CN117925713A - 一种增强型胞质表达系统和表达质粒及其应用 - Google Patents
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Abstract
本发明涉及一种胞质表达系统、包括该表达系统的表达质粒及其在增强胞质表达外源基因中的应用。本发明所述胞质表达系统在T7RNAP的胞质表达系统中的T7RNAP编码框的上游与细胞核启动子的下游之间插入有T7启动子的序列,通过为基于C3P3框架的T7RNAP表达系统引入自扩增机制,借助细胞核启动子驱动T7RNAP表达系统的表达,随之进入胞质可以识别胞质中外源蛋白表达载体和/或本发明所述胞质表达系统带有的T7启动子,既能启动外源蛋白表达载体的T7RNAP继续进行转录表达,促进外源蛋白表达又得以实现对自身表达载体进行正反馈表达,从而提高其表达量,继而显著地增强外源蛋白的表达水平。
Description
技术领域
本发明属于分子生物技术领域,涉及蛋白表达技术,具体是涉及一种增强型胞质表达系统和表达质粒及其应用。
背景技术
真核细胞mRNA的典型结构是具有5'末端的m7G帽和3'末端的poly(A)尾,这两个组分在mRNA的翻译和稳定性维持中发挥了重要作用,帽依赖性扫描机制是绝大多数拥有m7G帽和poly(A)尾的真核细胞mRNA进行翻译起始的经典方式。基于T7 RNA聚合酶(T7RNApolymerase,T7 RNAP)的胞质表达系统在哺乳动物细胞中所转录的mRNA无5'm7G帽结构,需引入内部核糖体进入位点(Internal ribosome entry site,IRES)元件以增强翻译功能实现蛋白表达。胞质表达系统无需质粒进入细胞核即可表达,可显著提高有效转染效率。然而,IRES介导的翻译水平远低于5'm7G。2019年, PH等(Nucleic Acids Res.2019)在T7RNAP的N端增加了非洲猪瘟病毒来源的NP868R加帽酶获得具有转录、加帽双功能的T7RNAP,基于双功能T7 RNAP的胞质表达系统(C3P3)无需借助IRES介导翻译,显示了优异的蛋白表达能力。然而,C3P3系统仍需要细胞核启动子表达双功能T7 RNAP,这样会导致其胞质中的含量可能未满足外源基因转录需求,存在提升空间。
发明内容
基于此,本发明的目的是提供增强型胞质表达系统、包括该表达系统的表达质粒及其在增强胞质表达外源基因中的应用。
本发明的第一个方面,是提供一种胞质表达系统,其为在T7 RNAP的胞质表达系统中的T7 RNAP编码框的上游与细胞核启动子的下游之间插入有T7启动子的序列。
在其中一些实施例中,所述的胞质表达系统从5’至3’依次包括以下元件的序列:细胞核启动子,T7启动子,非洲猪瘟病毒的加帽酶,T7 RNA聚合酶,bGHpA,poly A尾,核酶,T7终止子。
在其中的一些实施例中,所述T7启动子的序列是taatacgactcactatagg。
在其中一些实施例中,所述细胞核启动子选自CMV或CAG中的至少一种。
本发明的第二个方面,是提供一种胞质表达质粒patC3P3cag,其为表达质粒中插入有任一种上述胞质表达系统。
所述胞质表达质粒patC3P3cag,是携带双功能T7 RNAP的胞质表达系统(C3P3),且在在T7 RNAP编码框的上游与细胞核启动子的下游之间插入有T7启动子的序列。
本发明的第三个方面,是提供了所述胞质表达质粒patC3P3cag的制备方法,所述制备方法包括以下步骤:
S1、获得携带T7启动子和HDVRz-T7终止子片段的框架序列,所述框架序列如SEQID NO:1所示;
S2、获得携带CAG启动子编码基因的片段1,所述片段1的序列如SEQ ID NO:3所示,与所述框架序列通过重组连接,获得连接片段S2;
S3、构建patC3P3cag质粒:获得携带NP868R编码基因的片段2,所述片段2的序列如SEQ ID NO:4所示,将所述连接片段S2与所述片段2通过重组连接,重组连接产物转化感受态细胞,培养过夜,获得patC3P3cag质粒。
在其中一些实施例中,S1中包括以下步骤:以携带T7启动子和“HDVRz-T7终止子片段的质粒为模板,进行PCR扩增,所述扩增引物的序列如SEQ ID NO:8、SEQ ID NO:9所示,获得扩增产物;将如SEQ ID NO:2所示的T7RNAP编码基因重组连接,转化到感受态细胞,培养,酶切,即得所述框架序列。
在其中一些实施例中,上述步骤S2中的CAG启动子编码基因的片段序列如SEQ IDNO:3所示,所述CAG启动子编码基因的片段以pCAG-Puro-EGFP质粒为模板PCR扩增得到,所用引物对序列如SEQ ID NO:10、SEQ ID NO:11所示。
在其中一些实施例中,上述步骤S3中的NP868R编码基因的片段序列如SEQ ID NO:4所示,所述NP868R编码基因的片段以pUC57-NP868R质粒为模板PCR扩增得到,所用引物对序列如SEQ ID NO:12、SEQ ID NO:13所示。
本发明的第四个方面,是提供上述任一项所述的胞质表达系统或胞质表达质粒patC3P3cag在细胞质中进行外源基因中的应用,
或提供上述任一项所述的胞质表达系统或胞质表达质粒patC3P3cag在细胞质中增强表达外源蛋白中的应用。
本发明的第五个方面,是提供一种在细胞质中增强表达外源蛋白的方法,包括将上述胞质表达质粒patC3P3cag与外源蛋白的表达载体共转染至表达细胞,在表达细胞的细胞质中进行外源细胞的转录和表达。
本发明所述胞质表达系统通过巧妙地在T7 RNAP编码框的上游与细胞核启动子的下游之间插入有T7启动子的序列,由此为T7 RNAP的胞质表达系统引入自扩增机制,借助细胞核启动子,如CMV,CAG等驱动T7 RNAP的胞质表达系统的表达。我们发现,T7 RNAP的胞质表达系统对胞质中带有T7启动子的外源蛋白表达载体进行转录产生携带5'帽子结构的mRNA,能够实现对外源蛋白表达促进;同时,T7 RNAP的胞质表达系统还可识别胞质中存在的自身表达载体中的T7启动子,以实现对自身表达载体进行正反馈的转录和表达,从而提高其表达量,继而显著地增强外源蛋白的表达水平。
附图说明
