人白细胞介素10受体α基因真核表达及与JAK1蛋白的相互作用的检测

构建含人白介素10受体α(IL-10RA)基因的真核表达质粒p ENTER-IL-10RA-His,并在HEK293中进行真核表达,并用免疫共沉淀检测JAK1与IL10RA在细胞内的相互作用。在He La细胞中提取人总RNA,通过RT-PCR获得人IL10RA的基因全长,并将其克隆至真核表达载体p ENTER-His中。经PCR扩增、双酶切、测序鉴定后,将重组质粒p ENTER-IL-10RA-His转染至HEK293细胞中。免疫印迹法检测IL-10RA蛋白在HEK293细胞中的表达。结果显示,经PCR扩增和双酶切,测序鉴定质粒克隆正确。免疫印迹可见63 k D的目的蛋白。共同转染JAK1和IL-10RA的质粒,免疫印迹可见分别为133 k D和63 k D的目的条带,免疫共沉淀鉴定了JAK1和IL-10RA的相互作用。IL-10RA基因成功构建在p ENTER-His中,并在HEK293细胞中成功表达,并成功共转染JAK1和IL-10RA质粒,免疫共沉淀检测两者的相互作用。这为JAK1和IL-10RA相互作用的机制研究奠定基础。     

瞬时外向钾通道Kv4.3真核表达载体的构建

[目的]构建Kv4.3真核表达载体,观察其在真核细胞中表达情况。[方法]提取小鼠前额叶皮质RNA,通过RT-PCR和常规PCR技术扩增获得Kv4.3目的基因,利用EcoRⅠ和XbaⅠ限制性内切酶和T4 DNA连接酶将其克隆在pcDNA3.1载体中,并将构建好的重组质粒转染至HEK293细胞中,免疫印迹方法检测Kv4.3蛋白表达。[结果]成功扩增了Kv4.3基因(2 000 bp处有明显条带),先后经酶切和酶连后的重组体转化培养,提取质粒,再通过双酶切观察到2 000 bp处有Kv4.3目的条带和5 400 bp处有pcDNA3.1载体条带。将此重组质粒送去测序,结果显示基因测序结果与GenBank中Kv4.3序列一致。免疫印迹结果发现在HEK293细胞中,转染的重组质粒能够表达Kv4.3蛋白。[结论]成功构建了Kv4.3真核表达载体,其在HEK293细胞中可以稳定表达。     

非洲爪蟾基因MGC64236的克隆及初步分析

MGC64236基因是本实验室用脐静脉内皮细胞免疫的兔血清筛选非洲爪蟾cDNA文库而鉴定的一个功能未知的基因.本研究提取非洲爪蟾受精卵总RNA通过RT-PCR得到基因MGC64236的开放读码框651 bp、编码202个氨基酸;运用生物信息学研究工具进行分析,发现该基因编码的蛋白有3个潜在的跨膜域,有一保守的结构域DUF1370, 可能通过其胞内部分的磷酸化机制在介导细胞内外的信号转导中发挥重要作用;在非洲爪蟾胚胎各个发育时期用RT-PCR检测该基因的表达情况,发现在非洲爪蟾胚胎发育的几个重要时期该基因都有高表达,而在成体则特异地表达于脑和眼等神经组织;构建绿色荧光融合蛋白真核表达载体并转染HEK293细胞, 对MGC64236蛋白的亚细胞定位,发现MGC64236蛋白比较特异地表达在细胞膜.     

人白细胞介素2受体β(IL-2RB)基因真核表达及与JAK1的相互作用的检测

[目的]构建含人白介素2受体β(IL-2RB)基因的真核表达质粒p ENTER-IL-2RB-His,并在293T中进行真核表达,并用免疫共沉淀检测JAK1与IL-2RB在细胞内的相互作用。[方法]在Hela细胞中提取人总RNA,通过RT-PCR获得人IL-2RB的基因全长,并将其克隆至真核表达载体p ENTER-His中。经PCR克隆,双酶切、测序鉴定后,将重组质粒p ENTER-IL-2RB-His转染至293T细胞中。免疫印迹法检测不同时间点的IL2RΒ蛋白(24 h、36 h、48 h)在293T细胞中的表达,用免疫共沉淀检测JAK1和IL-2RB蛋白之间的相互作用。[结果]经PCR克隆、双酶切、测序鉴定质粒克隆正确。免疫印迹可见61 k Da的目的蛋白。共同转染JAK1和IL-2RB的质粒,免疫印迹可见分别为133 k Da和61 k Da的目的条带。[结论]成功获得IL-2RB基因全长,成功构建p ENTER-IL-2RB-His真核表达质粒,并在293T细胞中成功表达,随着时间的推移其表达量增高。免疫共沉淀可以检测到JAK1和IL-2RB两者的蛋白相互作用,这为下一步研究JAK1和IL-2RB这一对蛋白的相互作用的作用方式及作用机制奠定了基础。     

