杜氏盐藻kinesin家族蛋白dsKIF4 cDNA片段的克隆及其在鞭毛再生中的作用
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摘要
纤毛／鞭毛作为引发多条信号通路活化的重要细胞器,在肿瘤的发生中发挥重要作用,而鞭毛组装和肿瘤发生的分子机制目前还不清楚。鞭毛内运输机制((Intraflagellar transport, IFT)是目前研究比较清楚的鞭毛组装／解聚机制,驱动蛋白kinesin和马达蛋白dynein是鞭毛内运输系统的动力蛋白,它们的异常严重导致鞭毛组装/解聚的异常。而驱动蛋白kinesin家族蛋白在鞭毛组装过程中在鞭毛再生中发挥着重要作用。有研究表明kinesin-2在鞭毛组装过程中与货物蛋白结合沿着微管将鞭毛组分运输至鞭毛顶端。目前kinesin家族蛋白其他成员在鞭毛再生过程中的相关报道还很少。
杜氏盐藻(Dunaliella salina)是一种无细胞壁的单细胞真核绿藻,具有一对等长的鞭毛,长约13μm,具有典型的9+2结构。显微镜下即可观察到其鞭毛长度和运动性的变化。本课题组以无细胞壁具有双鞭毛的单细胞真核生物杜氏盐藻作为模式生物细胞器来研究纤毛在食管鳞癌发生中的作用。本研究的目的是扩增得到一个kinesin家族蛋白,并研究其在鞭毛再生过程中的作用。
方法
(1)通过设计简并引物扩增杜氏盐藻KIF4cDNA片段。
(2)采用5’巢式PCR和3’巢式PCR扩增dsKIF4cDNA片段全长。
(3)采用实时荧光定量PCR方法dsKIF4mRNA在鞭毛再生过程中的变化情况。
(4)构建dsKIF4保守序列的原核表达载体,诱导His-dsKIF4蛋白的表达,经
纯化后制备其抗体。
(5)统计学分析：荧光定量PCR结果分析,采用2-ΔΔCt法对目的基因的表达量(mRNA相对丰度)进行分析,△△Ct=△Ct实验组-△Ct对照组,△Ct目的基因=Ct目的基因-Ct内参基因,每组实验做三个平行,每个样品重复三次。然后应用SPSS18.0软件进行分析,并采用t检验和方差分析。P<0.05表示具有统计学意义。
结果
(1)根据KIF蛋白的保守性设计一对简并引物扩增得到一个720bp的杜氏盐藻KIF4cDNA片段。
(2)5'RACE和3'RACE分别得到一个1134bp和960bp的cDNA片段,经拼接后得到一个长度为2214bp的dsKIF4cDNA片段,编码734个氨基酸,与团藻的InvA Kinesin、衣藻的kinesin family protein以及人的KIF4C的同源性分别为56%,58%和42%。
(3)实时荧光定量PCR显示dsKIF4基因在鞭毛再生过程中表达量显著增高(P<0.05)。
(4)为了制备其抗体,我们选取包含马达结构域的476个氨基酸进行外源重组蛋白的表达。通过3mM IPTG37℃诱导3h得到分子量约为50kDa的重组蛋白,纯化后已制备其抗体。
结论
(1)本研究通过简并引物扩增得到一个长2214bp的杜氏盐藻kinesin家族蛋白KIF4的cDNA片段,编码734个氨基酸。
(2)实时荧光定量PCR显示dsKIF4基因在鞭毛再生过程中表达量显著增高,说明其参与鞭毛的再生过程。
(3)为了制备其抗体,我们选取包含马达结构域的476个氨基酸进行外源重组蛋白的表达。经表达纯化后已用于其抗体制备。
In mammals, most cell types have cilia, protruding structures involved in sensing mechanical and chemical signals from the extracellular environment, that act as major communication hubs for signaling controlling cell differentiation and polarity. It has been proposed that cancer cells with cilia disorder reduce or alter their response to extracellular cues that regulate growth and differentiation. But the machanism of cilia assembly regulation and its function in tumorigenesis are still unclear. Since the intraflagellar transport (IFT) was discovered in the biflagellate green alga Chlamydomonas, the mechanisms of flagellar/ciliary assembly and disassembly have aroused widespread concerns. Flagellar/ciliary assembly and disassembly, which determine the length of the flagella/cilia, require the IFT system composed of the molecular motors, kinesin-2and dynein and IFT particles to supply the necessary substances to the tip of flagella/cilia and to bring flagellar/ciliary turnover products back to the base. It is well known that kinesin-2plays a key role in flagellar/ciliary assembly, however, the role of other kinesin family protein members are still unclear.