图1为AutoC3P3系统的工作原理示意图,其中,CAGp:CAG启动子;T7p:T7启动子;T7t:T7终止子;NP868R:非洲猪瘟病毒的加帽酶;T7RNAP:T7RNA聚合酶;pA:poly A尾;Rbz:核酶。
图2为AutoC3P3系统的功能验证,其中,(A)质粒共转染24h的荧光显微镜照片;(B)质粒共转染24h的Gluc活性检测结果。
具体实施方式
为了便于理解本发明,下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。
下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Green和Sambrook主编的第四版《分子克隆实验指南》(Molecular Cloning:A Laboratory Manual)已于2013年出版,或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂,均为市售产品。
除非另有定义,本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
以下结合具体实施例对本发明作进一步详细的说明。
实施例1
1.胞质表达系统载体的构建
(1)构建patT7RNAP质粒:以实验保存的pT7-IRES-Nvsv质粒为模板PCR扩增(DNA聚合酶货号:P515-01,诺唯赞生物科技有限公司)携带T7启动子(taatacgactcactatagg,SEQID NO:14)和“HDVRz-T7终止子”片段的框架(如SEQ ID NO:1所示),所用引物对为F1(SEQID NO:6):gggtcggcatggcatctcca,R1(SEQ ID NO:7):cctatagtgagtcgtattaCTCGAGgcgatcgcatctctggaagatccgc;采用50μL扩增体系,质粒模板用量为10ng,反应退火温度为50℃,延伸时间为2min。以pcDNA4TO-optT7RNAP为模板PCR扩增T7RNAP编码基因的片段(如SEQ IDNO:2所示),所用引物对为F2(SEQ ID NO:8):
GtaatacgactcactatagggactcccgggctggcagcagggccccagcggcaccATGGGATCCatgaacaccatcaacatag,R2(SEQ ID NO:9):
tggagatgccatgccgacccTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTtgcgatgcaatttcctca;采用50μL扩增体系,质粒模板用量为10ng,反应退火温度为50℃,延伸时间为2min。将框架和片段用重组连接试剂盒(货号:C112,诺唯赞生物科技有限公司)连接。重组连接产物转化Top10感受态细胞,涂布到氨苄抗性的固体LB平板,37℃过夜培养,挑取单克隆到氨苄抗性的液体LB培养基内,37℃,200rmp震荡培养过夜,提质粒酶切鉴定,送测序分析,获得正确的patT7RNAP质粒。
SEQ ID NO:1为:
gggtcggcatggcatctccacctcctcgcggtccgacctgggcatccgaaggaggacgcacgtccactcggatggctaagggagagccact
agcataaccccttggggcctctaaacgggtcttgaggggttttttggcgatcgcatcgtcgaacggcaggcgtgcaaacttggcgtaatcatggt
catagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagt
gagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgg
ggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcac
tcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgta
aaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccga
caggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttct
cccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacc
ccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccact
ggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttg
gtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtt
tgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacg
ttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaa
cttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagat
aactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaac
cagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagta
gttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttccc
aacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgca
gtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcatt
ctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatca
ttggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagc
atcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaata
ctcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaatgggg
gttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccct
ttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggag
cagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgc
accatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaag
ggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttccca
gtcacgacgttgtaaaacgacggccagtgaattagaactcggtacgcgcggatcttccagagatgcgatcgcCTCGAGtaatacgactcactatagg(2870bp);
SEQ ID NO:2为:
GtaatacgactcactatagggactcccgggctggcagcagggccccagcggcaccATGGGATCCatgaacaccatcaacatagcca
agaatgatttctctgacattgaactggccgccatccctttcaacaccctggctgaccactacggggagagactcgccagggaacagttggccct
ggaacatgagtcttacgagatgggagaggcgcgcttcagaaagatgtttgaaaggcagcttaaggccggggaggtggcagacaacgctgcc
gccaagcctttgatcacgaccctgctgcccaaaatgattgcaagaatcaatgattggtttgaggaggtgaaagctaaacgcggcaagaggccaa
cagccttccagttcttacaggagatcaagccagaagccgtcgcctacatcacaatcaaaacgacgctggcctgcctcacctccgccgacaaca
caaccgtgcaggccgtggcctccgccataggccgggccatagaagatgaggccagattcggccggatcagggatctggaggctaagcatttc
aagaaaaacgtggaagagcagctcaacaagcgggtcggacacgtgtataagaaggcatttatgcaggtagtggaggcggatatgctcagcaa
aggcctgctgggcggcgaggcctggtcctcctggcacaaagaggacagcatccacgtgggtgtccgctgcattgaaatgcttattgaaagtac
aggcatggtgagccttcacagacagaatgctggggtggtcggccaagattcagaaactatcgagctcgccccagagtatgctgaggccatcgc
tacaagagccggggctctggccggaattagccccatgtttcagccttgtgttgtgccccccaagccctggactggaatcactggtggcggctatt
gggcgaacggaagaagacctctggccctggtgcgcacccacagcaaaaaggccctgatgaggtacgaagatgtctacatgccagaggtata
caaagctattaatatagcccagaacacagcctggaaaatcaacaagaaggtgttagctgtggctaatgtcatcacaaaatggaagcactgccca
gtggaggacatccctgccatagagcgggaggagctcccaatgaaaccagaagacatcgacatgaatcctgaagcactgaccgcctggaaga
gagcagcagctgctgtgtacagaaaggataaggcgagaaagagtcggcgcatcagcttagagttcatgttagaacaagctaataagttcgcca
accacaaggcgatctggtttccttacaacatggactggcgcggcagagtgtacgcggtcagcatgttcaacccccagggaaatgacatgacca
agggccttctgactctggccaaggggaaacctatcggaaaggagggctattactggctgaaaatccacggagccaactgtgccggcgtggata
aagttcctttcccagagcgaatcaagttcatagaagagaaccatgagaatatcatggcttgcgcgaagtcaccgctggaaaatacttggtgggcg
gagcaggactctcccttctgcttcttagccttctgttttgaatatgcgggtgtccagcaccatggcctgagctataactgcagcttgcctctggccttt
gatggctcttgcagcggcattcagcacttcagtgccatgctacgagatgaagtaggcgggcgggcagtgaatctgttgccctctgaaacagtcc
aagatatttacggtattgtggccaaaaaggtgaatgaaatcctacaggctgatgctattaacggaaccgacaatgaggtggtgactgtcactgatg
aaaacactggcgaaatctccgaaaaggttaagctgggcaccaaggctctggcaggccagtggcttgcctatggagtgactagatcggtgacca
agcgtagcgtgatgactctggcctacggcagcaaggaatttggcttccgccaacaggtgcttgaggataccattcaacccgccattgacagcgg
