Leu Lys His Phe Asn Pro Glu
210 215 220
Ser Trp Gln Asp Asp Leu Glu Asn Leu Tyr Leu Asp Thr Pro His His
225 230 235 240
Arg Gly Arg Ser Tyr His Asp Arg Lys Ser Lys Val Asp Leu Asp Arg
245 250 255
Leu Asn Asp Asp Val Lys Arg Tyr Ser Cys Thr Pro Arg Asn Tyr Ser
260 265 270
Val Asn Leu Arg Glu Glu Leu Lys Leu Thr Asn Val Val Phe Phe Pro
275 280 285
Arg Cys Leu Leu Val Gln Arg Cys Gly Gly Asn Cys Gly Cys Gly Thr
290 295 300
Val Asn Trp Lys Ser Cys Ala Cys Asn Ser Gly Lys Thr Val Lys Lys
305 310 315 320
Tyr His Glu Val Leu Lys Phe Glu Pro Gly His Phe Lys Arg Arg Gly
325 330 335
Arg Ala Lys His Met Ala Leu Val Asp Ile Gln Leu Asp His His Glu
340 345 350
Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro Pro Arg
355 360
<210> 3
<211> 1113
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgcaccggc tcatccttgt ctacacgcta gtctgcgcaa acttttgcag ctaccgggac 60
acctctgcca ccccgcagag cgcatctatc aaagctttgc gtaacgccaa cctcaggcga 120
gatgagagca atcacctcac agacttgtac cgaagagacg agaccatcca ggtgacagga 180
cacggccacg tgcagagtcc ccgcttccca aacagctacc ctcgcaacct gcttctgacc 240
tggcggctcc actcccagga gaaaacaagg atacagctag cctttgacaa tcagtttgga 300
ttagaggaag cggaaaatga tatctgtagg tatgattttg tagaagttga agacatatct 360
gaaaccagta ctgttattag aggacgatgg tgtggacaca aggaagttcc tccaaggata 420
acatcaagaa caaaccagat taaaataacg ttcaagtctg atgactactt tgtggctaaa 480
cctggattca agatttatta ttcttttgtg gaagatttcc aacctgcagc agcctcagag 540
accaactggg agtcagtcac aagctctatc tcaggggtat cctatcactc tccatcagta 600
acggacccca ctctcactgc ggatgctctg gacaaaacga ttgcagaatt tgatactgtg 660
gaagagctgc tcaagcactt caatcccgaa tcatggcaag acgatcttga gaatctgtat 720
ttggataccc ctcatcatcg aggcagatcg tatcatgaca ggaagtcaaa agttgacctg 780
gacaggctca acgatgatgt caagcgttac agttgcactc ccaggaatta ctccgtcaac 840
ttgagagaag agctgaagct taccaatgtg gtcttctttc cacgctgcct ccttgtgcag 900
cgctgcggag gaaactgtgg ctgtggaact gtcaactgga agtcctgtgc gtgcaattca 960
gggaaaactg tgaaaaagta tcacgaggtg ttaaagtttg aacctggcca tttcaagagg 1020
aggggcagag cgaagcacat ggctctcgtt gacatccagt tggatcatca tgagcggtgc 1080
gactgtatct gcagctcaag accacctcga taa 1113
<210> 4
<211> 1095
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgcaccggc tcatccttgt ctacacgcta gtctgcgcaa acttttgcag ctaccgggac 60
acctctgcca ccccgcagag cgcatctatc aaagctttgc gtaacgccaa cctcaggcga 120
gatgacttgt accgaagaga cgagaccatc caggtgacag gacacggcca cgtgcagagt 180
ccccgcttcc caaacagcta ccctcgcaac ctgcttctga cctggcggct ccactcccag 240
gagaaaacaa ggatacagct agcctttgac aatcagtttg gattagagga agcggaaaat 300
gatatctgta ggtatgattt tgtagaagtt gaagacatat ctgaaaccag tactgttatt 360
agaggacgat ggtgtggaca caaggaagtt cctccaagga taacatcaag aacaaaccag 420
attaaaataa cgttcaagtc tgatgactac tttgtggcta aacctggatt caagatttat 480
tattcttttg tggaagattt ccaacctgca gcagcctcag agaccaactg ggagtcagtc 540
acaagctcta tctcaggggt atcctatcac tctccatcag taacggaccc cactctcact 600
