|本期目录/Table of Contents|

[1]华俊豪,李 光,王义权*.文昌鱼中一个2A肽介导的多基因表达载体构建[J].厦门大学学报(自然科学版),2018,57(01):44-49.[doi:10.6043/j.issn.0438-0479.201702023]
 HUA Junhao,LI Guang,WANG Yiquan*.Construction of a 2A Peptide-linked Multicistronic Expression Vector for Amphioxus[J].Journal of Xiamen University(Natural Science),2018,57(01):44-49.[doi:10.6043/j.issn.0438-0479.201702023]
点击复制

文昌鱼中一个2A肽介导的多基因表达载体构建(PDF/HTML)
分享到:

《厦门大学学报(自然科学版)》[ISSN:0438-0479/CN:35-1070/N]

卷:
57卷
期数:
2018年01期
页码:
44-49
栏目:
研究论文
出版日期:
2018-01-26

文章信息/Info

Title:
Construction of a 2A Peptide-linked Multicistronic Expression Vector for Amphioxus
文章编号:
0438-0479(2018)01-0044-06
作者:
华俊豪李 光王义权*
厦门大学 生命科学学院,细胞应激生物学国家重点实验室,福建 厦门 361102
Author(s):
HUA JunhaoLI GuangWANG Yiquan*
State Key Laboratory of Cellular Stress Biology,School of Life Sciences,Xiamen University,Xiamen 361102,China
关键词:
多基因表达载体 2A肽 热激启动子 共表达 文昌鱼
Keywords:
multicistronic expression vector 2A peptide heat-shock promoter co-expression amphioxus
分类号:
Q 782
DOI:
10.6043/j.issn.0438-0479.201702023
文献标志码:
A
摘要:
2A肽(P2A)介导的多基因表达载体具有高裂解活性且上、下游基因等摩尔表达等优点,已广泛应用于动物转基因研究.文昌鱼(amphioxus)作为一种新兴的模式动物,尚无应用这种表达载体的报道,为此在pXT7转录系统基础上构建一个P2A介导的多基因表达载体.将体外转录的P2A介导的mRNA注入文昌鱼卵细胞并受精后,经激光共聚焦显微镜和蛋白质免疫印迹(Western blot)检测表明,该mRNA在文昌鱼胚胎中能够高效地翻译和剪切,并且在信号肽的作用下eGFP蛋白定位于细胞核中,而mCherry蛋白定位于细胞膜上,上、下游蛋白间的剪切效率达到91%; 进而构建了由文昌鱼热激蛋白基因启动子(BbHsp70)启动,并由P2A介导的多基因表达载体,实验证明其在热诱导和上、下游蛋白剪切方面均达到了预期效果.
Abstract:
The multicistronic expression vector mediated by 2A peptide(P2A)possesses advantages such as high cleavage efficiency,equimolar amounts of upstream and downstream gene expression,so that it has been widely applied to animal transgenic research.However,up to date,no multicistronic expression vector is available for the application in amphioxus,a promising new model animal for evolutionary development study,therefore we constructed a multicistronic expression vector harboring P2A based on pXT7 transcription system.After microinjecting mRNA transcribed in vitro into amphioxus eggs and fertilization,we observed the embryos under a laser confocal microscope and analyzed protein expression using Western blot assay.The results showed that the mRNA was translated and the P2A mediated protein was cleaved effectively in amphioxus embryos.Moreover,the eGFP protein was targeted to nucleus and mCherry protein to cell membrane under the role of corresponding signal peptides.The separation efficiency of the two proteins was about 91%.Furthermore,we constructed a heat-shock protein gene promoter(BbHsp70)mediated P2A multicistronic expression vector,and the experiments showed the efficiency of gene expression and protein separation achieved the desired results.The multicistronic expression vector mediated by 2A peptide(P2A)possesses advantages such as high cleavage efficiency,equimolar amounts of upstream and downstream gene expression,so that it has been widely applied to animal transgenic research.However,up to date,no multicistronic expression vector is available for the application in amphioxus,a promising new model animal for evolutionary development study,therefore we constructed a multicistronic expression vector harboring P2A based on pXT7 transcription system.After microinjecting mRNA transcribed in vitro into amphioxus eggs and fertilization,we observed the embryos under a laser confocal microscope and analyzed protein expression using Western blot assay.The results showed that the mRNA was translated and the P2A mediated protein was cleaved effectively in amphioxus embryos.Moreover,the eGFP protein was targeted to nucleus and mCherry protein to cell membrane under the role of corresponding signal peptides.The separation efficiency of the two proteins was about 91%.Furthermore,we constructed a heat-shock protein gene promoter(BbHsp70)mediated P2A multicistronic expression vector,and the experiments showed the efficiency of gene expression and protein separation achieved the desired results.

