《厦门大学学报（自然科学版）》

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.

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.

引言
1 材料与方法
2 结果与分析
3 讨论

图2 NLS-eGFP-P2A-mCherry-CAAX mRNA注射后在文昌鱼胚胎中的表达<br/>Fig.2 NLS-eGFP-P2A-mCherry-CAAX mRNA expression in amphioxus embryos after injection

图2 NLS-eGFP-P2A-mCherry-CAAX mRNA注射后在文昌鱼胚胎中的表达
Fig.2 NLS-eGFP-P2A-mCherry-CAAX mRNA expression in amphioxus embryos after injection

图1 PCR扩增NLS-eGFP-P2A-mCherry-CAAX目的片段<br/>Fig.1 PCR amplification of NLS-eGFP-P2A-mCherry-CAAX

图1 PCR扩增NLS-eGFP-P2A-mCherry-CAAX目的片段
Fig.1 PCR amplification of NLS-eGFP-P2A-mCherry-CAAX

图3 重组eGFP和mCherry蛋白在文昌鱼胚胎细胞中的分布<br/>Fig.3 Distribution of recombinant eGFP and mCherry proteins in amphioxus embryonic cells

图3 重组eGFP和mCherry蛋白在文昌鱼胚胎细胞中的分布
Fig.3 Distribution of recombinant eGFP and mCherry proteins in amphioxus embryonic cells

图4 文昌鱼胚胎8体节时期P2A剪切效率<br/>Fig.4 The efficiency of P2A cleavage in the 8-somite-stage embryos

图4 文昌鱼胚胎8体节时期P2A剪切效率
Fig.4 The efficiency of P2A cleavage in the 8-somite-stage embryos

图5 共聚焦显微镜观察BbHsp70启动的下游荧光蛋白表达<br/>Fig.5 Observation of the fluorescent protein expression initiated by BbHsp70 under a confocal microscope

图5 共聚焦显微镜观察BbHsp70启动的下游荧光蛋白表达
Fig.5 Observation of the fluorescent protein expression initiated by BbHsp70 under a confocal microscope

[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.

查看附录

备注

引言

1 材料与方法

2 结果与分析

3 讨论

学报简介

备注

引言

1 材料与方法

2 结果与分析

3 讨 论

学报简介

3 讨论