gcggatgctc tggacaaaac gattgcagaa tttgatactg tggaagagct gctcaagcac 660
ttcaatcccg aatcatggca agacgatctt gagaatctgt atttggatac ccctcatcat 720
cgaggcagat cgtatcatga caggaagtca aaagttgacc tggacaggct caacgatgat 780
gtcaagcgtt acagttgcac tcccaggaat tactccgtca acttgagaga agagctgaag 840
cttaccaatg tggtcttctt tccacgctgc ctccttgtgc agcgctgcgg aggaaactgt 900
ggctgtggaa ctgtcaactg gaagtcctgt gcgtgcaatt cagggaaaac tgtgaaaaag 960
tatcacgagg tgttaaagtt tgaacctggc catttcaaga ggaggggcag agcgaagcac 1020
atggctctcg ttgacatcca gttggatcat catgagcggt gcgactgtat ctgcagctca 1080
agaccacctc gataa 1095
<210> 5
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cgggatccgc caccatgcac cggctcatcc ttgtctac 38
<210> 6
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ccctcgagtt aagcgtagtc tgggacgtcg tatgggtatc gaggtggtct tgagctgc 58
<210> 7
<211> 191
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met His Arg Leu Ile Leu Val Tyr Thr Leu Val Cys Ala Asn Phe Cys
1 5 10 15
Ser Tyr Arg Asp Thr Ser Ala Thr Pro Gln Ser Ala Ser Ile Lys Ala
20 25 30
Leu Arg Asn Ala Asn Leu Arg Arg Asp Glu Ser Asn His Leu Thr Asp
35 40 45
Leu Tyr Arg Arg Asp Glu Thr Ile Gln Val Thr Gly His Gly His Val
50 55 60
Gln Ser Pro Arg Phe Pro Asn Ser Tyr Pro Arg Asn Leu Leu Leu Thr
65 70 75 80
Trp Arg Leu His Ser Gln Glu Lys Thr Arg Ile Gln Leu Ala Phe Asp
85 90 95
Asn Gln Phe Gly Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Tyr Asp
100 105 110
Phe Val Glu Val Glu Asp Ile Ser Glu Thr Ser Thr Val Ile Arg Gly
115 120 125
Arg Trp Cys Gly His Lys Glu Val Pro Pro Arg Ile Thr Ser Arg Thr
130 135 140
Asn Gln Ile Lys Ile Thr Phe Lys Ser Asp Asp Tyr Phe Val Ala Lys
145 150 155 160
Pro Gly Phe Lys Ile Tyr Tyr Ser Phe Val Glu Asp Phe Gln Pro Ala
165 170 175
Ala Ala Ser Glu Thr Asn Trp Glu Ser Val Thr Ser Ser Ile Ser
180 185 190
<210> 8
<211> 1088
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgcaccggc tcatccttgt ctacacgcta gtctgcgcaa acttttgcag ctaccgggac 60
acctctgcca ccccgcagag cgcatctatc aaagctttgc gtaacgccaa cctcaggcga 120
gatgagagca atcacctcac agacttgtac cgaagagacg agaccatcca ggtgacagga 180
cacggccacg tgcagagtcc ccgcttccca aacagctacc ctcgcaacct gcttctgacc 240
tggcggctcc actcccagga gaaaacaagg atacagctag cctttgacaa tcagtttgga 300
ttagaggaag cggaaaatga tatctgtagg tatgattttg tagaagttga agacatatct 360
gaaaccagta ctgttattag aggacgatgg tgtggacaca aggaagttcc tccaaggata 420
acatcaagaa caaaccagat taaaataacg ttcaagtctg atgactactt tgtggctaaa 480
cctggattca agatttatta ttcttttgtg gaagatttcc aacctgcagc agcctcagag 540
accaactggg agtcagtcac aagctctatc tcataacgga ccccactctc actgcggatg 600
ctctggacaa aacgattgca gaatttgata ctgtggaaga gctgctcaag cacttcaatc 660
ccgaatcatg gcaagacgat cttgagaatc tgtatttgga tacccctcat catcgaggca 720
gatcgtatca tgacaggaag tcaaaagttg acctggacag gctcaacgat gatgtcaagc 780
gttacagttg cactcccagg aattactccg tcaacttgag agaagagctg aagcttacca 840
