紫花苜蓿 DREB 1 基因的原核表达与蛋白纯化

构建 MsDREB1 基因的原核表达载体, 对表达产物进行鉴定和纯化, 为研究 DREB1 基因在植株中功能奠定基础。用冷酚法从紫花苜蓿提取 RNA, 并转化成 cDNA, 然后将其克隆至 pET32a 原核表达载体, 构建重组载体 pET32aDREB1 。用重组质粒转化大肠杆菌 BL21, IPTG 诱导表达后进行 SDS-PAGE 分析, 用 Ni-NTA 亲和层析柱对重组蛋白进行纯化。结果表明 , 原核表达载体构建正确 ; SDS-PAGE 分析, 出现了与 DNAMAN 预测的 43.2 kd 大小一致的蛋白条带;经分析重组蛋白纯化率达到 90%以上。     

阴道毛滴虫黏附蛋白65-3基因原核表达质粒的构建及表达

质粒 pMD-18T-ap65-3 经 Xho I 和 BamH I 双酶切, 1.0% 凝胶电泳后纯化回收阴道毛滴虫黏附蛋白 65-3 基因,定向克隆至原核表达载体 pET-32a( ), 构建原核表达质粒 pET32a-ap65-3. 重组质粒转化大肠埃希菌 JM109 感受态细胞中, 筛选阳性克隆, 进行PCR﹑酶切及测序鉴定正确后, 将重组质粒 pET32a-ap65-3 转化于大肠埃希菌 BL21 中,异丙基-β-D硫代半乳糖苷 (IPTG) 诱导重组蛋白表达后利用十二烷基磺酸钠一聚丙烯酰胺凝胶电泳 (SDS-PAGE) 和免疫印迹 (Western blot) 对重组蛋白进行分析鉴定. 本实验为研究阴道毛滴虫病的致病机制及蛋白生物学功能奠定了基础.     

立氏立克次体外膜蛋白H 基因片段的克隆表达与免疫原性分析

目的:克隆表达立氏立克次体(Rickettsia rickettsii)外膜蛋白H基因(ompH)片段并对其进行免疫原性分析。方法:采用PCR技术从立氏立克次体基因组中扩增ompH基因片段,将该基因片段与原核表达载体pET32a连接,构建重组原核表达质粒pET32a/ompH;将pET32a/ompH转入大肠杆菌细胞内,用IPTG诱导转化大肠杆菌表达目的基因。结果:获得长为327bp的ompH基因片段,SDS-PAGE分析发现pET32a/ompH转化菌表达了大小约27kDa蛋白,该蛋白与立氏立克次体免疫豚鼠血清及斑点热患者血清在免疫印迹分析中呈阳性反应,经该重组蛋白免疫血清中和后的立氏立克次体感染VERO活力减低。结论:pET32a/ompH转化的大肠杆菌表达了ompH基因片段,所产生的重组蛋白具有良好的免疫反应性及保护性。     

立氏立克次体外膜蛋白H基因片段的克隆表达与免疫原性分析

目的：克隆表达立氏立克次体（Rickettsia rickettsii）外膜蛋白H基因（ompH）片段并对其进行免疫原性分析。方法：采用PCR技术从立氏立克次体基因组中扩增ompH基因片段,将该基因片段与原核表达载体pET32a连接,构建重组原核表达质粒pET32a/ompH;将pET32a/ompH转入大肠杆菌细胞内,用IPTG诱导转化大肠杆菌表达目的基因。结果：获得长为327bp的ompH基因片段,SDS-PAGE分析发现pET32a/ompH转化菌表达了大小约27kDa蛋白,该蛋白与立氏立克次体免疫豚鼠血清及斑点热患者血清在免疫印迹分析中呈阳性反应,经该重组蛋白免疫血清中和后的立氏立克次体感染VERO活力减低。结论：pET32a/ompH转化的大肠杆菌表达了ompH基因片段,所产生的重组蛋白具有良好的免疫反应性及保护性。     

白念珠菌MP65和SAP2蛋白原核表达质粒的构建与表达

目的构建以白念珠菌基因MP65和SAP2为目的基因的原核表达质粒,IPTG诱导其表达融合蛋白,并对其免疫原性进行分析。方法PCR法自白念珠菌标准菌株获取MP65和SAP2基因,分别插入至原核表达载体pGEx．4T之和pET32a中;将重组表达质粒转染感受态EcoliBL-21,经IPTG诱导表达、纯化后,SDS—PAGE和Western blot分析。结果PCR法克隆出全长为1140bp的MP65和1197bp的SAP2基因,构建的原核表达质粒pGEX-4T-2-MP65及pET32a-SAP2,可分别表达出66KD左右的GST融合蛋白和His融合蛋白。结论成功获取了白念珠菌基因^伊65和SAP2,所构建的原核表达质粒在BL-21中成功表达;两种蛋白均有免疫原性。     