人白细胞介素2受体β(IL-2RB)基因真核表达及与JAK1的相互作用的检测北大核心

[目的]构建含人白介素2受体β(IL-2RB)基因的真核表达质粒p ENTER-IL-2RB-His,并在293T中进行真核表达,并用免疫共沉淀检测JAK1与IL-2RB在细胞内的相互作用。[方法]在Hela细胞中提取人总RNA,通过RT-PCR获得人IL-2RB的基因全长,并将其克隆至真核表达载体p ENTER-His中。经PCR克隆,双酶切、测序鉴定后,将重组质粒p ENTER-IL-2RB-His转染至293T细胞中。免疫印迹法检测不同时间点的IL2RΒ蛋白(24 h、36 h、48 h)在293T细胞中的表达,用免疫共沉淀检测JAK1和IL-2RB蛋白之间的相互作用。[结果]经PCR克隆、双酶切、测序鉴定质粒克隆正确。免疫印迹可见61 k Da的目的蛋白。共同转染JAK1和IL-2RB的质粒,免疫印迹可见分别为133 k Da和61 k Da的目的条带。[结论]成功获得IL-2RB基因全长,成功构建p ENTER-IL-2RB-His真核表达质粒,并在293T细胞中成功表达,随着时间的推移其表达量增高。免疫共沉淀可以检测到JAK1和IL-2RB两者的蛋白相互作用,这为下一步研究JAK1和IL-2RB这一对蛋白的相互作用的作用方式及作用机制奠定了基础。     

非受体型酪氨酸激酶JAK1基因真核表达质粒的构建与真核表达

[目的]构建非受体型酪氨酸激酶JAK1(nonreceptor tyrosine kinase-JAK1)基因真核表达质粒pcDNA4.0-HisJAK1,并转染293T细胞进行真核表达。[方法]从Hela细胞中提取总的RNA,通过RT-PCR获得JAK1基因全长c DNA序列,并将其克隆至真核表达载体pcDNA4.0-His中,构建重组质粒pcDNA4.0-His-JAK1。将真核重组表达质粒转染293T细胞,转染48 h后,在荧光显微镜下观察转染情况。免疫印迹法检测JAK1蛋白转染24 h、36 h与48 h在293T细胞中的表达。[结果]经双酶切与质粒PCR鉴定质粒克隆正确,测序鉴定序列中第2199位碱基A突变为G,为同义突变,不影响氨基酸序列。转染293T细胞48 h后,间接免疫荧光实验显示在293T细胞中检测到绿色荧光。免疫印迹检测可见大小约为133 kDa的目的蛋白。[结论]成功获得JAK1基因全长序列,并成功构建pcDNA4.0-HisJAK1真核重组表达质粒,并在真核细胞293T中获得有效表达,为进一步研究JAK1蛋白质相互作用奠定了基础。     

活化型及失活型SUMO1真核表达载体的构建及鉴定

目的:构建活化型及失活型SUMO1(small ubiquitin-related modifier 1)真核表达载体并鉴定其蛋白活性。方法:以野生型HA-SUMO1-pc DNA3.1为模板,采用PCR扩增目的基因,将其连接入克隆载体T vector,筛选阳性重组子;将目的基因酶切回收后亚克隆入真核表达载体pc DNA3.1。将阳性克隆转染HEK293细胞,免疫印迹鉴定底物蛋白与SUMO1的结合情况(即SUMO化水平)。结果:活化型及失活型SUMO1目的基因被扩增,并且亚克隆入pc DNA3.1,且测序结果与预期一致。免疫印迹分析显示,转染活化型SUMO1组检测到显著的蛋白SUMO化条带,而失活型组则无明显条带。结论:活化型及失活型SUMO1真核表达载体成功构建,并可于HEK293细胞高效表达。     

