HIV-1衣壳蛋白在转基因枸杞中表达的免疫组织化学定位

目的:研究转基因植物中重组蛋白的细胞定位,有助于进一步了解转基因枸杞中HIV-1衣壳(CA)蛋白融合蛋白的分泌表达途径。方法:利用含有MA4-CA融合基因的农杆菌转化枸杞,转化植株获得再生。采用免疫组织化学方法对转基因枸杞表达的CA融合蛋白进行初步定位。结果:免疫组织化学定位表明,在转基因枸杞愈伤组织中,HIV-1CA主要在细胞浆、细胞壁和细胞间隙中表达。结论:免疫组织化学结果初步证明了CA融合蛋白在转基因枸杞中的表达分布。     

转基因枸杞表达HIV-1壳体蛋白病毒样颗粒疫苗表达载体的构建

利用PCR技术扩增出人免疫缺陷病毒HIV-1MA₄-CA融合基因,将其克隆到pGEM-T载体中,测定其核苷酸序列,并推导其氨基酸序列。该基因全长为450bp,与已发表的HIV-1的全序列基因(AF324493)完全同源,编码一个含150个氨基酸残基的蛋白质。将该基因与分泌型表达载体pCAMBIA1305.2连接,同时将水稻中富含甘氨酸蛋白的信号肽序列(GRP)引入MA₄-CA融合基因,构建了含MA₄-CA基因的植物分泌表达载体pCAMBIA1305.2-MA₄-CA。     

HIV-1融合蛋白MA4-CA在大肠杆菌中的高效表达

将HIV-1MA4-CA融合基因克隆到高效表达载体pBV220中,将该重组表达载体转化大肠杆菌BL21,进行诱导表达。收集细菌,菌体裂解后进行SDS—PAGE检测。结果表明,成功地构建了含MA4-CA融合基因的表达载体pBV220-MA4-CA,该载体能在大肠杆菌中表达相对分子质量为16000并以包涵体形式存在的融合蛋白,此蛋白经洗涤后能够溶于8mol／L的尿素中。利用硫酸铵沉淀法进行初步纯化后即可得到纯度比较高的MA4-CA融合蛋白,为今后进一步的功能和应用研究打下了良好的基础。     

表达HIV壳体蛋白转基因枸杞悬浮细胞的培养与鉴定

枸杞是我国珍贵的中药材,利用枸杞作为转基因材料具有易于遗传操作,生物性状稳定的优点。将携有人类免疫缺陷病毒I型（HIV-1）壳体蛋白基因的植物表达载体导入根瘤农杆菌EHA105中,并通过农杆菌侵染枸杞叶片,诱导产生抗性愈伤组织,利用抗性愈伤组织作为材料进行悬浮细胞的培养并对转基因枸杞悬浮细胞鉴定。PCR结果表明已获得遗传转化的转基因枸杞悬浮细胞系。免疫组织化学检测结果表明HIV壳体蛋白已在转基因枸杞悬浮细胞中表达。     

转基因枸杞中蛋白酶体糜蛋白酶样活性的研究

利用提取的转基因枸杞和正常枸杞蛋白与蛋白酶体的特异性荧光底物Suc-LLVY-AMC室温孵育,于360nm（激发光）/460nm（发射光）波长下测定荧光值的方法,研究了转基因枸杞和正常枸杞中蛋白酶体糜蛋白酶活性的差异及其特异性抑制剂MG115对枸杞中蛋白酶体活性的影响。结果表明表达H IV壳体蛋白的转基因枸杞蛋白酶体活性是空载体对照转基因枸杞蛋白酶体活性的3.6倍,是正常枸杞的4.2倍。10μM的MG115对表达H IV壳体蛋白的转基因枸杞蛋白酶体活性抑制率为87%,携带空载体对照的转基因枸杞抑制率为74%,而对正常枸杞抑制率仅为8.6%;50μM的MG115抑制作用与10μM的相比没有明显变化。这一结果将为利用蛋白酶体抑制剂提高转基因枸杞悬浮细胞H IV-1CA含量的研究提供理论依据。     

MMP-9信号肽高效诱导PEX重组蛋白在COS7细胞中分泌表达

为了便于收集和纯化, 重组蛋白常需要引导至真核细胞外。蛋白能否分泌主要取决于其是否含有信号肽, 由于不同信号肽诱导蛋白分泌的效率不同,高效信号肽的筛选已成为生物工程领域提高重组蛋白产量的重要策略之一。为了筛选诱导MMP-2 C末端PEX在COS7细胞中高效分泌表达的信号肽,在PEX的N末端分别融合大鼠生长激素(rGH)、小鼠IgG κ链和人基质金属蛋白酶-9（matrix metalloproteinase 9, MMP-9）的信号肽并比较三种信号肽引导PEX分泌表达的效率。Western免疫印迹和ELISA蛋白定量检测表明MMP-9的信号肽引导PEX蛋白分泌的效率约为其它两种信号肽的两倍。利用Ni-NTA亲和柱对细胞培养基中的PEX进行纯化,蛋白产量约为1mg/L,纯化的PEX重组蛋白具有抑制鸡尿囊膜（chorioallantoic membrane,CAM）血管发生的作用。以上结果提示MMP-9的信号肽有效诱导具有生物活性的PEX重组蛋白在COS7细胞中分泌表达。     