参考文献/References:

[1] CHINNASAMY D,MILSOM M D,SHAFFER J,et al.Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI[J].Virology Journal,2006,3(1):1-16.
[2] KOMAR A A,HATZOGLOU M.Internal ribosome entry sites in cellular mRNAs:the mystery of their exis-tence[J].Journal of Biological Chemistry,2005,280(5):23425.
[3] SZYMCZAKWORKMAN A L,VIGNALI K M,VIGNALI D A.Design and construction of 2A peptide-linked multicistronic vectors[J].Cold Spring Harbor Protocols,2012,2012(2):199-204.
[4] DONNELLY M L L,LUKE G,MEHROTRA A,et al.Analysis of the aphthovirus 2A/2B polyprotein "cleavage" mechanism indicates not a proteolytic reaction,but a novel translational effect:a putative ribosomal "skip"[J].Journal of General Virology,2001,82(5):1013-1025.
[5] YAN J,WANG H,XU Q,et al.Signal sequence is still required in genes downstream of "autocleaving" 2A peptide for secretary or membrane-anchored expression[J].Analytical Biochemistry,2010,399(1):144-146.
[6] BERTRAND S,ESCRIVA H.Evolutionary crossroads in developmental biology:amphioxus[J].Development,2011,138(22):4819-4830.
[7] ZHANG Q J,SUN Y,ZHONG J,et al.Continuous culture of two lancelets and production of the second filial generations in the laboratory[J].J Exp Zool B Mol Dev Evol,2007,308(4):464-472.
[8] LI G,YANG X,SHU Z H,et al.Consecutive spawnings of Chinese amphioxus,Branchiostoma belcheri,in captivity[J].PLoS One,2012,7(12):e50838.
[9] LIU X,LI G,FENG J,et al.An efficient microinjection method for unfertilized eggs of Asian amphioxus Branchiostoma belcheri[J].Development Genes and Evolution,2013,223(4):269-278.
[10] KIM J H,LEE S R,LI L H,et al.High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines,zebrafish and mice[J].PLoS One,2011,6(4):e18556.
[11] LI G,SHU Z,WANG Y.Year-round reproduction and induced spawning of Chinese amphioxus,Branchiostoma belcheri,in laboratory[J].PLoS One,2013,8(8):e75461.
[12] LI G,ZHANG Q J,ZHONG J,et al.Evolutionary and functional diversity of green fluorescent proteins in cephalochordates[J].Gene,2009,446(1):41-49.
[13] LI D,LI G,WANG K,et al.Isolation and functional analysis of the promoter of the amphioxus Hsp70a gene[J].Gene,2012,510(1):39-46.
[14] 王慧,李光,王义权.文昌鱼Hedgehog基因敲除和突变体表型分析[J].遗传,2015,37(10):1036-1043.
[15] KOZMIKOVA I,KOZMIK Z.Gene regulation in amphioxus:an insight from transgenic studies in amphioxus and vertebrates[J].Marine Genomics,2015,24:159-166.
[16] KERRIGAN J J,XIE Q,AMES R S,et al.Production of protein complexes via co-expression[J].Protein Expression and Purification,2011,75(1):1-14.
[17] TIAN Y,LI W,WANG L,et al.Expression of 2A peptide mediated tri-fluorescent protein genes were re-gulated by epigenetics in transgenic sheep[J].Biochemical and Biophysical Research Communications,2013,434(3):681-687.

备注/Memo

备注/Memo:
收稿日期:2017-02-14 录用日期:2017-05-03
基金项目:国家自然科学基金(31372188,31471986,31672246)
*通信作者:wangyq@xmu.edu.cn
更新日期/Last Update: 1900-01-01