比格犬ERβ1293基因真核载体的构建及其在HEK293T细胞中的表达和定位

目的 观察比格犬Myc 标签ERβ1293重组真核表达载体在HEK293T细胞中的表达及定位.方法 以pEGFP-N1-ERβ1293重组真核表达载体为模板,PCR保真酶扩增得到ERβ1293基因编码区全长.Myc标签ERβ1293重组真核载体瞬时转染HEK293T细胞,运用蛋白质印迹技术（Western blotting）和间接免疫荧光技术（indirect immunofluorescence,IF）鉴定pcDNA3.1-Myc-ERβ1293在HEK293T细胞中的表达和定位情况.结果 成功构建pcDNA3.1-Myc-ERβ1293重组真核表达载体,转染至HEK293T细胞中.Western blotting检测有蛋白条带表达,共聚焦显微镜下观察IF处理后的转染细胞,荧光定位于细胞质.结论 前期实验得到比格犬ERβ剪切异构体ERβ1293编码区序列,缺失第四外显子,导致其与配体结合能力减弱或消失,因此目的 基因编码蛋白定位在细胞中发生变化.     

SCG10基因重组质粒的构建及蛋白表达和定位

目的构建SCG10真核表达载体并证实融合蛋白在细胞内表达及定位。方法以人胎脑cDNA文库为模板,PCR扩增SCG10全长编码基因,亚克隆至pEGFP-C1表达载体中。将构建的重组质粒测序并转染到人胚肾HEK293中,提取细胞蛋白进行Western blot检测。利用共聚焦激光扫描显微镜观察pEGFP-SCG10在HEK293细胞内定位。结果 SCG10全长基因序列克隆到了真核表达载体pEGFP-C1中,酶切鉴定片段大小540bp。Western blot检测到了融合蛋白表达,分子量约为48kD。pEGFP-SCG10在细胞内定位以细胞浆为主,在细胞核少量表达。结论成功构建了SCG10全长基因真核表达载体,pEGFP-SCG10蛋白主要定位于HEK293细胞浆内。     

小鼠GP73蛋白真核表达质粒的构建及其对mTOR转录水平的影响

目的:构建小鼠高尔基蛋白73(m GP73)的真核表达质粒,转染HEK293T细胞株验证重组质粒活性,并检测m GP73过表达情况下对小鼠肝癌细胞中mTOR m RNA水平的影响。方法:以实验室保存的H22小鼠肝癌细胞c DNA文库为模板,采用PCR技术扩增获得m GP73序列,将其插入pc DNA3.1-Flag-vector载体构建成重组质粒,转染HEK293T细胞后用蛋白免疫印迹检测融合蛋白的表达,并用q PCR检测在GP73过表达情况下H22细胞中mTOR转录水平的变化。结果:菌液PCR、重组质粒的双酶切结果及测序均表明重组质粒构建成功,蛋白免疫印迹表明m GP73蛋白在HEK293T细胞中获得表达,q PCR结果表明在m GP73过表达时mTOR的转录水平随之上升。结论:构建了pc DNA3.1-Flag-m GP73真核表达质粒,m GP73 m RNA过表达时mTOR的m RNA水平也上升,这为进一步研究m GP73在小鼠肝脏肿瘤转移过程中的作用奠定了基础。     

Expression of recombinant XVAX2 peptide-104 of Xenopus laevis and preparation of the antibody

An N-terminal polypeptide (XVAX2-P104) of the XVAX2 protein, which is involved in controlling the dorsoventral patterning of the retina in Xenopus laevis, was expressed and purified as a Histagged fusion protein (pHis-XVAX2-P104), and it was employed to generate an anti-XVAX2 antibody in New Zealand white rabbits. ELISA analysis shows that the titer of the antibody is as high as approximately 1:100,000. The antibody could specifically recognize the full-length XVAX2 protein in extracts of Xenopus embryos, as determined by Western Blotting.     

转录因子XBP1的融合表达、纯化及多克隆抗体的制备

人X盒结合蛋白 1(XBP 1)为一种转录因子 ,与多种肿瘤的发生、发展有密切关系 .XBP 1有2种剪切形式 ,即XBP 1S和XBP 1U .将这 2种剪切形式中的一段相同编码序列 (编码 82～ 14 7位氨基酸 )重组于谷胱甘肽S转移酶 (GST)融合蛋白表达载体pGEX KG中 ,构建成重组质粒pGST XBP 1(82～ 14 7位氨基酸 ) .将该重组质粒转化E .coliDH5α后 ,表达GST XBP 1(82～ 14 7位氨基酸 )融合蛋白 ,经谷胱甘肽 Sepharose 4B亲和层析获得纯化的融合蛋白 .用此融合蛋白免疫家兔制备多克隆抗体 .利用制备的抗体分别用Western印迹和免疫细胞化学检测XBP 1的 2种剪切形式在哺乳动物细胞中的表达 .结果表明 ,该抗体对XBP 1的 2种剪切形式均具有反应原性 ,效价高 ,特异性好 ,可以用于进一步研究XBP 1的功能     

