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。     

hnRNPA1基因在家蚕翅原基组织中的表达与定位分析

核不均一蛋白A1(hnRNPA1)是一个重要的RNA结合蛋白。本研究旨在获得家蚕hnRNPA1基因的cDNA,并对其在家蚕翅原基组织进行表达和定位分析。以家蚕幼虫期翅原基mRNA为模板通过反转录克隆家蚕BmhnRNPA1基因的全长cDNA,并对其进行生物信息学分析。构建pET32a-BmhnRNPA1蛋白表达载体,表达且纯化得到BmhnRNPA1重组蛋白,并制备该蛋白多克隆抗体,通过实时荧光定量RT-PCR、Western blot和免疫组化方法检测BmhnRNPA1在家蚕幼虫和蛹翅原基组织中的表达与定位。克隆得到了家蚕hnRNPA1基因的全长cDNA片段,其开放阅读框(ORF)序列为951 bp,编码316个氨基酸,预测分子量为34.98 kDa,等电点为5.15。编码蛋白在第18～90个氨基酸和109～181个氨基酸处为保守的RRM结构域。系统进化分析显示,家蚕hnRNPA1与小菜蛾hnRNPA1的亲缘关系最近。QRT-PCR结果显示,BmhnRNPA1在家蚕幼虫和蛹期的翅原基组织中均有表达,且在蛹期第3天的表达量达到峰值。Western blot进一步证实了实验结果。免疫组化分析结果显示,该蛋白存在于翅原基组织中,并定位于细胞核内。家蚕BmhRNPA1具有两个RNA结合结构域,属于hnRNPs家族,定位于细胞核内,表明其可能参与mRNA的选择性剪接作用。本研究结果为进一步探索该基因的功能提供了基础。     

猪雌激素受体关联受体a基因的克隆、表达及其对脂肪细胞脂质聚集的影响

雌激素受体关联受体α(Estrogen-related receptor α,ERRα)是调控机体能量代谢的关键转录调控因子,其在白色脂肪组织中的作用尚不清楚。本研究旨在通过touch down-PCR方法克隆猪ERRα基因的ORF序列;通过Western blotting和细胞免疫荧光染色法分析其在猪各组织及成熟脂肪细胞中的表达模式;利用ERRα特异性抑制剂XCT790处理原代培养的猪成熟脂肪细胞,探讨其对成熟脂肪细胞甘油三酯聚集的影响。结果显示,所克隆的猪ERRα基因ORF序列长1269bp(GenBank Accession No.FJ446485,尚未公布),编码422个氨基酸,其核苷酸和氨基酸序列与其他物种高度同源;ERRα蛋白高表达于猪白色脂肪组织(White adipose tissue,WAT)、肾脏以及心脏中,在脾脏中表达量较低;细胞免疫荧光化学染色显示,ERRα蛋白广泛分布于脂肪细胞的细胞核和胞浆中;XCT790在10μmol/L浓度时显著抑制了ERRα蛋白的表达和成熟脂肪细胞中甘油三酯的聚集。本研究将为有效调控体脂沉积提供新的靶点和理论参考。     

尼罗罗非鱼Ly75基因的克隆、原核表达和多克隆抗体制备

[目的]获得尼罗罗非鱼淋巴细胞抗原75(OnLy75)基因的cDNA序列和多克隆抗体。[方法]采用RACE技术克隆OnLy75基因的cDNA全序列,通过DNAMAN、MEGA等软件分析其序列特征;构建OnLy75的原核表达载体,并对其重组蛋白进行原核诱导表达;将纯化后的OnLy75重组蛋白免疫新西兰大耳白兔,并通过Western Blot技术检测多克隆抗体的效果。[结果]OnLy75的cDNA序列全长为6 632 bp,开放阅读框5 199 bp,编码1 732个氨基酸。其OnLy75重组蛋白主要存在于包涵体中;将重组蛋白纯化后免疫新西兰大耳白兔,成功获得了兔抗OnLy75多克隆抗体。用该多抗对尼罗罗非鱼不同器官进行Western Blot内源性检测,结果显示该蛋白在精巢中有丰富的表达。[结论]兔抗OnLy75多克隆抗体的成功制备,为进一步深入研究OnLy75在尼罗罗非鱼组织中的定位及功能研究提供了工具。     