人神经生长因子信号肽介导β-内啡肽的分泌表达

目的:构建人神经生长因子信号肽与人β-内啡肽融合基因的真核表达载体,研究人神经生长因子信号肽介导β-内啡肽的分泌表达.方法:取得人基因组后,PCR 法获取人的神经生长因子信号肽部分序列及人β-内啡肽序列;通过 SOE-PCR法将两段 DNA 序列连接,然后插入到真核表达载体内,测序正确后扩增转染级的真核表达载体.表达载体脂质体法转染 NIH3T3细胞,转染后 48-72h 收集细胞及培养上清,RT-PCR 法检测融合基因的转录.RIA 法测定细胞外β-内啡肽的浓度.结果:成功构建全人源的分泌型表达β-内啡肽的真核表达载体,DNA 序列经测序完全符合实验设计;融合基因能够顺利地得到转录并进行表达翻译,在细胞培养上清中可检测到其产物.结论:构建的真核表达载体能够分泌表达人β-内啡肽,提示人神经生长因子信号肽序列能够发挥其介导蛋白产物分泌表达的作用.     

犬细小病毒VP2蛋白在真核细胞中的分泌表达及特性

摘要:【目的】利用真核细胞分泌表达犬细小病毒VP2蛋白和研究其特性。【方法】为构建犬细小病毒（Canine parvovirus, CPV）VP2基因的真核分泌型表达载体,首先通过酶切从含有人CD5信号肽序列的质粒中将CD5信号肽基因片段切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2基因,并将其插入到pcDNA3.1- CD5sp载体中CD5信号肽的下游,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经磷酸钙介导转染293T细胞,使其在真核细胞中进行分泌表达,并通过ELISA检测表达的VP2蛋白与犬转铁蛋白受体（TfR）结合的活性。【结果】序列分析结果表明,本实验构建的犬细小病毒VP2基因真核分泌型表达载体结构正确,将该表达载体转染的293T细胞,在培养基中通过Western-blot检测到有VP2重组蛋白的存在。经ELISA检测表明表达的重组VP2蛋白具有与犬转铁蛋白受体结合的活性。【结论】 利用人的CD5信号肽实现了犬细小病毒VP2蛋白在真核细胞中的分泌表达,表达的VP2蛋白具有与犬转铁蛋白受体结合的活性。     

hK-Fc融合蛋白的改良、表达及其生物活性的分析

为了延长人激肽释放酶(hK)的血清半衰期,提高分泌蛋白的产率,制备了重组激肽释放酶-IgG1 Fc融合蛋白(hK'-Fc)。采用PCR扩增hK基因和IgG1的Fc序列,用鼠源信号肽序列替换hK基因原有的信号肽序列,构建改良型融合蛋白hK'-Fc以及天然型融合蛋白hK-Fc的表达载体,转染中国仓鼠卵巢细胞(CHO)细胞,筛选稳定分泌融合蛋白的细胞株,通过Western blotting鉴定信号肽改造效果,利用Protein A+G亲合层析柱纯化融合蛋白,酶学实验检测融合蛋白的体外活性。结果表明:成功构建了pcDNA-hK'-Fc以及pcDNA-hK-Fc重组表达载体;获得了稳定表达融合蛋白的细胞株,产量达11mg/L以上;信号肽改造后融合蛋白的分泌效率提高约5～10倍;融合蛋白能水解其特异性的底物S-2266,具有生物学活性。本研究为进一步探讨融合蛋白的体内半衰期打下了坚实基础,也为研制治疗脑梗塞疗效更好的第二代hK蛋白和其他药用蛋白的改良提供新的线索。     

编码山羊补体C3d与口蹄疫病毒VP1融合蛋白重组质粒的构建及表达

旨在构建含分子佐剂山羊补体C3d基因的O型口蹄疫病毒VP1基因真核表达质粒。克隆山羊C3d基因, 通过linker(G4S)2将3拷贝C3d基因串联; 克隆羊源O型口蹄疫病毒VP1基因, 通过linker(G4S)2与3拷贝C3d基因相连, 构建重组质粒pUC19-VP1-C3d3。将VP1-C3d3融合基因亚克隆入含有分泌表达信号肽tPA序列的pcDNA3.1(+)CMV启动子下游, 构建重组真核表达质粒pcDNA3.1-tPA-VP1-C3d3。在脂质体介导下, 将pcDNA3.1-tPA -VP1-C3d3转染HeLa细胞。间接免疫荧光分析表明, VP1- C3d3在HeLa细胞中获得了瞬时表达, Western blot分析证实转染的阳性细胞能分泌预期大小(133 kD)的融合蛋白。重组质粒pcDNA3.1-tPA-VP1-C3d3为研制以羊补体C3d为分子佐剂的口蹄疫新型疫苗奠定了基础。     