人Lrp蛋白在细胞中的定位及LPS对其表达的影响

为了对脂多糖应答基因(lrp)的功能进行深入的研究,用镍离子螯合柱(Ni-NT)纯化后的 全长Lrp蛋白免疫新西兰大白兔制备多克隆抗体并吸附去除非特异性反应成分.Western 印迹表明,吸附纯化后的抗体可以与Lrp蛋白特异结合,并有较高免疫印迹滴度,为Lrp功能研究提供了重要的工具.激光共聚焦扫描荧光显微镜检测显示Lrp主要位于细胞核膜周围.Western印迹、RT-PCR以及细胞免疫组化染色结果都表明,用LPS刺激后,lrp在人HEK293 和U937细胞内的表达均有明显的上升.结果提示,Lrp可能与对Lrp介导的反应有关.     

The protocadherin PAPC establishes segmental boundaries during somitogenesis in xenopus embryos

BACKGROUND: One prominent example of segmentation in vertebrate embryos is the subdivision of the paraxial mesoderm into repeating, metameric structures called somites. During this process, cells in the presomitic mesoderm (PSM) are first patterned into segments leading secondarily to differences required for somite morphogenesis such as the formation of segmental boundaries. Recent studies have shown that a segmental pattern is generated in the PSM of Xenopus embryos by genes encoding a Mesp-like bHLH protein called Thylacine 1 and components of the Notch signaling pathway. These genes establish a repeating pattern of gene expression that subdivides cells in the PSM into anterior and posterior half segments, but how this pattern of gene expression leads to segmental boundaries is unknown. Recently, a member of the protocadherin family of cell adhesion molecules, called PAPC, has been shown to be expressed in the PSM of Xenopus embryos in a half segment pattern, suggesting that it could play a role in restricting cell mixing at the anterior segmental boundary. RESULTS: Here, we examine the expression and function of PAPC during segmentation of the paraxial mesoderm in Xenopus embryos. We show that Thylacine 1 and the Notch pathway establish segment identity one segment prior to the segmental expression of PAPC. Altering segmental identity in embryos by perturbing the activity of Thylacine 1 and the Notch pathway, or by treatment with a protein synthesis inhibitor, cycloheximide, leads to the predicted changes in the segmental expression of PAPC. By disrupting PAPC function in embryos using a putative dominant-negative or an activated form of PAPC, we show that segmental PAPC activity is required for proper somite formation as well as for maintaining segmental gene expression within the PSM. CONCLUSIONS: Segmental expression of PAPC is established in the PSM as a downstream consequence of segmental patterning by Thylacine 1 and the Notch pathway. We propose that PAPC is part of the mechanism that establishes the segmental boundaries between posterior and anterior cells in adjacent segments.     

Immuno-Localization of DEAD Family Proteins in Germ Line Cells of Xenopus Embryos.

In order to investigate whether a vasa -like protein is present in germ line cells of Xenopus , antibodies were produced which react specifically with synthetic oligopeptides of sequences from near the N- or C-termini or with one including the DEAD box of the Drosophila vasa protein.
Only the antibody against the oligopeptide including the DEAD box reacted strongly with germ plasm (GP) or with cytoplasm of germ line cells of Xenopus embryos by immunofluorescence microscopy. By immunoelectron microscopy, the antibody was demonstrated to react with the GP-specific structure, germinal granules, in cleaving embryos, and with their derivatives in the germ line cells of embryos at stages extending from gastrula to feeding tadpole. It also reacted with mitochondria not only in the GP and the germ line cells but also in somatic cells, and with myofibrils in muscle cells. By Western blotting, the antibody was shown to react with several bands of Mr 42–69 ± 10³ in protein samples from Xenopus embryos. In samples from Drosophila ovaries, it reacted with a Mr 71 ± 10³ band which was probably the vasa protein. This indicates the possibility that Xenopus embryos contain several DEAD family proteins. One of these is present on germinal granules, resembling the vasa protein on polar granules of Drosophila .     

