CDK2蛋白质分子结构与其入核转运过程关系的初步分析

应用重组技术构建野生型及缺失型CDK2基因的真核表达载体,分别使野生型及缺失型(2DK2蛋白与增强型绿色荧光蛋白(Enhanced-green Fluorescent Protein,EGFP)形成融合蛋白。通过脂质体介导的方法将载体转染人宫颈癌细胞系HeLa和中华仓鼠卵巢细胞系CHO,经过细胞周期同步化处理后于荧光显微镜下观察EGFP的亚细胞定位以示踪野生型及缺失型CDK2基因的表达。结果表明,野生型CDK2基因的表达产物定位于细胞核．而两种缺失型CDK2基因分别编码的CDK2蛋白N-端1～201及98～298多肽均主要定位于细胞质。以上结果提示,CDK2蛋白序列中不含有与核定位直接相关的信号,其入核过程可能是由其N-端1～97及202～298多肽范围内的部分氨基酸共同形成高级结构,并依赖此高级结构与其他含有入核信号的蛋白形成复合物,从而被带动进入细胞核的     

核基质结合区的位置对转基因表达的影响

为研究核基质结合区 (MAR）序列不同插入位置对转基因表达作用的影响,PCR扩增人β 珠蛋白MAR分别插入到含氯霉素乙酰转移酶（chloramphenicol acetyltransferase,CAT）报告基因真核表达载体pCATG表达盒两侧、5′端及3′端.酶切鉴定后,用阳离子聚合物转染CHO细胞,G418筛选出阳性细胞克隆,ELISA分析CAT基因的表达水平,半定量PCR分析CAT基因相对拷贝数.结果表明,表达盒两侧含MAR序列的载体能提高介导的转基因表达水平平均提高10.4倍,5′端含MAR序列的载体表达水平平均提高3.9倍,3′端含MAR序列的载体反而降低转基因表达水平.5′端含MAR序列的表达载体其转基因相对拷贝数高于其它两组载体的基因拷贝数,转基因表达量与基因拷贝数不成正比.     

人SDCT2蛋白亚细胞定位信号分析

为了确定人高亲和力钠离子依赖性二羧酸共转运蛋白（high-affinity sodium-dependent dicarboxylate co-transporter, SDCT2,NaDC3）在细胞内的定位,构建了SDCT2与增强型绿色荧光蛋白（EGFP）的融合蛋白表达载体,并转染肾小管上皮细胞LLC-PK1,激光共聚焦显微镜观察显示,SDCT2蛋白主要定位于细胞的基底侧膜上.同时将SDCT2-EGFP融合基因mRNA显微注射到爪蟾卵母细胞中表达,可见融合蛋白的绿色荧光仅分布在细胞膜上.为了进一步确定该蛋白质的亚细胞定位信号序列,将SDCT2基因的N端及C端分别缺失,并构建缺失突变体与EGFP的融合蛋白表达载体,将它们转染到LLC-PK1中,观察SDCT2 缺失体在细胞内的分布情况.结果显示,N端缺失的SDCT2蛋白主要位于细胞质中,顶膜和基底侧膜上也有表达;C端缺失的SDCT2蛋白主要位于基底侧膜上,顶膜几乎没有表达,细胞质中表达很少.免疫组化结果也显示,SDCT2只表达于人近端肾小管上皮细胞的基底侧膜.这表明SDCT2蛋白的N端序列对其亚细胞定位是必需的,人SDCT2蛋白的基底膜定位信号位于N端序列中.     

EGFP/SDCT1融合蛋白表达、亚细胞定位信号分析、组织分布及电生理功能研究

从正常人肾中克隆低亲和力钠离子依赖二羧酸共转运蛋白 1(sodium-dependent dicarboxylate co-transporter 1, SDCT1, NADC1)全长基因, 并将其和N端及C端缺失突变的SDCT1基因分别插入增强型绿色荧光蛋白基因(EGFP)表达载体中构建EGFP/SDCT1融合蛋白真核表达载体, 然后将它们转染到人肾小管上皮细胞HKC中表达并用激光共聚焦显微镜观察融合蛋白的亚细胞定位情况以确定其定位信号. 双重PCR分析证实融合基因已整合到细胞基因组中, Western blot显示融合基因已在细胞中得到表达. 共聚焦显微镜分析显示正常人SDCT1蛋白主要定位于细胞膜上, 与生物信息学的预测结果一致, 而C端缺失的SDCT1基因转染的细胞, 其绿色荧光位于细胞质, N端缺失基因转染的细胞, 其绿色荧光主要位于细胞膜上. 将体外转录的融合基因mRNA显微注射到爪蟾卵母细胞中表达并用双电极电压钳技术记录细胞跨膜电流, 结果在卵细胞膜上测定出了Na⁺内向电流. 免疫组化结果显示SDCT1主要表达于人近端肾小管上皮细胞的管腔侧, 而在远端肾小管、集合管、肾间质和肾小球中未见SDCT1的表达. 上述研究表明, 正常人SDCT1蛋白定位于近端肾小管上皮细胞的管腔侧膜上, SDCT1蛋白的C端部分对于其合成后的迁移及靶向定位是必需的, 人SDCT1蛋白的细胞膜定位序列可能位于其C端部分.     