aaagggcctcatgttcacacagcccaaccaagccgccggatacatggccaagctcatctgggagagcgtatctgtcacggtggtggccgccgt
ggaggcaatgaactggctgaagagcgctgctaagctcctggcagctgaagtgaaggacaagaaaaccggagaaatcctgagaaaaaggtgt
gctgttcactgggtgactcctgacgggttccctgtttggcaggagtacaagaagcccatccagacaagactaaatctcatgttcctgggtcaattc
cggctgcagccaaccattaacaccaacaaagacagtgagattgatgcccataagcaggaaagtggtatcgcgcctaactttgtgcactcccagg
acggctcccatctgagaaagacagtcgtgtgggcccacgagaaatatggcatcgagagcttcgccctgatccatgactccttcgggaccatccc
agctgacgccgccaacctcttcaaagcagtgagagaaaccatggtggacacatacgaaagctgtgacgtgctggccgatttttatgaccagtttg
ctgatcaactgcacgaaagtcagctggacaaaatgcctgccctgcctgccaaaggaaacctgaatcttagagacattttggagagtgacttcgcc
ttcgcctgataatctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgcc
ttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcaAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAgggtcggcatggcatctcca(2990bp)。
(2)构建patT7RNAPcag质粒:XhoⅠ单酶切patT7RNAP质粒获得框架,其中酶切体系为20μL,酶用量为1μL,质粒用量为500-1000ng,37℃孵育2h,以pCAG-Puro-EGFP质粒为模板PCR扩增携带CAG启动子编码基因的片段(如SEQ ID NO:3所示),所用引物对为F3(SEQ IDNO:10):cagagatgcgatcgc CTCGAGATccgttacataacttacgg,R3(SEQ ID NO:11):tagtgagtcgtattaCTCGAGggtgg caccggtccaacctg;采用50μL扩增体系,质粒模板用量为10ng,反应退火温度为50℃,延伸时间为2min。将框架和片段通过同源重组酶重组连接。重组连接产物转化Top10感受态细胞,涂布到氨苄抗性的固体LB平板,37℃过夜培养,挑取单克隆到氨苄抗性的液体LB培养基内,37℃,200rmp震荡培养过夜,提质粒酶切鉴定,送测序分析,获得正确的patT7RNAPcag质粒。
SEQ ID NO:3为:
cagagatgcgatcgcCTCGAGATccgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgt
caatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgcc
aagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagtacatgaccttatgggactttcctacttggcagtacatc
tacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttat
ttattttttaattattttgtgcagcgatgggggcggggggggggggggggcggggcgaggggcggggcggggcgaggcggagaggtgcgg
cggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggc
gggcgggagtcgctgcgcgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactc
ccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcaggttggaccggtgccaccCTCGAGtaatacgactcacta(834bp)。
(3)构建patC3P3cag质粒:BamHI单酶切patT7RNAPcag质粒获得框架,酶切体系为20μL,酶用量为1μL,质粒用量为500-1000ng,37℃孵育2h。以pUC57-NP868R质粒为模板PCR扩增携带NP868R编码基因的片段(如SEQ ID NO:4所示),所用引物对为F4(SEQ ID NO:12):cccagcggcaccATGGGATC CATGGCTAGCCTGGACAACCTG,R5(SEQ ID NO:13):gttgatggtgttcatGGATCC GCCCCCGCCGCTGCCGCCCCCGCCGTTCTTTCTCAGGC;采用50μL扩增体系,质粒模板用量为10ng,反应退火温度为50℃,延伸时间为2min。将框架和片段通过同源重组酶重组连接。重组连接产物转化Top10感受态细胞,涂布到氨苄抗性的固体LB平板,37℃过夜培养,挑取单克隆到氨苄抗性的液体LB培养基内,37℃,200rmp震荡培养过夜,提质粒酶切鉴定,送测序分析,获得正确的patC3P3cag质粒。
SEQ ID NO:4为:
cccagcggcaccATGGGATCCATGGCTAGCCTGGACAACCTGGTGGCTAGATATCAGAGATGC
TTCAACGATCAGAGCCTGAAGAACAGCACCATCGAGCTGGAGATCAGATTTCAGCAGAT
CAACTTCCTGCTGTTCAAGACCGTGTACGAGGCCCTGGTGGCCCAAGAGATCCCTAGCA