Dunaliella salina (D. salina) is a unicellular biflagellate eukaryotic alga without cell wall, which has a pair of same length flagella of13μm. The flagella of D. salina were employed as model organelle to investigate the cilia assemly mechanism. In this study, a kinesin family protein gene KIF4cDNA of D.salina was cloned and its effect on flagellar regeneration was investigated.
Methods
(1) A pair of degenerate primers was designed to clone the kinesin family protein dsKIF4by reverse transcription PCR.
(2)3'and5'RACE nested PCR was employed to obtain the full length of dsKIF4.
(3) The relative abundance of dsKIF4mRNA was detected during flagellar regeneration by real-time PCR.
(4) To investigate the effect of dsKIF4in flagellar regeneration at protein level a prokaryotic expression vector was constructed and the fusion protein was induced by IPTG and it antibody was being prepared after purification of the fusion protein.
Results
(1) A720bp cDNA fragment of D. salina was obtained by reverse transcription PCR using a pair of degenerate primers.
(2) A1134bp and a960bp cDNA fragment were obtained by3'and5'RACE nested PCR respectively and then a2214bp cDNA was obtained after splice, which encoded736amino acids. The induced protein shared high homology with kinesin family protein from other species such as InvA Kinesin from Volvox carteri (56%)> kinesin family protein from Chlamydomonas reinhardtii (58%) and KIF4C from human (42%) and was named dsKIF4as it contained a KIF4motor domain.
(3) The relative abundance of dsKIF4mRNA was increased during flagellar regeneration, demonstrating that dsKIF4might be involved in flagellar regeneration.
(4) The fused protein of dsKIF4was heterogenous expressed in E. coli and the His-dsKIF4protein was expressed with molecular weight of～50kDa. The anti-dsKIF4has been preparing after his-dsKIF4was purified.
Conlusion
A cDNA fragment of720bp was obtained by reverse transcription PCR using a pair of degenerate primers. And then a2214bp cDNA fragement was cloned by3' and5' RACE nested PCR, which encoded736amino acids. The induced protein shared high homology with kinesin family protein from other species and was named dsKIF4as it contained a KIF4motor domain. The relative abundance of dsKIF4mRNA was detected by real-time PCR and the results showed that it was increased during flagellar regeneration, demonstrating that dsKIF4might be involved in flagellar regeneration. In addition, in order to investigate the effect of dsKIF4in flagellar regeneration at protein level, we the fused protein of dsKIF4was heterogenous expressed in E. coli and the His-dsKIF4protein was expressed with molecular weight of～50kDa. The anti-dsKIF4has been preparing after his-dsKIF4was purified.
杜氏盐藻(Dunaliella salina)是一种无细胞壁的单细胞真核绿藻,具有一对等长的鞭毛,长约13μm,具有典型的9+2结构。显微镜下即可观察到其鞭毛长度和运动性的变化。本课题组以无细胞壁具有双鞭毛的单细胞真核生物杜氏盐藻作为模式生物细胞器来研究纤毛在食管鳞癌发生中的作用。本研究的目的是扩增得到一个kinesin家族蛋白,并研究其在鞭毛再生过程中的作用。
方法
(1)通过设计简并引物扩增杜氏盐藻KIF4cDNA片段。
(2)采用5’巢式PCR和3’巢式PCR扩增dsKIF4cDNA片段全长。
(3)采用实时荧光定量PCR方法dsKIF4mRNA在鞭毛再生过程中的变化情况。
(4)构建dsKIF4保守序列的原核表达载体,诱导His-dsKIF4蛋白的表达,经
纯化后制备其抗体。
(5)统计学分析：荧光定量PCR结果分析,采用2-ΔΔCt法对目的基因的表达量(mRNA相对丰度)进行分析,△△Ct=△Ct实验组-△Ct对照组,△Ct目的基因=Ct目的基因-Ct内参基因,每组实验做三个平行,每个样品重复三次。然后应用SPSS18.0软件进行分析,并采用t检验和方差分析。P<0.05表示具有统计学意义。
结果
(1)根据KIF蛋白的保守性设计一对简并引物扩增得到一个720bp的杜氏盐藻KIF4cDNA片段。
(2)5'RACE和3'RACE分别得到一个1134bp和960bp的cDNA片段,经拼接后得到一个长度为2214bp的dsKIF4cDNA片段,编码734个氨基酸,与团藻的InvA Kinesin、衣藻的kinesin family protein以及人的KIF4C的同源性分别为56%,58%和42%。
(3)实时荧光定量PCR显示dsKIF4基因在鞭毛再生过程中表达量显著增高(P<0.05)。
(4)为了制备其抗体,我们选取包含马达结构域的476个氨基酸进行外源重组蛋白的表达。通过3mM IPTG37℃诱导3h得到分子量约为50kDa的重组蛋白,纯化后已制备其抗体。
结论
(1)本研究通过简并引物扩增得到一个长2214bp的杜氏盐藻kinesin家族蛋白KIF4的cDNA片段,编码734个氨基酸。
(2)实时荧光定量PCR显示dsKIF4基因在鞭毛再生过程中表达量显著增高,说明其参与鞭毛的再生过程。
(3)为了制备其抗体,我们选取包含马达结构域的476个氨基酸进行外源重组蛋白的表达。经表达纯化后已用于其抗体制备。