TransgenicLycium barbarum L. Established as HIV capsid protein expression system

A DNA fragment (MA₄-CA) encoding HIV CA viral like particles (VLPs) was transferred intoLycium barbarum L. byAgrobacterium tumefaciens-mediated transformation. PCR amplification analysis of DNA from transgenic plants confirmed the presence of the fusion gene under the control of promoter p35S. CA protein was identified in transformed leaf extracts by enzyme-linked immunosorbent assay (ELISA), indicating that a transgenicLycium barbarum L. expression system can produce immunogenic CA. Supported by the Natural Science Foundation of China, Nos. 30271215 and 30471621     

Production and secretion of recombinant thaumatin in tobacco hairy root cultures

Production of recombinant proteins in plant cell or organ cultures and their secretion into the plant cell culture medium simplify the purification procedure and increase protein yield. In this study, the sweet-tasting protein thaumatin I was expressed and successfully secreted from tobacco hairy root cultures. The presence of an ER signal peptide appears to be crucial for the secretion of thaumatin: without an ER signal peptide, no thaumatin was detectable in the spent medium, whereas inclusion of the ER signal peptide calreticulin fused to the N terminus of thaumatin led to the secretion of thaumatin into the spent medium of hairy root cultures at concentrations of up to 0.21 mg/L. Extracellular thaumatin levels reached a maximum after 30 days (stationary phase) and the subsequent decline was linked to the rapid increase of proteases in the medium. Significant amounts of thaumatin were trapped in the apoplastic space of the root cells. The addition of polyvinylpyrrolidone and sodium chloride into the culture medium led to an increase of extracellular thaumatin amounts up to 1.4 and 2.63 mg/L, respectively. Thaumatin production compares well with yields from other transgenic plants, so that tobacco hairy roots can be considered an alternative production platform of thaumatin.     

Production of recombinant proteins in plant root exudates.

The large-scale production of recombinant proteins in plants is limited by relatively low yields and difficulties in extraction and purification. These problems were addressed by engineering tobacco plants to continuously secrete recombinant proteins from their roots into a simple hydroponic medium. Three heterologous proteins of diverse origins (green fluorescent protein of jellyfish, human placental alkaline phosphatase [SEAP], and bacterial xylanase) were produced using the root secretion method (rhizosecretion). Protein secretion was dependent on the presence of the endoplasmic reticulum signal peptide fused to the recombinant protein sequence. All three secreted proteins retained their biological activity and, as shown for SEAP, accumulated in much higher amounts in the medium than in the root tissue.     

Production and secretion of a heterologous protein by turnip hairy roots with superiority over tobacco hairy roots

A fully contained and efficient heterologous protein production system was designed using Brassica rapa rapa (turnip) hairy roots. Two expression cassettes containing a cauliflower mosaic virus (CaMV) 35S promoter with a duplicated enhancer region, an Arabidopsis thaliana sequence encoding a signal peptide and the CaMV polyadenylation signal were constructed. One cassette was used to express the green fluorescent protein (GFP)-encoding gene in hairy roots grown in flasks. A stable and fast-growing hairy root line secreted GFP at >120 mg/l culture medium. GFP represented 60 % of the total soluble proteins in the culture medium. Turnip hairy roots retained sustainable growth and stable GFP production over 3 years. These results were superior to those obtained using tobacco hairy roots.     

Exploratory studies on CA-15L,an anti-HIV active HIV-1 capsid fragment

Utilizing overlapping fragment peptide libraries covering the whole sequence of an HIV-1 capsid (CA) protein with the addition of an octa-arginyl moiety, we had previously found several peptides with anti-HIV-1 activity. Herein, among these potent CA fragment peptides, CA-15L was examined because this peptide sequence overlaps with Helix 7, a helix region of the CA protein, which may be important for oligomerization of the CA proteins. A CA-15L surrogate with hydrophilic residues, and its derivatives, in which amino acid sequences are shifted toward the C-terminus by one or more residues, were synthesized and their anti-HIV activity was evaluated. In addition, its derivatives with substitution for the Ser149 residue were synthesized and their anti-HIV activity was evaluated because Ser149 might be phosphorylated in the step of degradation of CA protein oligomers. Several active compounds were found and might become new anti-HIV agents and new tools for elucidation of CA functions.     

枸杞的组织培养及植株再生的条件优化

目的:探讨枸杞组织培养及其植株再生条件的优化。方法:应用MS培养基为基本培养基,以各种不同激素配比进行枸杞愈伤诱导、分化诱导及根的诱导。结果:以枸杞叶片为外植体,利用2,4-D(2,4-二氯苯氧基乙酸)与KT(细胞分裂素)不同配比诱导出了愈伤组织。利用6-BA(6-苄基腺嘌呤)与NAA(α-萘乙酸)不同浓度的配比组合,成功地进行了杞再生芽诱导及根系诱导。结论:以MS培养基为基本培养基,并采用各种激素的不同配比,可以优化枸杞植株的再生条件。