CCATCAGCCACAGCATCAGATGCATCAAGAAGGTGCACCATGAGAACCACTGCAGAGA
GAAGATCCTGCCTAGCGAGAACCTGTACTTCAAGAAGCAGCCCCTGATGTTCTTCAAGT
TCAGCGAGCCCGCTAGCCTGGGCTGCAAGGTGAGCCTGGCCATCGAGCAGCCCATCAG
AAAGTTCATCCTGGACAGCAGCGTGCTGGTGAGACTGAAGAACAGAACCACCTTCAGA
GTGAGCGAGCTGTGGAAGATCGAGCTGACCATCGTGAAGCAGCTGATGGGCAGCGAGG
TGAGCGCCAAGCTGGCCGCCTTCAAGACCCTGCTGTTCGACACCCCCGAGCAGCAGAC
CACCAAGAACATGATGACCCTGATCAACCCCGACGACGAGTACCTGTACGAGATCGAGA
TCGAGTACACCGGCAAGCCCGAGAGCCTGACCGCCGCCGACGTGATCAAGATCAAGAA
CACCGTGCTGACCCTGATCTCCCCCAACCACCTGATGCTGACCGCCTACCACCAAGCCA
TCGAGTTCATCGCTAGCCACATCCTGAGCAGCGAGATCCTGCTGGCTAGAATCAAGAGC
GGCAAGTGGGGCCTGAAGAGACTGCTGCCCCAAGTGAAGAGCATGACCAAGGCCGACT
ACATGAAGTTCTACCCCCCCGTGGGCTACTACGTGACCGACAAGGCCGACGGCATCAGA
GGCATCGCCGTGATCCAAGACACACAGATCTACGTGGTGGCCGATCAGCTGTACAGCCT
GGGCACCACCGGCATCGAGCCCCTGAAGCCCACCATCCTGGACGGCGAGTTCATGCCC
GAGAAGAAGGAGTTCTACGGCTTCGACGTGATCATGTACGAGGGCAACCTGCTGACAC
AGCAAGGCTTCGAGACAAGAATCGAGAGCCTGAGCAAGGGCATCAAGGTGCTGCAAG
CCTTCAACATCAAGGCCGAGATGAAGCCCTTCATCAGCCTGACAAGCGCCGACCCCAAC
GTGCTGCTGAAGAACTTCGAGAGCATCTTCAAGAAAAAGACAAGACCCTACAGCATCG
ACGGCATCATCCTGGTGGAGCCCGGCAACAGCTACCTGAACACCAACACCTTCAAGTG
GAAGCCCACCTGGGACAACACCCTGGACTTCCTGGTGAGAAAGTGCCCCGAAAGCCTG
AACGTGCCCGAGTACGCCCCCAAGAAGGGCTTCAGCCTCCATCTGCTGTTCGTGGGCAT
CAGCGGCGAGCTGTTCAAGAAGCTGGCCCTGAACTGGTGCCCCGGCTACACCAAGCTG
TTCCCCGTGACACAGAGAAATCAGAACTACTTCCCCGTGCAGTTTCAGCCTAGCGACTT
CCCCCTGGCCTTCCTGTACTACCACCCCGACACAAGCAGCTTCAGCAACATCGACGGCA
AGGTGCTGGAGATGAGATGCCTGAAGAGAGAGATCAACTACGTGAGATGGGAGATCGT
GAAGATCAGAGAGGACAGACAGCAAGACCTGAAGACCGGCGGCTACTTCGGGAACGA
TTTCAAGACCGCCGAACTGACCTGGCTGAACTACATGGACCCCTTCAGCTTCGAGGAGC
TGGCCAAGGGCCCTAGCGGCATGTACTTCGCCGGCGCCAAGACCGGCATCTACAGAGCT
CAGACAGCCCTGATCTCCTTCATCAAGCAAGAGATCATTCAGAAGATCAGCCATCAGAG
CTGGGTGATCGACCTGGGCATCGGCAAGGGCCAAGACCTGGGCAGATACCTGGACGCC
GGCGTGAGACACCTGGTGGGCATCGACAAGGATCAGACCGCCCTGGCCGAACTGGTGT
ACAGAAAGTTCAGCCACGCCACCACAAGACAGCACAAGCACGCCACCAACATCTACGT
GCTGCACCAAGACCTGGCCGAGCCCGCCAAGGAGATCAGCGAGAAGGTCCACCAAATC
TACGGCTTCCCCAAGGAGGGCGCTAGCAGCATCGTGAGCAACCTGTTCATCCACTACCT
GATGAAGAACACACAGCAAGTGGAGAACCTGGCCGTGCTGTGCCACAAGCTGCTGCAG
CCCGGCGGCATGGTGTGGTTCACCACCATGCTGGGCGAGCAAGTGCTGGAGCTGCTGC
ACGAGAACAGAATCGAGCTGAACGAGGTGTGGGAGGCTAGAGAGAACGAGGTGGTGA
AGTTCGCCATCAAGAGACTGTTCAAGGAGGACATCCTGCAAGAGACCGGCCAAGAGAT
CGGCGTGCTGCTGCCCTTCAGCAACGGCGACTTCTACAACGAGTACCTGGTGAACACCG
CCTTCCTGATCAAGATCTTCAAGCACCACGGCTTCAGCCTGGTGCAAAAGCAGAGCTTC
AAGGACTGGATTCCCGAGTTTCAGAACTTCTCCAAGTCCCTGTACAAGATCCTGACCGA
GGCCGACAAGACCTGGACAAGCCTGTTCGGCTTCATCTGCCTGAGAAAGAACGGCGGGGGCGGCAGCGGCGGGGGCGGATCCatgaacaccatcaac(2670bp)。
2.胞质表达系统AutoC3P3的功能性验证