猪Gli1基因的克隆、表达谱分析及脂肪组织特异性表达载体的构建

Hedgehog(Hh)信号通路对动物脂肪沉积具有抑制作用,并且从果蝇到脊椎动物具有高度保守性,但在家猪研究中鲜见报道。文章选择家猪Hh通路的转录激活因子Gli1进行研究,通过RT-PCR结合RACE技术,首次获得家猪Gli1基因cDNA全长,利用Real-time PCR对家猪Gli1基因在不同组织中的表达丰度进行了分析,并构建了真核表达载体和脂肪组织特异性表达载体。结果表明:猪Gli1基因cDNA全长3 576 bp,基因组序列全长10 715 bp,共12个外显子,编码1 106个氨基酸。生物信息学分析表明,猪Gli1为不稳定亲水性蛋白,不具有跨膜结构域和信号肽序列,但具有锌指结构与核定位序列。对7个物种的Gli1蛋白序列和基因组序列相似性进行分析,发现各物种间序列相似性均在80%以上,说明Gli1在物种间高度保守。组织表达谱分析表明,Gli1仅在成体猪舌组织中表达;在家猪脂肪组织发育进程中,Gli1仅在出生1周的猪脂肪组织中检测到微弱表达,但1月龄及3月龄猪脂肪组织中均检测不到表达,由此推断猪Gli1表达与脂肪组织发育呈负相关。最后,将猪Gli1编码区克隆到真核表达载体pIRES2-EGFP,体外转染实验证明该载体能够正确表达猪Gli1,另外还构建了脂肪组织特异性表达载体,为构建脂肪组织特异性转基因动物奠定基础。     

沙门菌毒力基因spvB的原核表达及其多克隆抗体的制备

目的:构建沙门菌毒力基因spvB的原核表达载体,诱导表达纯化SpvB蛋白并以其为抗原免疫小鼠,制备多克隆抗体。方法:利用生物信息学软件对SpvB进行分析,选取抗原性较高、易表达的氨基酸序列作为克隆序列,以携带spvB基因的鼠伤寒沙门菌为模板,PCR扩增目的片段后与原核表达载体pET28a(+)连接;将质粒pET28a-SpvB转化大肠埃希菌BL21(DE3)后诱导表达并纯化。目的蛋白免疫小鼠,制备抗SpvB多克隆抗体,Western blot检测抗体特异性。结果:成功构建spvB原核表达载体,经IPTG诱导结果显示,重组蛋白表达且主要存在于包涵体中,将纯化后的蛋白免疫小鼠Western blot检测血清中抗体与SpvB特异性结合。结论:获得具有免疫原性的SpvB蛋白及其多克隆抗体,为进一步研究该基因的功能奠定基础。     

彩鲫Ran基因全长cDNA的克隆及其组织表达特异性分析

根据Ran的保守区序列设计简并引物,结合SMART、cDNA合成及RACE-PCR技术,克隆到彩鲫(Carassius auratus color variety)的Ran基因的cDNA全长,其编码区全长为648bp,编码215个氨基酸。采用BIAST程序在NCBI数据库中对其进行同源基因搜索,结果表明,其推测的编码氨基酸序列与斑马鱼和鲑鱼的Ran基因编码的氨基酸序列的同源性分别高达98％和97％。还对其编码区全长序列进行了原核表达,以经纯化的表达蛋白免疫家兔,制备出了具有较高特异性的多克隆抗体。采用Western blotting进行的组织特异性分析表明,Ran蛋白在卵巢、精巢和肾中表达,在心、脑、肝、脾和肌肉中不表达。本工作为采用免疫耗竭法、免疫共沉淀、体外系统模拟等方法进一步研究鱼类．Ran基因的生理功能及分离鉴定其结合蛋白提供了良好基础。     

大鼠NeuroD2基因的克隆、真核表达载体的构建及其在小鼠MSCs细胞中的表达

克隆大鼠NeuroD2基因,旨在构建p IRES2-Ac GFP1-ND2真核表达载体并检测其在小鼠MSCs中的表达。采用RTPCR技术克隆大鼠NeuroD2基因,分析该蛋白质结构、功能域、细胞定位及与其他物种同源性;构建pIRES2-AcGFP1-ND2表达载体并转染小鼠骨髓间充质干细胞(MSCs);采用Real-time PCR、免疫荧光及Western blot技术检测重组质粒在小鼠MSCs中的表达。结果表明,大鼠NeuroD2基因CDS区全长1 149 bp,共编码382个氨基酸,属于b HLH家族,二级结构以α-螺旋和无规卷曲为主,空间结构呈现近似"螺旋-环-螺旋(HLH)"结构,主要分布在细胞核,氨基酸序列与家鼠(99%)、罗猴(98%)、人(97.7%)同源性较高;Real-time PCR结果表明转染组NeuroD2基因表达量显著高于对照组(P0.05);免疫荧光及Western blot结果显示转染组NeuroD2蛋白表达量显著高于对照组(P0.05)。成功克隆大鼠NeuroD2基因并构建了pIRES2-AcGFP1-ND2真核表达载体。     