重组酶Exo、Beta和Gam在大肠杆菌中的表达与纯化

目的:构建exo、beta和gam基因的原核表达质粒,在大肠杆菌中表达、制备重组酶Exo、Beta和Gam。方法:将exo、beta和gam基因分别构建在原核表达载体pEGKG和pET28a上,分别转化大肠杆菌BL21(DE3)和DH5α,经IPTG诱导后,在大肠杆菌中获得可溶性表达蛋白,用柱层析方法纯化蛋白。结果:构建了原核表达质粒pET28a-exo、pET28a-beta、pET28a-gam和pEGKG-exo、pEGKG-beta、pEGKG-gam;SDS-PAGE结果表明重组酶融合蛋白His-Exo、GST-Exo、His-Beta、GST-Beta、His-Gam、GST-Gam得到可溶性表达;用His标签抗体和GST标签抗体通过Western印迹方法检测到纯化后的融合蛋白。结论:在大肠杆菌中诱导表达了λ噬菌体重组酶,并纯化获得了一定量的纯度较好的蛋白,为下一步制备检测用多克隆抗体奠定了基础。     

猪链球菌2型溶血素基因与38KDa蛋白基因克隆及联合表达

目的:研究有效的猪链球菌病疫苗。方法:以四川株猪链球菌2型基因组DNA为模板分别扩增溶血素基因和38KDa基因主要功能区基因;并经连接、克隆及酶切鉴定。分别构建原核表达载体pET32a-Sly、pET32a-38KDa,提取阳性克隆质粒分别进行双酶切并纯化,通过PCR串联两片断,将目的片断定向克隆到表达载体pET32a中,经测序正确后,重组质粒转化入大肠杆菌BL21 (DE3),用IPTG进行诱导表达。结果:重组菌菌体裂解物经SDS-PAGE电泳可检测到相对分子量约为60Ku的重组蛋白,表达产物经纯化后,免疫印迹法(Western-blotting)证实该重组蛋白可以与SS2阳性血清发生特异性反应。结论:本研究为重组蛋白的免疫保护应用奠定基础。     

Lys_(10)-PAI-1融合蛋白在大肠杆菌中的表达、纯化和凝胶阻滞实验

用PCR方法构建 10个赖氨酸与纤溶酶原激活剂抑制物 1的融合基因Lys10 PAI 1,并克隆于pET2 8a(+ )和pET32a(+ )原核表达载体 .将重组表达质粒pET32 PAI和pET2 8 PAI转化大肠杆菌BL2 1(DE3) .IPTG诱导后可获得分子量分别为 6 3kD和 4 3kD的目的蛋白 ,表达蛋白占菌体蛋白2 0 %以上 ,大多数重组蛋白以不溶形式存在 .表达产物经变性、复性、超滤、透析和亲和层析等步骤 ,可以得到纯化的Trx·PAI 1和rPAI 1重组蛋白 .Western印迹结果表明 ,目的蛋白具有人PAI 1的抗原性 .凝胶阻滞实验发现 ,纯化的重组蛋白在一定条件下 ,可以与质粒结合 ,使质粒的迁移率明显改变 .研究结果表明 ,Trx·PAI 1和rPAI 1有希望成为受体介导基因转移的配体     

分选酶A在pET32a(+)原核表达载体中的表达和鉴定

旨在pET32a(+)原核表达载体中表达金黄色葡萄球菌(Staphylococcus aureus)中的转肽酶分选酶(SrtA)并进行鉴定.以含有pET22-srtA质粒为模板,设计并合成引物,PCR扩增得到SrtA△N24和SrtA△N59基因,经过BamH Ⅰ、Xho Ⅰ酶切,克隆入表达载体pET32a(+)中,构建重组载体pET32a-SrtA△N24及pET32a-SrtA△N59,并转化入大肠杆菌BL21(DE3).经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blotting对表达产物分别进行分析和鉴定.然后对重组质粒在大肠杆菌BL21(DE3)中的表达条件进行了优化.结果显示重组载体pET32a-SrtA△N24和pET32a-SrtA△N59分别表达出相对分子量为约42 kD和37 kD的融合蛋白,经SDS-PAGE和Westem blotting检测显示其分子量与预期的大小相符合.成功构建了重组质粒pET32a-SrtA△N24和pET32a-SrtA△N59,并且在大肠杆菌BL21(DE3)中获得了高效融合表达.     

