植物抗体及其应用

20世纪80年代末科学家将抗体基因转入烟草表达，标志着植物抗体的诞生。人类既可以以植物为生物反应器异源表达和生产具有药用及商业价值的蛋白质；也可用抗体在植物体中进行免疫调节，以研究植物生理代谢机制，或增加植物抵抗病虫害的能力。本文旨对植物抗体的优越性、表达生产及其在医药学、植物抗病及代谢等领域的进展进行综述和讨论。     

植物表达抗体的研究进展

植物抗体(plantibody)是利用基因工程技术在植物中表达或生产抗体, 即将编码全抗体或抗体片段的基因导入植物,在植物中表达出具有功能性识别抗原及结合特性的全抗体或部分抗体片段, 是抗体基因工程研究的一个新领域, 它有如下优点: (1)高表达量。在植物中表达抗体, 重组抗     

基因工程抗体的原核高效表达

基因工程抗体具有生产简单、价格低廉和容易获得稀有抗体等优点而日益受到广泛关注 ,是当前制备治疗性抗体的主要手段。抗体表达量低依然是制约其大规模生产和应用的主要因素 ,通过对表达载体、宿主细胞和表达条件等进行改造与优化 ,将有可能获得基因工程抗体的高效表达     

PIWIL4的克隆及其抗体制备和初步应用

目的 制备兔抗人PIWIL4的多克隆抗体,鉴定其特异性,并应用该抗体检测内源性PIWIL4在人各细胞系中的表达差异及细胞定位.方法 构建原核表达质粒pGEX-5X-1-PIWIL4,转化大肠杆菌BL21,PIWIL4蛋白经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达.融合蛋白通过切胶纯化后免疫家兔制备抗体血清.以间接酶联免疫吸附试验(ELISA)检测抗体效价,Western blot鉴定抗体特异性及检测PIWILA在各细胞系中的表达差异,免疫荧光染色观察PIWIL4的细胞定位.结果 成功构建原核表达质粒,表达并纯化PIWIL4蛋白.免疫大白兔后得到PIWIL4多克隆抗体,ELISA检测抗体效价为1:20 000,Western blot和免疫组化确定抗体具有高度特异性,并成功地应用该抗体检测到PIWIL4在人多种细胞系中的表达差异及细胞定位.结论 PIWIL4蛋白及其多克隆抗体的成功制备,为进一步研究PIWILA的生物学功能奠定了基础.     

PIWIL4的克隆及其抗体制备和初步应用

目的 制备兔抗人PIWIL4的多克隆抗体,鉴定其特异性,并应用该抗体检测内源性PIWIL4在人各细胞系中的表达差异及细胞定位.方法 构建原核表达质粒pGEX-5X-1-PIWIL4,转化大肠杆菌BL21,PIWIL4蛋白经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达.融合蛋白通过切胶纯化后免疫家兔制备抗体血清.以间接酶联免疫吸附试验(ELISA)检测抗体效价,Western blot鉴定抗体特异性及检测PIWILA在各细胞系中的表达差异,免疫荧光染色观察PIWIL4的细胞定位.结果 成功构建原核表达质粒,表达并纯化PIWIL4蛋白.免疫大白兔后得到PIWIL4多克隆抗体,ELISA检测抗体效价为1:20 000,Western blot和免疫组化确定抗体具有高度特异性,并成功地应用该抗体检测到PIWIL4在人多种细胞系中的表达差异及细胞定位.结论 PIWIL4蛋白及其多克隆抗体的成功制备,为进一步研究PIWILA的生物学功能奠定了基础.     

兔抗人Rig-I蛋白N端CARD结构域多克隆抗体的制备与鉴定

目的:制备人维甲酸诱导基因I(hRig-I)N端CARD结构域(hRig-I-N)多克隆抗体.方法:从逆转录病毒载体MigRI-hRig-I中扩增hRig-I基因N端CARD结构域852 bp长度的基因片段,克隆入pGEX-4T3中,转化大肠杆菌BL21(DE3),在IPTG诱导下,表达GST-hRig-I-N融合蛋白,将表达的融合蛋白纯化,免疫家兔制备抗体,并以间接ELISA法测定抗体效价,以Western blot、细胞免疫荧光方法鉴定抗体的特异性.结果:在大肠杆菌中成功表达融合蛋白GST-hRig-I-N,ELISA法检测抗体的效价达到1:125 000,Western blot及细胞免疫荧光检测结果显示抗体可与原核及真核表达的hRig-I-N蛋白特异结合.结论:制备了高效价、高特异性的hRig-I-N抗体,为进一步研究hRig-I-N的功能奠定了基础.     

