Purification of monoclonal antibody from tobacco extract using membrane-based bioseparation techniques

Transgenic plants offer a promising system for large-scale production of therapeutic proteins such as monoclonal antibodies (mAbs). This paper describes a membrane-based process suitable for purification of a humanized mAb expressed in tobacco. Most monoclonal antibody purification schemes rely on the use of Protein A as the affinity ligand for antibody capture. The main objective of our work was to develop non-Protein A-based purification methods to avoid some of the problems and limitations associated with this ligand, e.g. cost, immunotoxicity, and antibody aggregation during elution. Ion exchange membrane chromatography (IEMC) was used for primary capture and preliminary purification of the mAb from tobacco juice. Hydrophobic interaction membrane chromatography (HIMC) was then used for high-resolution purification, followed by ultrafiltration for polishing, desalting and buffer exchange. Using this scheme, both high mAb purity (single peak in size exclusion chromatogram, i.e., ca. 100% purity) and high recovery (77% of mAb spiked into the tobacco extract) were achieved. Membrane chromatography is generally considered unsuitable for resolving bound proteins by gradient elution and is therefore commonly used in the bind and elute mode with a single-step change of mobile phase. We show that the gradient elution process in the HIMC step can be optimized to increase the resolution and thereby obtain product of high purity.     

Separation,identification, and structural characterization of sucrose ester isomers from tobacco

Sucrose esters (SEs) are crucial tobacco smoke flavor precursors and play a significant role in tobacco's functionality. Due to their structural complexity, the separation and analysis of SEs in tobacco remain a major challenge, and massive structures of SEs have not yet been fully identified. In this study, the fractions enriched in SEs were obtained from oriental and flue-cured tobacco through a series of pretreatments, and two types of SEs (Types I and II) were distinguished by liquid chromatography-tandem mass spectrometry (LC-MSⁿ) analysis, with Type II SEs newly characterized in tobacco. Five groups of main SEs were further purified using preparative high-performance LC (HPLC) coupled to an evaporative light scattering detector, and their structures were characterized by nuclear magnetic resonance spectrometry techniques including ¹H, ¹³C, correlation spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. By combining LC-MSⁿ and nuclear magnetic resonance spectrometry, the structures of eight SE isomers were finally proposed, of which four were newly identified. These findings further enhance the understanding of the structural diversity of SEs in tobacco, serving as a valuable reference for future research on the elucidation, synthesis, and metabolism of SEs.     

不同作物茬口对烟草大田农艺性状及烟草品质的影响

在烟草种植过程中,白菜茬、玉米茬、红薯茬和芝麻茬等茬口对烟草的品质影响较大.测定4种茬口种植烟草后的株高、茎围、叶片数、烟草叶面积、化学质量、品质等指标的差异.结果表明,红薯茬和芝麻茬烟草田间长势比较好,株高比较高,茎围粗壮,叶面积要远远高于白菜茬和玉米茬;从质量性状来看,红薯茬和芝麻茬能显著提高烟叶可溶性糖、还原糖和烟碱含量,并且能降低烟叶含氯量;从施木克值、钾氯比、氮碱比这些品质指数及产量、产值来看,烟草以红薯茬和芝麻茬较好,白菜茬次之,玉米茬最差.     

烟草制品及烟气中重金属检测方法的研究进展

从烟草制品及其烟气的前处理方法和检测方法两方面对烟草中重金属含量测定的研究进展进行了综述。重点介绍了湿法消解、微波消解、萃取和悬浮进样技术等样品前处理方法,以及原子光谱法、质谱法和液相色谱法等测定方法。列举了不同方法的灵敏度、准确性和适用范围,对各方法的优缺点进行了比较,并对烟草中重金属检测方法的发展进行了展望。     

Porphyrin light-harvesting arrays constructed in the recombinant tobacco mosaic virus scaffold

We have demonstrated the construction of multiple porphyrin arrays in the tobacco mosaic virus (TMV) supramolecular structures by self-assembly of recombinant TMV coat protein (TMVCP) monomers, in which Zn-coordinated porphyrin (ZnP) and free-base porphyrin (FbP) were site-selectively incorporated. The photophysical properties of porphyrin moieties incorporated in the TMV assemblies were also characterized. TMV-porphyrin conjugates employed as building blocks self-assembled into unique disk and rod structures under the proper conditions as similar to native TMV assemblies. The mixture of a ZnP donor and an FbP acceptor was packed in the TMV assembly and showed energy transfer and light-harvesting activity. The detailed photophysical properties of the arrayed porphyrins in the TMV assemblies were examined by time-resolved fluorescence spectroscopy, and the energy transfer rates were determined to be 3.1-6.4x10(9) s(-1). The results indicate that the porphyrins are placed at the expected positions in the TMV assemblies.     