In mammals, most cell types have cilia, protruding structures involved in sensing mechanical and chemical signals from the extracellular environment, that act as major communication hubs for signaling controlling cell differentiation and polarity. It has been proposed that cancer cells with cilia disorder reduce or alter their response to extracellular cues that regulate growth and differentiation. But the machanism of cilia assembly regulation and its function in tumorigenesis are still unclear. Since the intraflagellar transport (IFT) was discovered in the biflagellate green alga Chlamydomonas, the mechanisms of flagellar/ciliary assembly and disassembly have aroused widespread concerns. Flagellar/ciliary assembly and disassembly, which determine the length of the flagella/cilia, require the IFT system composed of the molecular motors, kinesin-2and dynein and IFT particles to supply the necessary substances to the tip of flagella/cilia and to bring flagellar/ciliary turnover products back to the base. It is well known that kinesin-2plays a key role in flagellar/ciliary assembly, however, the role of other kinesin family protein members are still unclear.
Dunaliella salina (D. salina) is a unicellular biflagellate eukaryotic alga without cell wall, which has a pair of same length flagella of13μm. The flagella of D. salina were employed as model organelle to investigate the cilia assemly mechanism. In this study, a kinesin family protein gene KIF4cDNA of D.salina was cloned and its effect on flagellar regeneration was investigated.
Methods
(1) A pair of degenerate primers was designed to clone the kinesin family protein dsKIF4by reverse transcription PCR.
(2)3'and5'RACE nested PCR was employed to obtain the full length of dsKIF4.
(3) The relative abundance of dsKIF4mRNA was detected during flagellar regeneration by real-time PCR.
(4) To investigate the effect of dsKIF4in flagellar regeneration at protein level a prokaryotic expression vector was constructed and the fusion protein was induced by IPTG and it antibody was being prepared after purification of the fusion protein.
Results
(1) A720bp cDNA fragment of D. salina was obtained by reverse transcription PCR using a pair of degenerate primers.
(2) A1134bp and a960bp cDNA fragment were obtained by3'and5'RACE nested PCR respectively and then a2214bp cDNA was obtained after splice, which encoded736amino acids. The induced protein shared high homology with kinesin family protein from other species such as InvA Kinesin from Volvox carteri (56%)> kinesin family protein from Chlamydomonas reinhardtii (58%) and KIF4C from human (42%) and was named dsKIF4as it contained a KIF4motor domain.
(3) The relative abundance of dsKIF4mRNA was increased during flagellar regeneration, demonstrating that dsKIF4might be involved in flagellar regeneration.
(4) The fused protein of dsKIF4was heterogenous expressed in E. coli and the His-dsKIF4protein was expressed with molecular weight of～50kDa. The anti-dsKIF4has been preparing after his-dsKIF4was purified.
Conlusion
A cDNA fragment of720bp was obtained by reverse transcription PCR using a pair of degenerate primers. And then a2214bp cDNA fragement was cloned by3' and5' RACE nested PCR, which encoded736amino acids. The induced protein shared high homology with kinesin family protein from other species and was named dsKIF4as it contained a KIF4motor domain. The relative abundance of dsKIF4mRNA was detected by real-time PCR and the results showed that it was increased during flagellar regeneration, demonstrating that dsKIF4might be involved in flagellar regeneration. In addition, in order to investigate the effect of dsKIF4in flagellar regeneration at protein level, we the fused protein of dsKIF4was heterogenous expressed in E. coli and the His-dsKIF4protein was expressed with molecular weight of～50kDa. The anti-dsKIF4has been preparing after his-dsKIF4was purified.
引文
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