PS1/GFP融合蛋白对PS1的亚细胞定位的初步研究

PS1基因突变与早发家族性老年痴呆有密切联系.构建pEGFP-C1-PS1以及pEGFP-N2-PS1融合基因表达载体,于HEK293和CHO细胞系中表达PS1/GFP融合蛋白,以GFP绿色荧光作为PS1的亚细胞定位信号,通过SPOTII以及CONFOCAL显微镜进行观察,初步获得PS1全长蛋白在细胞中定位的部分信息,即PS1定位于细胞核膜,细胞质内有不均匀的分布,少量存在于细胞-细胞接触处的细胞膜上.     

家蚕二分浓核病毒NS1蛋白的表达及定位

[目的]在Bm N细胞中表达家蚕二分浓核病毒(Bombyx mori bidensovirus,Bm BDV)非结构蛋白NS1,并分析其亚细胞定位。[方法]在病毒非结构蛋白NS1基因5'端加上kozak序列、3'端融合Flag标签序列;将重组序列克隆至昆虫细胞表达载体pIBV5/His上,转染BmN细胞,通过Western blot和免疫荧光检测NS1蛋白的表达和亚细胞定位。[结果]PCR和酶切鉴定显示重组表达载体构建正确;Western blot检测到一条大小约37 kDa的特异条带,免疫荧光分析显示表达的蛋白主要定位于细胞核中。[结论]构建的真核表达质粒能在BmN细胞中稳定表达NS1蛋白,该蛋白主要定位在细胞核中。     

PS1/GFP融合蛋白对PS1的亚细胞定位与功能的初步研究

PS1基因突变与早发家族性老年痴呆有密切联系。本文构建pEGFP-C1-PS1以及pEGFP-N2-PS1融合基因表达载体,于HEK293和CHO细胞系中表达PS1/GFP融合蛋白,以GFP绿色荧光作为PS1的亚细胞定位信号,通过SPOTII以及CONFOCAL显微镜进行观察,初步获得PS1全长蛋白在细胞中定位的部分信息,即本实验条件下,PS1定位于细胞核膜,细胞质内有不均匀的分布,少量存在于细胞-细胞接触处的细胞膜上。     

荧光蛋白标签对gD囊膜蛋白在BHK-21细胞亚细胞定位的影响

【目的】探究荧光蛋白标签对马疱疹病毒I型(Equine herpes virus type 1,EHV-1)gD囊膜蛋白亚细胞定位的影响。【方法】以EHV-1基因组为模板利用PCR扩增gD全基因,分别克隆至pAcGFP1-C1和p Ds Red2-N1质粒,构建p Ac-GFP-gD(GFP-gD)和p Ds-gD-Red(gD-Red)重组质粒;将GFP基因插入gD基因信号肽序列之后并克隆至PVAX-1质粒,构建PVAX-S-GFP-gD’(S-GFP-gD’)重组质粒;将Flag标签序列与gD囊膜蛋白N端序列融合后并克隆至p VAX-1表达载体,构建p VAX-Flag-gD(Flag-gD)重组质粒。将4种不同重组真核表达质粒分别转染BHK-21细胞,通过激光共聚焦显微镜对不同融合蛋白gD进行亚细胞定位。【结果】成功构建4种不同的融合蛋白gD真核表达载体;在BHK-21细胞单独表达时,不同融合蛋白gD绝大部分都定位于高尔基体,极少量定位于细胞核内。【结论】不同插入位点的荧光蛋白标签对gD囊膜蛋白亚细胞定位无明显影响,这对今后研究其它蛋白亚细胞定位提供参考。     