白喉毒素无毒突变体CRM197基因的克隆与表达

目的构建白喉毒素(Diphtheria toxin)无毒突变体CRM197(Cross-reacting materials 197)的原核表达载体,并在大肠杆菌中表达重组蛋白。方法以白喉杆菌(ATCC39255)基因组DNA为模版,采用聚合酶链式反应(Polymerase chain reaction,PCR)扩增CRM197基因,插入表达载体pET11b中,构建重组原核表达质粒pET11b-CRM197。经双酶切及测序鉴定正确后,重组质粒被转化入大肠杆菌Rosetta 2(DE3)pLysS,IPTG诱导表达,表达产物经SDS-PAGE和Western blot进行鉴定。结果重组表达质粒经双酶切及测序鉴定,结果表明与预期一致;表达的重组蛋白相对分子质量约58 000,并可与鼠抗CRM197单克隆抗体特异性结合。结论已成功构建了重组原核表达载体pET11b-CRM197,重组的CRM197蛋白在大肠杆菌中得到了表达,为以该重组突变体作蛋白载体制备结合疫苗奠定了基础。     

Neuritin Attenuates Cognitive Function Impairments in Tg2576 Mouse Model of Alzheimer's Disease

Neuritin, also known as CPG15, is a neurotrophic factor that was initially discovered in a screen to identify genes involved in activity-dependent synaptic plasticity. Neuritin plays multiple roles in the process of neural development and synaptic plasticity, although its binding receptor(s) and downstream signaling effectors remain unclear. In this study, we found that the cortical and hippocampal expression of neuritin is reduced in the brains of Alzheimer''s disease (AD) patients and demonstrated that viral-mediated expression of neuritin in the dentate gyrus of 13-month-old Tg2576 mice, an AD animal model, attenuated a deficit in learning and memory as assessed by a Morris water maze test. We also found that neuritin restored the reduction in dendritic spine density and the maturity of individual spines in primary hippocampal neuron cultures prepared from Tg2576 mice. It was also shown that viral-mediated expression of neuritin in the dentate gyrus of 7-week-old Sprague-Dawley rats increased neurogenesis in the hippocampus. Taken together, our results demonstrate that neuritin restores the reduction in dendritic spine density and the maturity of individual spines in primary hippocampal neurons from Tg2576 neurons, and also attenuates cognitive function deficits in Tg2576 mouse model of AD, suggesting that neuritin possesses a therapeutic potential for AD.     

Expression and purification of recombinant human neuritin from <Emphasis Type="Italic">Pichia pastoris</Emphasis> and a partial analysis of its neurobiological activity in vitro

Neuritin (also known as candidate plasticity gene 15 (cpg15)) is a neurotrophic factor that was recently discovered in a screen aimed at identifying genes involved in activity-dependent synaptic plasticity. Neuritin plays multiple roles in both neural development (Chen et al. Zhonghua Yan Ke Za Zhi 46:978–983 2010; Corriveau et al. J Neurosci 19:7999-8008 1999; Lee and Nedivi J Neurosci 22:1807-1815 2002) and synaptic plasticity (Fujino et al. Gene Dev 25:2674-2685 2011; Leslie and Nedivi Prog 14 Neurobiol 94:223-237 2011; Loebrich and Nedivi Physiol Rev 89:1079 2009). In this study, to produce bioactive, soluble recombinant human neuritin protein, a portion of NRN1 was cloned into the Pichia pastoris expression vector pPIC9K. The recombinant vector was then transformed into the methylotrophic yeast strain P. pastoris GS115, and a shaking flask method and His-tag purification strategy were utilized to express and purify neuritin protein. The resulting protein had a molecular mass of approximately 11 kDa, and subsequent functional analyses indicated that the purified neuritin promoted neurite outgrowth from embryonic chicken dorsal root ganglions, while also prolonging the survival of these ganglions, and from PC12 cells. These findings suggest that neuritin was expressed effectively in vitro and that this protein may play a role in stimulating neurite outgrowth of both dorsal root ganglions and PC12 cells. This study provides a novel strategy for the large-scale production of bioactive neuritin, which will enable further study of the biological function of this protein.     