(1)实验设计:使用pgluGFPpsT7质粒(其序列如SEQ ID NO:5所示)作为外源基因表达载体进行功能验证。pgluGFPpsT7采用T7启动子驱动高斯荧光素酶(Gluc)和绿色荧光蛋白(GFP)的表达,缺乏细胞核表达启动子,只能依赖T7RNA聚合酶进行转录。通过与patC3P3cag共转染细胞,测得Gluc的活性和GFP的荧光强度可便捷地评估AutoC3P3介导的外源基因表达水平。patC3P3cag质粒转染后部分进入细胞核,部分滞留在细胞质。细胞核内的patC3P3cag质粒在CAG启动子驱动下转录出NP868R-T7 RNAP的编码mRNA,随后该mRNA出核在胞质中翻译成NP868R-T7 RNAP融合蛋白。胞质中的NP868R-T7 RNAP识别胞质中滞留patC3P3cag质粒的T7启动子,形成正反馈回路持续驱动自身的表达。同时NP868R-T7 RNAP识别胞质中pgluGFPpsT7质粒的T7启动子,驱动报告基因的表达(图1)。
SEQ ID NO:5为:
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagac
aagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccata
tgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgat
cggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacg
acgttgtaaaacgacggccagtgaattagaactcggtacgcgcggatcttccagagatgcgatcgcCTCGAGtaatacgactcactatagg
gaATGGGATCCATGgtgaatggcgtgaaggtgctgtttgccctgatctgcattgctgtggctgaggccaagcccacagagaacaatg
aggacttcaacattgtggctgtggcctccaactttgccaccaccgacctggatgccgacaggggcaagctgcctggcaagaagctgcccctgg
aggtgctgaaggagatggaggccaatgccaggaaggctggctgcaccaggggctgcctgatctgcctgtcccacatcaagtgcacccccaa
gatgaagaagttcatccctggccggtgccacacctatgagggcgacaaggagtctgcccagggcggcattggcgaggccattgtggacatcc
ctgagatccctggcttcaaggacctggagcccatggagcagttcattgcccaggtggacctgtgtgtggactgcaccaccggctgcctgaagg
gcctggccaatgtgcagtgctctgacctgctgaagaagtggctgccccagagatgtgccacctttgcctccaaaatccagggccaggtggaca
agatcaagggcgctggcggcgatgacaccggatccggcgccaccaacttctccctgctgaagcaggctggcgatgtggaggagaaccctgg
gcccATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT
GGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATA
CGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAAC
ACTTGTCACTACTTTCACTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAA
CGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTT
TTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCC
TTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGAC
ACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAG
AATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACT
AGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAA
CCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACA
TGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACA
AATAGTAAACGCGTTTATAAgtctagagggcccgtttaaacccgctgatcagcctcgactagcataaccccttggggcctctaa
acgggtcttgaggggttttttggcgatcgcatcgtcgaacggcaggcgtgcaaacttggcgtaatcatggtcatagctgtttcctgtgtgaaattgtt
atccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttg
cgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggc
gctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatcca
cagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgttttt
ccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgt
ttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttct
catagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgc
cttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgagg
tatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagtta
ccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaa
aaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatca
aaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaat
cagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccat
ctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcg
cagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacg
ttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcc
cccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcac
tgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccga
gttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaa
ctctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggt
gagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattga
agcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaatgggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc(4195bp)。
(2)实验方案:将pgluGFPpsT7报告载体分别同AutoC3P3载体(patC3P3cag)和C3P3载体(pC3P3cag)共转染293T细胞,采用Lipo8000转染试剂(货号:C0533-1.5mL,碧云天生物科技有限公司),24h后比较报告基因的表达水平。
(3)实验步骤:将生长状态良好的293T细胞铺至12孔板过夜培养,待细胞汇合度达80-90%后,使用Lipo8000进行质粒共转,设置单独报告质粒转染组,每组三个复孔。即共分成3个组别进行,每孔的转染质粒组成分别为a.转染patC3P3cag质粒2μg和pgluGFPpsT7质粒1μg共转、b.pC3P3cag质粒2μg和pgluGFPpsT7质粒1μg共转,及c.单独pgluGFPpsT7质粒组1μg。转染并培养24h,收集细胞培养上清使用荧光照度计检测Gluc活性,即按组分别取50μL细胞上清和稀释的腔肠素混匀后使用荧光照度计检测Gluc活性并读取数值,在荧光显微镜下观察并拍照记录GFP的表达情况。
(4)实验结果:patC3P3cag质粒共转组的GFP阳性细胞数量(图2A)及Gluc表达量(图2B)均优于pC3P3cag质粒共转组,说明相同实验条件下,AutoC3P3介导的报告基因表达能力明显由于C3P3系统。自扩增机制的引入可显著增强C3P3系统的外源基因表达能力。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种胞质表达系统,其特征在于,在T7 RNAP的胞质表达系统中的T7RNAP编码框的上游与细胞核启动子的下游之间插入有T7启动子的序列。
2.根据权利要求1所述的胞质表达系统,其特征在于,所述胞质表达系统从5’至3’依次包括以下元件的序列:细胞核启动子,T7启动子,非洲猪瘟病毒的加帽酶,T7 RNA聚合酶,bGHpA,poly A尾,核酶,T7终止子。
3.根据权利要求2所述的胞质表达系统,所述细胞核启动子选自CMV或CAG中的至少一种。
4.一种胞质表达质粒patC3P3cag,其特征在于,所述胞质表达质粒patC3P3cag为表达质粒中插入有权利要求1-3任一项所述的胞质表达系统。
5.权利要求4所述的胞质表达质粒patC3P3cag的制备方法,其特征在于,所述制备方法步骤包括如下:
S1、获得携带T7启动子和HDVRz-T7终止子片段的框架序列,所述框架序列如SEQ IDNO:1所示;
S2、获得携带CAG启动子编码基因的片段1,所述片段1的序列如SEQ ID NO:3所示,与所述框架序列通过重组连接,获得连接片段S2;
S3、构建patC3P3cag质粒:获得携带NP868R编码基因的片段2,所述片段2的序列如SEQID NO:4所示,将所述连接片段S2与所述片段2通过重组连接,重组连接产物转化感受态细胞,培养过夜,获得patC3P3cag质粒。
6.根据权利要求5所述的制备方法,其特征在于,S1中包括以下步骤:以携带T7启动子和“HDVRz-T7终止子片段的质粒为模板,进行PCR扩增,所述扩增引物的序列如SEQ ID NO:8、SEQ ID NO:9所示,获得扩增产物;将如SEQ ID NO:2所示的T7RNAP编码基因重组连接,转化到感受态细胞,培养,酶切,即得所述框架序列。
7.根据权利要求5所述的制备方法,其特征在于,所述感受态细胞是Top10感受态细胞。
8.权利要求1-3任一项所述的胞质表达系统或权利要求4所述的胞质表达质粒patC3P3cag在细胞质中进行外源基因中的应用。
9.权利要求1-3任一项所述的胞质表达系统或权利要求4所述的胞质表达质粒patC3P3cag在细胞质中增强表达外源蛋白中的应用。
10.一种在细胞质中增强表达外源蛋白的方法,其特征在于,包括将权利要求4所述胞质表达质粒patC3P3cag与外源蛋白的表达载体共转染至表达细胞,在表达细胞的细胞质中进行外源细胞的转录和表达。
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