人前蛋白转化酶枯草溶菌素9的cDNA克隆和表达及多克隆抗体的制备

目的 构建人前蛋白转化酶枯草溶菌素9(pcsk9)的原核表达载体并诱导其表达,制备其蛋白的多克隆抗体.方法 聚合酶链式反应扩增人工合成的pcsk9 cDNA序列;扩增产物经TA克隆、双酶切、连接,构建pET-28b-pcsk9表达载体;将重组质粒转化人大肠杆菌表达菌BL21(DE3)的感受态细胞中,诱导表达;用纯化检测过的目的蛋白免疫新西兰兔,最后采集全血收集血清,获得多克隆抗体,并对该抗体进行免疫印迹及效价测定.结果 PET-28b-pcsk9原核表达载体构建成功,用诱导表达的目的蛋白免疫动物,收集血清获得多克隆抗体.对多克隆抗体进行纯化,免疫印迹检测结果证明自制多克隆抗体特异性良好,效价测定为1∶13 200.结论 成功构建了pcsk9原核表达载体pET-28b-pcsk9,且获得了其蛋白的多克隆抗体.     

Ficolin-A的克隆、蛋白表达和抗体的制备与鉴定

目的: 克隆小鼠ficolin-A(mouse ficolin-A)基因, 构建在真核及原核表达载体, 并在大肠杆菌中表达和鉴定其蛋白, 并制备其抗体, 以进一步用于研究小鼠ficolin-A的功能.方法: 用RT-PCR的方法从新生7 d的C57BL/6小鼠肝脏中运用GeneRacer kit扩增ficolin-A cDNA片段, 并将该片段分别插入pVAX-1真核表达载体及pGEX-KG 原核表达载体中, 实现插入基因的融合, 在IPTG的诱导下在大肠杆菌中表达.用GST-Sepharose 4B的柱子对表达的融合蛋白进行纯化, 用SDS-PAGE和Western blot对表达产物进行鉴定.制备ficolin-A的多克隆抗体并对其效价进行测定.结果: 成功地构建了pVAX-1-ficolin-A真核表达载体及pGEX-KG-ficolin-A原核表达载体, 并在大肠杆菌中获得高效的表达, 表达产物的相对分子质量(Mr)同预期值相一致.并且成功制备了多克隆抗体.结论: 成功地构建了重组表达载体pVAX-1-ficolin-A及pGEX-KG-ficolin-A, 并在E.coli BL21中表达了ficolin-A蛋白, 为下一步研究小鼠ficolin-A的功能奠定了基础.     

组织因子人-鼠嵌合抗体真核表达载体的构建及鉴定

目的:构建组织因子人-鼠嵌合抗体的真核共表达载体pCN40-V_L-V_H,并转染中国仓鼠卵巢细胞(CHO)中进行稳定表达,以克服鼠源单克隆抗体(mAb)用于临床的局限性.方法:从分泌鼠抗人的组织因子mAb的杂交瘤细胞株(克隆号:mTA)中抽提总RNA,经RT-PCR扩增mAb V_L和V_H的基因序列,构建重组真核表达载体pCN40-V_L-V_H,并进行酶切鉴定和测序.通过磷酸钙沉淀法转染CHO细胞,以终浓度800 μg/L G418筛选阳性细胞,通过间接ELISA、Western blot检测细胞培养上清中嵌合抗体的表达量、活性及特异性.结果:成功构建了抗人组织因子嵌合抗体真核共表达载体pCN40-V_L-V_H并在CHO细胞中表达.ELISA、Western blot证实,表达的抗组织因子嵌合抗体为人鼠嵌合抗体,该抗体能与重组人TF抗原特异性的结合且表达量为51.25 mg/L.结论:成功地构建并在真核细胞中表达了抗人组织因子嵌合抗体,为进一步的研究奠定了基础.     