猪I-FABP基因的分子克隆与组织特异性表达分析

小肠型脂肪酸结合蛋白对长链脂肪酸具有高度的亲和力,参与脂肪酸的吸收和细胞内转运。利用cDNA末端快速扩增（RACE）技术并结合同源克隆策略,克隆到了编码猪小肠型脂肪酸结合蛋白基因（I-FABP）的全长cDNA序列（GenBank接受号：AY960624）,并对系统发育关系等进行了生物信息学分析。猪I-FABP基因的cDNA序列全长614 bp,其中包括399bp的开放式读码框（ORF）,43bp的5’末端非编码区（5’URT）和172bp的3’末端非编码区（3’URT）,编码132个氨基酸残基蛋白,在氨基酸水半上与其他物种的I-FABP具有高度的同源性。以邻接法（Neigbor-Joining,NJ）所构建的系统发育关系表明,猪I-FABP与其他物种的,I-FABP属于同一类群,且与人的遗传距离最近。Northern杂交和半定量RT—PCR分析发现,猪I-FABP在猪体组织中出现约620bp大小的转录本,且在猪体组织中广泛存在,但在小肠组织中表达量最为丰富。     

松墨天牛Mfe2基因的克隆、原核表达和表达谱分析

【目的】Mfe2为过氧化物酶体多功能酶类型2(peroxisomal multifunctional enzyme type 2)基因,在脂肪酸的β氧化中起到了极其重要的作用。本研究旨在获得和鉴定松墨天牛Monochamus alternatus的Mfe2基因,原核表达其编码蛋白,并分析其在该虫不同虫态和成虫不同组织中的表达模式。【方法】采用在载体与目的片段之间设计具有重叠区引物的快速克隆方法克隆松墨天牛Mfe2基因,并对克隆所得序列进行生物信息学分析;IPTG诱导原核表达,并以Western blot结果验证Mfe2基因表达的融合蛋白的成功表达;使用RT-q PCR技术分析这一基因在天牛不同虫态和成虫不同组织中的表达谱。【结果】克隆到松墨天牛Mfe2基因,并命名为Ma Mfe2(Gen Bank登录号:MF944109),其ORF序列全长为2 148 bp,编码716个氨基酸,预测分子量大小为77.56 k D。生物信息分析显示,Ma Mfe2具有AICARFT_IMPCHas保守结构域序列,与光肩星天牛Anoplophora glabripennis过氧化物酶体多功能酶2氨基酸序列一致性最高(为91%)。Western blot检测显示大肠杆菌Escherichia coli BL21(DE3)中Ma Mfe2表达的蛋白与抗体有特异性结合。发育表达谱结果表明,Ma Mfe2在松墨天牛蛹期表达量最高,在卵和幼虫期表达量较低;组织表达谱结果表明,在成虫不同组织中,Ma Mfe2在脂肪体中表达量最高,在前胸中表达量最低。【结论】松墨天牛Mfe2基因Ma Mfe2编码过氧化物酶体多功能酶,在不同虫态和成虫组织中存在差异表达。本研究为进一步探究该基因在松墨天牛中的功能奠定了基础。     

Identification of a novel PP2C-type mitochondrial phosphatase

A novel phosphatase has been cloned and partially characterized. It has a mitochondrial leader sequence and its amino acid sequence places it in the PP2C family like two known mitochondrial phosphatases. Western blot analysis of subcellular fractions and confocal microscopy of 3T3L1 preadipocytes expressing the GFP-tagged protein confirm its mitochondrial localization. Western blot analysis indicates that the protein is expressed in several mouse tissues, with highest expression in brain, heart, liver, and kidney. The recombinant protein exhibits Mn(2+)-dependent phosphoserine phosphatase activity against the branched-chain alpha-keto acid dehydrogenase complex, suggesting the enzyme may play a role in regulation of branched chain amino acid catabolism. Whether there are other mitochondrial substrates for the enzyme is not known.     

重组肺炎链球菌表面黏附素A(rPsaA)的融合表达及抗原性分析

为探索一种大量表达功能性重组肺炎链球菌表面黏附素A(rPsaA)的方法,用PCR和基因重组技术将基因psaA的先导序列替换为伯氏疏螺旋体外膜蛋白A(OspA)信号肽基因序列,并在大肠杆菌中表达。表达产物用SDS-PAGE检测,Western blot分析抗原性。结果表明,rPsaA表达量明显提高且有较好的抗原性。     

Construction of a Novel <Emphasis Type="Italic">Pichia pastoris</Emphasis> Cell-Surface Display System Based on the Cell Wall Protein Pir1