NGF与Neuritin在脑外伤伴四肢骨折病人血清中的表达及意义

目的：通过检测脑外伤患者、脑外伤合并骨折患者、骨折患者及正常人外周血中NG3F,neuritin不同时间段的表达,根据其含量的变化,判断与骨折愈合速度的相关性,寻找骨折愈合的关键因子。方法：收集单纯脑外伤、单纯骨折患者各80例、脑外伤合并骨折患者60例、健康体检人群20例。选取外伤后3天、10天、2周抽取所有实验对象的静脉血,应用ELISA技术测定标本中NGF与Neuritin的含量。结果：损伤后每个时间段里,患者血清中NGF、neuritin含量有不同程度升高,均高于正常对照组,其中又以脑外伤合并骨折组最高。血清NGF在骨折合并脑外伤组伤后第3天含量明显升高,为（0．86±0．21）,伤后10天为（1．47±0．29）。14天为（2．07±0．21）,脑外伤合并四肢骨折组neuritin血清含量在伤后第3天略有升高为（83．47±18．85）,10天（108．50±31．65）,2周（91．86±21．12）．脑外伤合并骨折病人血清NGF、neuritin表达明显高于其他对照组,差别均有统计学意义（P〈0．05）。结论：脑外伤合并骨折病人血清NGF、neuritin表达明显高于其他对照组,说明与骨折愈合有着密切的相关性,两种因子可能在骨折愈合修复过程中共同起作用,从而,推测可能是脑外伤后骨折愈合加速的重要因素。     

Characterization of neuritin as a novel angiogenic factor

Neuritin (NRN1), a neurotrophic factor, plays an important role in neurite growth and neuronal survival. In this study, we identify a new function of neuritin as a novel angiogenic factor in vitro and in vivo. Recombinant neuritin protein had no effect on the proliferation and adhesion of human umbilical vein endothelial cells (HUVEC), but it dose-dependently increased endothelial cell migration. Furthermore, overexpression of neuritin significantly promoted tumor angiogenesis, and surprisingly, it inhibited tumor growth in a xenograft tumor model. Thus, our results indicate that neuritin may act as an important angiogenic factor and serve as a potential target for cancer therapy.     

Neuritin Activates Insulin Receptor Pathway to Up-regulate Kv4.2-mediated Transient Outward K+ Current in Rat Cerebellar Granule Neurons

Neuritin is a new neurotrophic factor discovered in a screen to identify genes involved in activity-dependent synaptic plasticity. Neuritin also plays multiple roles in the process of neural development and synaptic plasticity. The receptors for binding neuritin and its downstream signaling effectors, however, remain unclear. Here, we report that neuritin specifically increases the densities of transient outward K⁺ currents (I_A) in rat cerebellar granule neurons (CGNs) in a time- and concentration-dependent manner. Neuritin-induced amplification of I_A is mediated by increased mRNA and protein expression of Kv4.2, the main α-subunit of I_A. Exposure of CGNs to neuritin markedly induces phosphorylation of ERK (pERK), Akt (pAkt), and mammalian target of rapamycin (pmTOR). Neuritin-induced I_A and increased expression of Kv4.2 are attenuated by ERK, Akt, or mTOR inhibitors. Unexpectedly, pharmacological blockade of insulin receptor, but not the insulin-like growth factor 1 receptor, abrogates the effect of neuritin on I_A amplification and Kv4.2 induction. Indeed, neuritin activates downstream signaling effectors of the insulin receptor in CGNs and HeLa. Our data reveal, for the first time, an unanticipated role of the insulin receptor in previously unrecognized neuritin-mediated signaling.     

Exogenous neuritin treatment improves survivability and functions of Schwann cells with improved outgrowth of neurons in rat diabetic neuropathy

Pathogenesis and treatment for diabetic neuropathy are still complex. A deficit of neurotrophic factors affecting Schwann cells is a very important cause of diabetic neuropathy. Neuritin is a newly discovered potential neurotrophic factor. In this study, we explored the effect of exogenous neuritin on survivability and functions of diabetic Schwann cells of rats with experimental diabetic neuropathy. Diabetic neuropathy was induced in rats. 12‐week diabetic rats contrasted with non‐diabetic normal rats had decreased levels of serum neuritin and slowed nerve conduction velocities (NCVs). Schwann cells isolated from these diabetic rats and cultured in high glucose showed reduced cell neuritin mRNA and protein and supernatant neuritin protein, increased apoptosis rates, increased caspase‐3 activities and progressively reduced viability. In contrast, exogenous neuritin treatment reduced apoptosis and improved viability, with elevated Bcl‐2 levels (not Bax) and decreased caspase‐3 activities. Co‐cultured with diabetic Schwann cells pre‐treated with exogenous neuritin in high glucose media, and diabetic DRG neurons showed lessened decreased neurite outgrowth and supernatant NGF concentration occurring in co‐culture of diabetic cells. Exogenous neuritin treatment ameliorated survivability and functions of diabetic Schwann cells of rats with diabetic neuropathy. Our study may provide a new mechanism and potential treatment for diabetic neuropathy.     