釉成熟蛋白Amelotin抗体的制备和初步鉴定

目的:表达并纯化小鼠釉成熟蛋白(Amelotin), 制备多克隆抗体, 并进行初步鉴定.方法:RT-PCR获得Amelotin成熟肽片段, 构建pET32a-Amelotin, IPTG诱导表达Amelotin, 纯化后免疫新西兰大白兔制备多克隆抗体, ELISA检测抗体滴度.Western blot和免疫组织化学鉴定抗体特异性.结果:成功构建了Amelotin重组表达载体pET32a-Amelotin,表达的重组蛋白纯化后免疫新西兰大白兔, 得到的多克隆抗体ELISA显示抗体效价可以达到1:12 800.Western blot分析表明该抗体能特异结合Amelotin, 免疫组化分析表明自制的抗体能特异的与Amelotin相互作用.结论:以纯化的Amelotin为免疫原, 成功地制备了效价及特异性较高的兔抗Amelotin抗体,为进一步建立简便的Amelotin检测方法及研究Amelotin生物学功能奠定了良好的基础.     

Recombinant helical plant virus-based nanoparticles for vaccination and immunotherapy

Plant virus-based nanoparticles (PVNs) are self-assembled capsid proteins of plant viruses, and can be virus-like nanoparticles (VLPs) or virus nanoparticles (VNPs). Plant viruses showing helical capsid symmetry are used as a versatile platform for the presentation of multiple copies of well-arrayed immunogenic antigens of various disease pathogens. Helical PVNs are non-infectious, biocompatible, and naturally immunogenic, and thus, they are suitable antigen carriers for vaccine production and can trigger humoral and/or cellular immune responses. Furthermore, recombinant PVNs as vaccines and adjuvants can be expressed in prokaryotic and eukaryotic systems, and plant expression systems can be used to produce cost-effective antigenic peptides on the surfaces of recombinant helical PVNs. This review discusses various recombinant helical PVNs based on different plant viral capsid shells that have been developed as prophylactic and/or therapeutic vaccines against bacterial, viral, and protozoal diseases, and cancer.     

PF-IND: probability algorithm and software for separation of plant and fungal sequences

The separation of plant and fungal sequences in EST pools by bioinformatic methods is difficult because of sequence similarities between plants and fungi, lack of enough sequence information, and the short length of the isolated fragments. An algorithm and software that utilize the differences in codon usage bias to discriminate between plant and fungal sequences are described. The software (PF-IND) includes five pairs of fungi and their host plants that can be used to analyze a large number of related species. Analysis of a sequence provides an arbitrary value that defines the likelihood that a sequence will be a fungal or a plant gene. The software can distinguish between homologous fungal and plant genes and it helps identify the correct reading frame of unknown expressed sequence tags (ESTs) for which BLAST analyses do not provide clear information. Short sequences of 100-150 bp can be analyzed with high confidence. PF-IND analysis of 100 sequences derived from fungal infected plants identified the origin of 94 sequences. Only 66 sequences were identified by a BLASTX analysis of the same 100 ESTs. Overall, PF-IND is a novel bioinformatic tool aimed at assisting the research of fungus-plant interactions.     

Surface colonization by Azospirillum brasilense SM in the indole-3-acetic acid dependent growth improvement of sorghum

The key to improving plant productivity is successful bacterial-plant interaction in the rhizosphere that can be maintained in the environment. The results presented here confirm Azospirillum brasilense strain SM as a competent plant growth promoting bacterium over mid- and long-term associations with sorghum. This study establishes that plant growth can be directly correlated with the associated bacterium's indole-3-acetic acid (IAA) production capability as IAA over-expressing variants, SMp30 and SMΔi3-6 fared better than the wild type strain. The auxin antagonist, p-chlorophenoxy isobutyric acid confirmed the role of bacterial IAA in plant growth promotion and verified the presence of larger amount of IAA available to the seeds on inoculation with IAA over-expressing mutants. Microscopic analysis identified the bacterial association at root tips, root-shoot junction and elongation zone and their surface colonizing nature. Scanning electron microscopy identified larger number of root hairs and extensive exopolysaccharide covering in comparison to untreated ones. In addition, vibroid-shaped Azospirilla attached by means of fibrillar material were dispersed along the elongation zone. The notable difference with IAA over-expressing variants was enhanced number of root hairs. Thus, the variant strains may be more efficient surface colonizers of the sorghum root and used as superior bio-inoculants for improving plant productivity.     