A novel system based on Pir1 from Saccharomyces cerevisiae was developed for cell-surface display of heterologous proteins in Pichia pastoris with the alpha-factor secretion signal sequence. As a model protein, enhanced green fluorescence protein (EGFP) was fused to the N-terminal of the mature peptide of Pir1 (Pir1-a). The expression of fusion protein EGFP-Pir1-a was irregular throughout the P. pastoris cell surface per detection by confocal laser scanning microscopy. A truncated sequence containing only the internal repetitive sequences of Pir1-a (Pir1-b) was used as a new anchor protein in further study. The fusion protein EGFP-Pir1-b was expressed uniformly on the cell surface. The fluorescence intensity of the whole yeast was measured by spectrofluorometer. Western blot confirmed that the fusion proteins were released from cell walls after mild alkaline treatment. The results indicate that a Pir1-based system can express proteins on the surface of P. pastoris and that the fusion proteins do not affect the manner in which Pir1 attaches to the cell wall. The repetitive sequences of Pir1 are required for cell wall retention, and the C-terminal sequence contributes to the irregular distribution of fusion proteins in P. pastoris.     

双委夜蛾气味结合蛋白AdisOBP6的原核表达抗体制备及表达谱分析

【目的】本研究旨在对双委夜蛾Athetis dissimilis气味结合蛋白OBP6进行原核表达、抗体制备以及表达谱分析,便于今后对AdisOBP6功能展开研究。【方法】利用生物信息学软件分析AdisOBP6蛋白结构特征;利用通过原核表达获得的重组蛋白4次免疫新西兰大白兔,制备AdisOBP6抗体;采用荧光定量PCR和Western blot技术检测AdisOBP6在双委夜蛾雌雄成虫触角、不同时期精巢以及受精卵和未受精卵中的表达情况。【结果】同源建模预测显示,AdisOBP6具有6个保守半胱氨酸残基和7个α-螺旋结构,并 折叠成一个结合口袋。原核表达结果显示,在20℃下0.5 mmol/L IPTG诱导重组蛋白表达量最多、最稳定。4次免疫重复中,抗体效价分别超过1∶512 000, 1∶512 000, 1∶512 000和1∶64 000。Westernblot检测获得的抗体与AdisOBP6蛋白能够特异性结合。荧光定量PCR结果显示,AdisOBP6在双委夜蛾精巢中的表达量远高于在触角中的,在幼虫期精巢中AdisOBP6就已经开始表达,在蛹期精巢中表达量低于在幼虫期的, 成虫羽化后精巢中的表达量逐渐进入高峰,在未受精卵和受精卵中几乎不表达或表达量极低。Western blot分析发现,AdisOBP6蛋白也主要在精巢中表达,在雌雄成虫触角、受精卵和未受精卵中表达量很低。【结论】本研究实现了AdisOBP6的原核表达,成功地制备了抗体;并证实AdisOBP6在双委夜蛾精巢中大量表达,成虫羽化后表达量达到高峰,推测AdisOBP6可能参与了授精过程。本研究结果为今后进一步研究AdisOBP6的功能奠定了基础。     

Molecular cloning,expression patterns and subcellular localization of porcine <Emphasis Type="Italic">TMCO1</Emphasis> gene

The product of transmembrane and coiled-coil domains 1 (TMCO1) gene is a member of DUF841 superfamily of several eukaryotic proteins with unknown function. The partial DNA sequence of porcine TMCO1 was first cloned with a pig 567 bp ORF encoding 188 amino acids. By tissues expression analysis, the TMCO1 was found highly expressed in the liver, kidney and heart. The porcine TMCO1 protein was subsequently demonstrated to localize in the mitochondrion by confocal fluorescence microscopy. This data provides an important basis for conducing further studies on the functions and regulatory mechanisms underlying the role of TMCO1 gene.     

Confocal quantification of cis-regulatory reporter gene expression in living sea urchin

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.     

Molecular structure, expression analysis and functional characterization of APRIL (TNFSF13) gene in bat (Vespertilio superans Thomas)

A proliferation-inducing ligand (APRIL) is a novel member of the tumor necrosis factor (TNF) superfamily, which is involved in immune regulation. In the present study, the full-length cDNA of APRIL (designated bAPRIL) from bat was cloned using RT-PCR and its biological activities have been characterized. The open reading frame (ORF) of this cDNA consists of 753 bases, encoding a protein of 250 amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other, known APRIL homologs. Real-time quantitative PCR (qPCR) analysis indicated that bAPRIL mRNA was predominantly expressed in bat lymphoid tissue spleen. The SUMO-bsAPRIL was efficiently expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. Laser scanning confocal microscopy analysis showed that bsAPRIL could bind to its receptors on B cells. In vitro, MTT assays indicated that bsAPRIL could promote the survival/proliferation of mouse splenic B cells grown with anti-mouse IgM. These findings indicate that bsAPRIL plays an important role in the survival and proliferation of B cells and has functional cross-reactivity among mammalians. The present findings may provide valuable information for research into the immune system of the bat.     

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.