核定位信号分析示踪载体——pGST-EGFP的构建及功能鉴定

目的 构建谷胱甘肽转硫酶（GST）与EGFP相融合的新型蛋白质示踪载体--pGST-EGFP,以用于蛋白质细胞亚定位信号序列的深入分析.方法 以质粒pEGFP-N1为骨架,融合从pGEX-2TK载体中扩增的GST编码序列,构建成pGST-EGFP融合表达质粒;再插入人工合成的已知核定位蛋白SV40的核定位序列（NLS）,构建成pGST-EGFP-SV40 NLS作为阳性对照;另外,构建小分子量蛋白TNNI2在pGST-EGFP的融合表达质粒.将对照pEGFP-N1和各重组质粒分别用脂质体介导,瞬时转染HeLa细胞,荧光显微镜下观察蛋白的核定位情况.结果 单独表达的EGFP呈全细胞分布,而GST-EGFP融合蛋白只存在于细胞浆;SV40 NLS能将GST-EGFP融合蛋白带进细胞核.虽然TNNI2-EGFP融合蛋白的细胞亚定位呈现核内丰度更高的特点,但TNNI2-GST-EGFP融合蛋白仅限定于胞浆分布,提示TNNI2不能主动定位到细胞核中.结论 成功构建了蛋白质细胞亚定位示踪载体--pGST-EGFP.作为核定位信号分析系统,其对小分子蛋白细胞亚定位的示踪效果优于传统的pEGFP载体,更适用于科研工作中小分子量蛋白质核定位信号序列的研究.     

大麦黄矮病毒GAV株系ORF4基因在杆状病毒-昆虫细胞系统中的表达及亚细胞定位

根据已报道的大麦黄矮病毒GAV株系(BYDV-GAV)相关基因序列,利用RT-PCR方法获得ORF4基因。在杆状病毒-昆虫细胞系统中,成功表达了ORF4和GFP(绿色荧光蛋白)的融合蛋白(GFP:ORF4),Western blot检测到目的蛋白的表达。利用激光共聚焦显微镜观察其在细胞中的积累和亚细胞分布,发现ORF4基因编码的17kD蛋白(P4)能进入细胞核,并在细胞核膜上聚集。通过对ORF4基因编码的P4蛋白的N端和C端缺失突变结合蛋白质的结构预测分析,鉴定出N端α螺旋结构对于P4蛋白的核膜定位是必需的。这些结果为进一步研究ORF4基因在黄矮病毒GAV系统侵染中的生物学功能奠定了基础。     

Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein.

We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.     

In vivo analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori using recombinant baculovirus as vector

In order to investigate the functional signal peptide of silkworm fibroin heavy chain (FibH) and the effect of N- and C-terminal parts of FibH on the secretion of FibH in vivo, N- and C-terminal segments of fibh gene were fused with enhanced green fluorescent protein (EGFP) gene. The fused gene was then introduced into silkworm larvae and expressed in silk gland using recombinant AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) as vector. The fluorescence of EGFP was observed with fluorescence microscope. FibH-EGFP fusion proteins extracted from silk gland were analyzed by Western blot. Results showed that the two alpha helices within N-terminal 163 amino acid residues and the C-terminal 61 amino acid residues were not necessary for cleavage of signal peptide and secretion of the fusion protein into silk gland. Then the C-terminal 61 amino acid residues were substituted with a His-tag in the fusion protein to facilitate the purification. N-terminal sequencing of the purified protein showed that the signal cleavage site is between position 21 and 22 amino acid residues.     

Protein Purification with C-Terminal Fusion of Maltose Binding Protein

For affinity-chromatography-based purification of proteins that are prone to abnormal termination of translation or that may not be modified at their N-termini, affinity tags are needed which can be fused to the C-terminus. In this publication we describe that maltose binding protein (MBP) fused to the C-terminus of the plant photoreceptor phytochrome B allows purification of the fusion protein via amylose affinity chromatography. After overexpression in yeast a 125-fold enrichment could be achieved. The spectral properties of phytochrome B were not impaired by the fusion and purification. These results demonstrate that not only the widely used N-terminal fusions of MBP but also C-terminal fusions can be employed for protein purification.     

Synthesis of an artificial gene coding for thymosin alpha1 and its expression in Escherichia coli as a hybrid with human tumor necrosis factor]

Chemical-enzymatic synthesis and cloning in Escherichia coli of an artificial gene encoding the immunoactive peptide thymosin alpha 1 have been carried out. Recombinant plasmids were constructed which contain fusion genes coding for hybrids of human tumour necrosis factor (TNF) and thymosin alpha 1 as N- or C-terminal part of the hybrid protein. In the C-terminal hybrid protein, TNF and thymosin alpha 1 are linked through a methionine residue, thus allowing for thymosin alpha 1 to be cleaved off the rest of the hybrid protein with cyanogen bromide. In case of the N-terminal hybrid protein, the linker between the thymosin alpha 1 and TNF sequences is the acid-labile dipeptide Asp-Pro. Expression of the hybrid genes in E. coli and properties of the recombinant proteins were studied. The N-terminal hybrid protein was secreted into periplasmic space, in contrast with the C-terminal hybrid protein, which formed insoluble aggregates inside bacterial cells. Procedures for the isolation of both hybrid proteins were developed. The N-terminal hybrid protein displayed full biological activity in the cytotoxic assay on the mouse fibroblast L-929 whereas the C-terminal hybrid protein proved to be much less active. Treatment of the hybrid protein TNF-thymosin alpha 1 with cyanogen bromide lead to a mixture of two polypeptides, from which thymosin alpha 1 was purified to homogeneity by simple chromatographic procedures.     