Neuritin Attenuates Neuronal Apoptosis Mediated by Endoplasmic Reticulum Stress In Vitro

Neuritin is an extracellular glycophosphatidylinositol-linked protein that promotes neuronal survival, differentiation, function, and repair, but the exact mechanism of this neuroprotective effect remains unclear. Meanwhile, endoplasmic reticulum stress (ERS) induced apoptosis is attracting increased attention. In this work, we hypothesized that neuritin inhibited ERS to protect cortical neurons. To check this hypothesis, we exposed primary cultured cortical neurons to oxygen and glucose deprivation (OGD) for 45 min followed by reperfusion (R) to activate ERS. We then performed resuscitation for 6, 12, 24, and 48 h. ERS-related factors such as glucose-regulated protein 78 (GRP78), caspase-12 and CHOP were detected by Western blotting and quantitative real-time polymerase chain reaction assay. Apoptosis was assessed by Annexin V binding and propidium iodide staining. Ultrastructural changes of endoplasmic reticulum were observed under a transmission electron microscope. Results showed that GRP78 expression significantly increased at 12, 24, and 48 h and peaked at 24 h. Caspase-12 and CHOP expression significantly increased in a time-dependent manner at 12, 24, and 48 h. GRP78, caspase-12 and CHOP expression as well as apoptosis rate of primary cultured neurons and the ultrastructural changes of endoplasmic reticulum in the OGD/R?+?neuritin group significantly improved compared with the OGD/R group. In conclusion, the neuroprotection function of neuritin may be involved in ERS pathways.     

Neuritin在大鼠脑外伤合并骨折过程中的作用研究

目的：研究脑外伤合并股骨骨折的骨痂中Neuritin的表达及血清中变化,探讨中枢神经系统损伤加速对骨折愈合的作用,为临床难治性骨创伤提供新的理论依据。方法：取96只雄性SD大鼠随机分成：A组正常组8只、B组单纯骨折组40只、C组单纯脑外伤组8只及D组骨折合并脑外伤组40只,做股骨骨折和采用脑损伤液压装置建脑损伤模型,术后3、7、14、21、28天取血离心,ELISA法测血清中的Neuritin值。分时间处死B组和D组,取骨痂做切片,行免疫组织化学染色,观测各时间点骨痂中Neuritin的变化和骨折愈合情况。结果：①免疫组织化学染色：骨折愈合过程中血管内皮细胞、软骨细胞及成骨细胞的胞浆中有阳性表达,并显色强。D组1w至3w时的Neuritin阳性细胞百分数均高于B组有显著性统计意义（P〈0．05）,但4周时（P〉0．05）。②血清浓度值：A组值（81．37±1．37）,B组、C组、D组3天（94．94±3．77107．28±3．46118．35±1．43）逐渐升高,2周达到高峰（110．18±1．48131．89±3．26161．48±1．46）,然后下降,3d至3w时各组有显著性统计意义（P〈0．05）,4周下降为略高于正常（P〉0．05）。结论：血清和骨痂中Neuritin的表达显著升高,提示Neuritin可能是大鼠股骨骨折合并脑损伤时促进骨折修复的重要因素一。     

弗氏2a志贺氏菌2457T株YciD蛋白的融合表达和纯化

目的：原核表达重组弗氏2a志贺氏菌2457T株YciD蛋白,为其功能研究奠定基础。方法：用PCR方法从弗氏2a志贺氏菌2457T株染色体中扩增YciD蛋白编码序列,经过纯化、酶切后克隆到原核表达载体pET32a中,构建重组载体pET32a-yciD,转化大肠杆菌BL21（DE3）菌株获得工程菌株,对其表达和纯化条件进行优化;利用Western Blot检测融合蛋白的表达。结果：构建了YciD蛋白的融合表达载体,并在大肠杆菌中获得高效表达;经Ni-NTA亲和层析柱纯化获得了高纯度的YciD蛋白;Western Blot表明,此蛋白可与His标签抗体反应,表明获得了目的蛋白。结论：在原核表达系统中表达、纯化弗氏2a志贺氏菌2457T株YciD蛋白,为进一步对其进行功能研究奠定了基础。