atgtggtctt ctttccacgc tgcctccttg tgcagcgctg cggaggaaac tgtggctgtg 900
gaactgtcaa ctggaagtcc tgtgcgtgca attcagggaa aactgtgaaa aagtatcacg 960
aggtgttaaa gtttgaacct ggccatttca agaggagggg cagagcgaag cacatggctc 1020
tcgttgacat ccagttggat catcatgagc ggtgcgactg tatctgcagc tcaagaccac 1080
ctcgataa 1088
<210> 9
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccaagaagtc ggtggacaag aa 22
<210> 10
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cggtcattgt cactggtcaa c 21
<210> 11
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gtgccagttt cgatccgtag a 21
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggccagcatc gtgtagatga 20
<210> 13
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggaggtggtg atagccggta t 21
<210> 14
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tgggtaatcc atagagccca g 21
<210> 15
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tgggaacctg gaagcttgtc tc 22
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gaattccacg cccagtttga 20
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
tggcgtagca ggaagtctga 20
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tgcctccatt gggataaatg tc 22
Claims (10)
1.一种绵羊血小板源性生长因子PDGFD,其特征在于,所述血小板源性生长因子PDGFD包括PDGFD-T1和PDGFD-T2中的一种或两种;
所述PDGFD-T1的氨基酸序列如SED ID NO:1所示,所述PDGFD-T2的氨基酸序列如SEDID NO:2所示。
2.一种编码权利要求1所述的血小板源性生长因子PDGFD的核酸,其特征在于,所述的核酸的核苷酸序列如SED ID NO:3~4所示。
3.一种含有权利要求2所述核酸的慢病毒表达载体。
4.一种含有权利要求3所述慢病毒表达载体的重组慢病毒。
5.一种宿主细胞,其特征在于,所述宿主细胞含有权利要求2所述核酸、权利要求3所述慢病毒表达载体或权利要求4所述的重组慢病毒。
6.一种抑制动物脂肪沉积的产品,其特征在于,所述产品的有效成分包括权利要求1所述血小板源性生长因子PDGFD、权利要求2所述核酸、权利要求3所述慢病毒表达载体、权利要求4所述的重组慢病毒或权利要求5所述宿主细胞。
7.权利要求1所述血小板源性生长因子PDGFD、权利要求2所述核酸、权利要求3所述慢病毒表达载体、权利要求4所述的重组慢病毒或权利要求5所述宿主细胞在制备抑制动物脂肪沉积的产品中的应用。
8.根据权利要求7所述的应用,其特征在于,所述产品抑制前体脂肪细胞分化成熟。
9.权利要求1所述血小板源性生长因子PDGFD、权利要求2所述核酸、权利要求3所述慢病毒表达载体、权利要求4所述的重组慢病毒或权利要求5所述宿主细胞在制备CEBPα抑制剂、PPARγ抑制剂、FAS抑制剂、FABP4抑制剂或LPL抑制剂中的应用。
10.权利要求1所述血小板源性生长因子PDGFD、权利要求2所述核酸、权利要求3所述慢病毒表达载体、权利要求4所述的重组慢病毒或权利要求5所述宿主细胞在制备改善动物肉质产品中的应用。
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1325407A (zh) * | 1998-11-10 | 2001-12-05 | 路德维格癌症研究所 | 血小板衍生生长因子d、其编码dna及其应用 |
WO2007108643A1 (en) * | 2006-03-21 | 2007-09-27 | Purimed Co., Ltd. | New gene and polypeptides of platelet derived growth factor b |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN1325407A (zh) * | 1998-11-10 | 2001-12-05 | 路德维格癌症研究所 | 血小板衍生生长因子d、其编码dna及其应用 |
WO2007108643A1 (en) * | 2006-03-21 | 2007-09-27 | Purimed Co., Ltd. | New gene and polypeptides of platelet derived growth factor b |
Non-Patent Citations (3)
Title |
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"platelet-derived growth factor D isoform X2 [Ovis aries", 《GENBANK》 * |
"platelet-derived growth factor D isoform X3 [Ovis aries]", 《GENBANK》 * |
Y. ARTEMENKO等: "Anti-adipogenic effect of PDGF is reversed by PKC inhibition", 《JOURNAL OF CELLULAR PHYSIOLOGY》, vol. 204, pages 646 - 653 * |
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