Improved RNA extraction and one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in plant extracts

A procedure was developed for simultaneous detection of plant RNA viruses and of plant RNA, as a control. RT-PCR amplification with primers designed for the detection of the plant mRNAs encoding malate dehydrogenase (MDH) and the large subunit of ribulose bisphosphate carboxylase oxygenase (RubiscoL) was used for the development of a plant extraction procedure that consistently yields extracts that can be amplified. The control amplification was used successfully on extracts from cane, leaf and/or bud tissues from grapevine, apple, raspberry, strawberry, peach, apricot, plum and wheat. Multiplex RT-PCR conditions were established for the simultaneous detection in grapevine extracts of either arabis mosaic virus, rupestris stem pitting associated virus and malate dehydrogenase mRNA, or grapevine virus A, grapevine virus B, grapevine leafroll associated virus-3, and RubiscoL mRNA.     

Direct gene transfer via polyethylene glycol

Summary Polyethylene glycol can be used to induce DNA uptake into plant protoplasts. Procedures for isolation, culture and transformation ofN. tabacum protoplasts are described and can be adapted for other dicot and monocot species. Criteria for proof of transformation are discussed.     

A flexible apparatus for continuous recording of water uptake by plants

An apparatus for the continuous and automatic recording of water uptake by small plants or single shoots is described. The root system of the plant (or plants) to be investigated is submerged in a water supply vessel. The water decrease in the vessel is detected, and the water is replaced by pumping new water into the vessel in a stepwise fashion. The apparatus can be used for measuring the total amount of water taken up, and the rate of water uptake. A water uptake of less than 0·1 mm³ can be reliably determined in the present applications. An example from measurements of cyclic uptake of water by one single oat plant is given.     

Forensic botany: using plant evidence to aid in forensic death investigation

Forensic botany is still an under-utilized resource in forensic casework, although it has been used on occasion. It is an area of specialty science that could include traditional botanical classification of species, DNA, or materials evidence (trace and transfer evidence), crime mapping or geo-sourcing, all dependent on the specific case application under consideration. Critical to the evaluation of plant evidence is careful collection, documentation, and preservation for later scientific analysis. This article reviews proper procedures and recent cases where botanical evidence played a role in establishing either manner or time of death. Plant evidence can be useful for determining if a death was due to an accident, suicide, or homicide, or what time of year burial may have taken place. In addition, plant evidence can be used to determine if a crime scene is a primary or secondary scene and to locate missing bodies.     

Therapeutic Efficacy of the Neuroprotective Plant Adaptogen in Neurodegenerative Disease (Parkinson’s Disease as an Example)

Therapeutic efficacy of the plant neuroprotector Phytomix-40 in Parkinson’s disease was demonstrated. This preparation consists of the components from extracts of 40 plants, including some adaptogens (ginseng, eleutherococcus, Rhodiola rosea, etc.). The preparation normalized immune, antioxidant, and hormonal parameters in patients. The neuroprotective plant adaptogen can be used in complex therapy for Parkinson’s disease for improving its efficacy.     

The field efficacy of garlic extract against Dermanyssus gallinae in layer farms of Babol,Iran

Dermanyssus gallinae (Acari: Mesostigmata) is the most important hematophagous ectoparasite in layer farms in many countries. The reproduction rate of the parasite is rapid and can be completed in a week under favorable conditions. The parasite has direct and indirect effects on birds. It can also act as a vector for some important pathogens. Many researchers have investigated the effects of essential oils, plant extracts, oriental medicinal plant extracts, and silica against red mite. They can be used as killing agents or repellents. In the present study, the effect of garlic (Allium sativum) extract was investigated for controlling red mite infestation in a layer farm in Babol, North of Iran. Our results showed that the extract was effective and we obtained a 96 % success after two successive sprays.     

The mutational consequences of plant transformation

Plant transformation is a genetic engineering tool for introducing transgenes into plant genomes. It is now being used for the breeding of commercial crops. A central feature of transformation is insertion of the transgene into plant chromosomal DNA. Transgene insertion is infrequently, if ever, a precise event. Mutations found at transgene insertion sites include deletions and rearrangements of host chromosomal DNA and introduction of superfluous DNA. Insertion sites introduced using Agrobacterium tumefaciens tend to have simpler structures but can be associated with extensive chromosomal rearrangements, while those of particle bombardment appear invariably to be associated with deletion and extensive scrambling of inserted and chromosomal DNA. Ancillary procedures associated with plant transformation, including tissue culture and infection with A tumefaciens, can also introduce mutations. These genome-wide mutations can number from hundreds to many thousands per diploid genome. Despite the fact that confidence in the safety and dependability of crop species rests significantly on their genetic integrity, the frequency of transformation-induced mutations and their importance as potential biosafety hazards are poorly understood.