萃取分光光度法快速测定烟草及制品中烟碱的研究

用酸为浸取剂,铬兰黑为显色剂,ＣＨ２Ｃｌ２ 萃取液,建立了快速简便、灵敏度高的测定烟草及制品中烟碱含量的萃取分光光度法．测量的线性范围为０～３３μｇ／ｍ ｌ．     

风油精在烟草染色体标本制备中的应用

采用风油精法制得的烟草染色体标本和用秋水仙溶液预处理制片比较,获得了较多的染色体长度适中的早中期同步分裂相,片子干净,染色体分散性较好,可显示部分染色体Ｃ 带,并且风油精在染色体制片过程中更安全,便于广泛应用。     

新型潜香化合物3,6-二甲基-2,5-吡嗪二甲酸二薄荷醇酯的热裂解产物

为开发新型高温释放型烟用香料,以2,3,5,6-四甲基吡嗪和薄荷醇为原料,经过酯化反应制备了3,6-二甲基-2,5-吡嗪二甲酸二薄荷醇酯(DPAME).采用在线热裂解-气相色谱-质谱联用技术(Py-GC-MS)在空气氛围和不同的温度(300、600和900 ℃)下,对DPAME进行热裂解研究,裂解产物经GC-MS进行了定性和半定量分析.结果表明,DPAME在300 ℃下裂解产生了多种有致香效果的醛类、薄荷烯和薄荷醇等;在600 ℃和900 ℃下裂解产生了烯烃类、烷基吡嗪、薄荷醇和薄荷烯等香味物质,并且吡嗪类的种类和相对含量在这两个温度下明显增加.结合DPAME的热裂解产物分析和卷烟感官评吸结果,初步推测了其可能的裂解机理.采用该方法可以方便、快速地分离鉴定物质的热裂解产物,为该物质在烟草中的加香应用提供理论依据.     

烟草行业CIMS集成化支撑保障体系

分析了烟草行业信息化建设当前存在的问题,提出采用CIMS技术实现烟草行业现代集约化管理.基于烟草行业CIMS的应用系统集成模式构建行业CIMS集成支撑保障体系,它包括行业集成支撑体系和行业集成保障体系.行业集成支撑体系负责行业CIMS应用系统的数据集成、应用服务集成和业务过程集成,行业集成保障体系负责为行业CIMS应用系统集成提供标准化体系与安全体系等保障机制,保证系统的安全高效率运行.实际应用扩充了CIMS内涵,将其集成范围从传统的制造企业扩展到工商贸,直至烟草整个行业,行业集成数据中心提供标准化管理体系、信息安全管理体系与行业集成技术支撑体系三大类功能,是行业集成的支撑保障体系,为烟草行业各级部门的集约化管理服务.     

热裂解-气相色谱-质谱联用技术研究多羟基吡嗪的热裂解行为

采用热裂解-气相色谱-质谱联用技术（Py—GC-MS）,选择不同温度,在空气存在的条件下对烟叶重要组分多羟基吡嗪进行了热裂解挥发性成分分析,结果表明：该方法具有较好的重复性（相对标准偏差〈1．1％）;不同温度下挥发性的热裂解产物不同;该化合物的热裂解能够产生吡嗪类化合物,而且,随着热裂解温度的升高吡嗪类化合物的含量增加。在挥发性的热裂解产物中,在300℃时吡嗪类化合物只占8．35％,吡啶类化合物占19．07％;在600℃时吡嗪类化合物占16．96％,吡啶类化合物占30．58％;在900℃时吡嗪类化合物占21．61％,吡啶类化合物占27．08％。