Xenopus paraxial protocadherin inhibits Wnt/β-catenin signalling via casein kinase 2β

Xenopus paraxial protocadherin (PAPC) regulates cadherin-mediated cell adhesion and promotes the planar cell polarity (PCP) pathway. Here we report that PAPC functions in the Xenopus gastrula as an inhibitor of the Wnt/β-catenin pathway. The intracellular domain of PAPC interacts with casein kinase 2 beta (CK2β), which is part of the CK2 holoenzyme. The CK2α/β complex stimulates Wnt/β-catenin signalling, and the physical interaction of CK2β with PAPC antagonizes this activity. By this mechanism, PAPC restricts the expression of Wnt target genes during gastrulation. These experiments identify a novel function of protocadherins as regulators of the Wnt pathway.     

Subcellular localization analysis of bovine foamy virus Borf1 protein

The Borf1 protein is encoded by an immediate-early gene of the bovine foamy virus (BFV) and plays a key role in the viral life cycle. Borf1 is a DNA binding protein which can transactivate both the long terminal repeat (LTR) and the internal promoter (IP) of BFV by specifically binding to the transactivation responsive element (TRE). To analyze the subcellular localization of Borf1 during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borf1 serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borf1 protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borf1 in HeLa cells that was transfected with Borf1. Moreover, the immuno-fluorescence assay also showed that the localization of Borf1 during the infection and transfection of BFV was identical.     

Subcellular Localization Analysis of Bovine Foamy Virus Borfl Protein

The Borfl protein is encoded by an immediate-early gene of the bovine foamy virus （BFV） and plays a key role in the viral life cycle. Borfl is a DNA binding protein which can transactivate both the long terminal repeat （LTR） and the internal promoter （IP） of BFV by specifically binding to the transactivation responsive element （TRE）. To analyze the subcellular localization of Borfl during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borfl serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borfl protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borfl in HeLa cells that was transfected with Borfl. Moreover, the immuno-fluorescence assay also showed that the localization of Borfl during the infection and transfection of BFV was identical.     

An anti-peptide antibody that recognizes the dopamine D2 receptor from bovine striatum.

The bovine dopamine D2 receptor was purified by wheat-germ-agglutinin-Sepharose chromatography and affinity chromatography, using the D2-receptor-specific agonist N-0434. Purification yields a preparation with a major protein band of 95 kDa. In order to ascertain the identity of this protein, polyclonal antibodies against the dopamine D2 receptor have been raised using synthetic peptides based on the predicted amino acid sequence of the cloned D2 receptor. For the initial screening of these antibodies, three fusion proteins consisting of beta-galactosidase and receptor fragments were constructed. One antiserum reacted strongly with the corresponding D2 receptor fusion protein, both on Western blots and in immunoprecipitation experiments. In each case, recognition was inhibited by competition with free peptide. On Western blots of partially purified receptor preparations from bovine striatum, the antiserum specifically recognized a 95-kDa glycoprotein. From similar preparations, the antiserum precipitated a substantial proportion of active D2 receptor, as determined by a decrease in [3H]spiperone binding in the supernatant. Active receptor could be released from the immunoprecipitate by addition of free peptide. Immunocytochemical analysis of cells transiently transfected with DNA coding for the D2 receptor showed specific staining of transfected cells. The antibody raised against a sequence in the third intracellular loop is able to shift the affinity of the receptor for dopamine from high to low, indicating that the antiserum may be interfering with receptor-GTP-binding-protein interactions.     

小鼠睾丸特异性基因Vad1.2重组蛋白的构建及多克隆抗体制备

目的：构建小鼠睾丸特异性基因Vad1.2重组蛋白,制备多克隆抗体。方法：将小鼠睾丸组织Vad1.2转录本行RT-PCR扩增,通过DNA重组技术插入克隆载体p ET15b,进行酶切及DNA序列分析。将表达载体转化入大肠杆菌BL21（DE3）RIL感受态细胞,10 mmol/L IPTG诱导表达重组蛋白,通过10%SDS-PAGE、Western blotting及质谱分析进行鉴定。随后对重组Vad1.2蛋白进行纯化和进一步鉴定。最后,制备兔抗小鼠Vad1.2多克隆抗体,并通过Vad1.2-EGFP质粒转染GC-2spd（ts）细胞对其特异性进行验证。结果：大肠杆菌BL21（DE3）RIL细胞中诱导表达并纯化的小鼠Vad1.2重组蛋白构建正确并经Western blotting和质谱分析得到证实。所制备的多克隆抗体可特异性识别GC-2spd（ts）细胞中过表达的Vad1.2-EGFP融合蛋白。结论：成功构建小鼠Vad1.2重组蛋白,并制备多克隆抗体,为Vad1.2基因的进一步研究奠定了实验基础。