Construction of recombinant S-layer proteins (rSbsA) and their expression in bacterial ghosts--a delivery system for the nontypeable Haemophilus influenzae antigen Omp26

This study has investigated the feasibility of a combination of recombinant surface layer (S-layer) proteins and empty bacterial cell envelopes (ghosts) to deliver candidate antigens for a vaccine against nontypeable Haemophilus influenzae (NTHi) infections. The S-layer gene sbsA from Bacillus stearothermophilus PV72 was used for the construction of fusion proteins. Fusion of maltose binding protein (MBP) to the N-terminus of SbsA allowed expression of the S-layer in the periplasm of Escherichia coli. The outer membrane protein (Omp) 26 of NTHi was inserted into the N-terminal and C-terminal regions of SbsA. The presence of the fused antigen Omp26 was demonstrated by Western blot experiments using anti-Omp26 antisera. Electron microscopy showed that the recombinant SbsA maintained the ability to self-assemble into sheet-like and cylindrical structures. Recombinant E. coli cell envelopes (ghosts) were produced by the expression of SbsA/Omp26 fusion proteins prior to gene E-mediated lysis. Intraperitoneal immunization with these recombinant bacterial ghosts induced an Omp26-specific antibody response in BALB/c mice. These results demonstrate that the NTHi antigen, Omp26, was expressed in the S-layer self-assembly product and this construct was immunogenic for Omp26 when administered to mice in bacterial cell envelopes.     

Ribosomes with large synthetic N-terminal extensions of protein S15 are active in vivo

The genes for ribosomal proteins S4, S13 or S15 were fused with the gene for staphylococcal protein A, or derivatives thereof (2A'-7A'). The gene fusions were introduced into Escherichia coli strains, mutated in the corresponding ribosomal protein gene, by transformation. These mutated ribosomal proteins cause a phenotype that can be complemented. Thus, the phenotype of the transformants was tested and the ribosomal proteins were analyzed. The S4 N-terminal fusion protein severely disturbed growth of both the mutant and the wild-type strains. The S13 C-terminal fusion protein was proteolyzed close to the fusion point, giving a ribosomal protein moiety that could assemble into the ribosome normally. S15 N-terminal fusion proteins complemented a cold-sensitive strain lacking protein S15 in its ribosomes. These fused proteins were assembled into active ribosomes. The position of S15 in the 30S ribosomal subunit is well known. Therefore, in structural studies of the ribosome in vivo, the S15 fusion proteins can be used as a physical reporter for S15.     

Expression of EGFP/SDCT1 fusion protein, subcellular localization signal analysis, tissue distribution and electrophysiological function study

A main pathway for energy ATP production inhuman body is by tricarboxylic acid cycle (Krebs cy-cle). Sodium-dependent dicarboxylate co-transporterprotein (SDCT, NaDC, NaC) is an organic aniontransporter protein family responsible for trans-mem- for 30 s, and extension 72℃ for 2 min; followed bybrane transport of Krebs cycle intermediate metabolite final extension 72℃ for 7 min. PCR products weresuch as succinate and citrate. They predominantly lo- …     

TLS-ERG leukemia fusion protein inhibits RNA splicing mediated by serine-arginine proteins

The translocation liposarcoma (TLS) gene is fused to the ETS-related gene (ERG) in human myeloid leukemia, resulting in the generation of a TLS-ERG protein. We demonstrate that both TLS and the TLS-ERG leukemia fusion protein bind to RNA polymerase II through the TLS N-terminal domain, which is retained in the fusion protein; however, TLS recruits members of the serine-arginine (SR) family of splicing factors through its C-terminal domain, whereas the TLS-ERG fusion protein lacks the ability to recruit SR proteins due to replacement of the C-terminal domain by the fusion partner ERG. In transient-transfection assays, the TLS-ERG fusion protein inhibits E1A pre-mRNA splicing mediated by these TLS-associated SR proteins (TASR), and stable expression of the TLS-ERG fusion protein in K562 cells alters the splicing profile of CD44 mRNA. These results suggest that TLS fusion proteins may lead to cellular abnormalities by interfering with the splicing of important cellular regulators.