纳米抗体研究进展

纳米抗体是由骆驼科动物缺失轻链的天然重链抗体的可变区(VHH)组成的单域抗体,其可变区的相对分子质量约为15×10~3。与传统抗体相比,纳米抗体具有相对分子质量小、亲和力高、稳定性高、溶解性好、免疫原性低、穿透力强、人源化简单等优势。基于纳米抗体的特性以及骆驼天然重链抗体的VHH单域抗体的特殊结构,使其在基础医学研究以及疾病诊断和治疗方面具有广阔的应用前景。     

纳米抗体异源表达的研究进展

羊驼体内存在天然缺失轻链的重链抗体(HcAb),其单域抗原结合片段也称为VHH或纳米抗体(nanobody,Nb),是目前已知的最小抗原结合片段。与传统抗体相比,纳米抗体具有分子量小(12~15kDa)、稳定性好和免疫原性低等特点,这些特点使得VHH在基础研究、诊断及治疗上具有极大的应用价值,目前已有多种纳米抗体进入了临床研究阶段。综述了近年来VHH在革兰氏阴性菌(大肠杆菌)、革兰氏阳性菌(芽孢杆菌和乳酸杆菌)、酵母、丝状真菌、昆虫细胞、哺乳动物细胞和植物细胞中异源表达的研究现状,包括表达系统、宿主、载体特点、载体构建方式及产量等;从分子水平、表达水平和理性设计三个层面探讨了纳米抗体产量提高的策略,以期为纳米抗体研究者提供借鉴和思路。     

双特异性纳米抗体的研究进展及其应用

从纳米抗体的研究进展,双特异性纳米抗体在感染类疾病、肿瘤以及免疫系统疾病治疗领域的研究成果、研究热点及发展前景等方面综述了双特异性纳米抗体的研究进展并分析了未来可能的发展方向。首先比较了纳米抗体与全长单克隆抗体之间的差异并阐述了双特异性纳米抗体具备的独特优势;继而概括了双特异性纳米抗体的发展历程,并对新冠病毒的中和性抗体、CAR-T细胞治疗和免疫检查点治疗抗体等研究热点进行了深入分析;最后对双特异性纳米抗体的应用前景进行分析,指出双特异性纳米抗体能够克服目前全长抗体治疗过程中存在的缺陷,成为一种极具发展潜力的抗体形式。     

纳米抗体及其在诊断检测中的研究进展

骆驼血液中存在天然缺失轻链的重链抗体,克隆该重链抗体的可变区得到最小的抗原结合片段,即纳米抗体(Nanobody,Nb)。Nb的单域性质使其较普通抗体具有一些独特性能,比如高度水溶性和构象稳定性,较强的抗原亲合力和优良的组织穿透能力,容易体外表达和人源化改造修饰等,Nb的以上持性使其在诊断检测领域展现出广阔的应用前景。尽管Nb的应用开发已经取得前所未有的成功,技术上仍然有待进一步优化,其中包括噬菌体纳米抗体库构建以及Nb的胶体金标记分析等技术。文中简单介绍Nb的研究进展,并从Nb制备、在疾病的体外检测以及体内肿瘤无创伤影像诊断领域的应用3个方面,讨论进一步提高Nb亲合力和人源化改造等优化Nb分子特征的策略,分析Nb在疾病检测诊断应用中存在的问题,并提出一些积极的应对方案。     

基于重链抗体构建的单域抗体研究进展

在骆驼血清中存在天然的缺失轻链的重链抗体(heavy chainantibody ,HCAb) ,克隆重链抗体的可变区构建的只由一个重链可变区组成的单域抗体称为VHH抗体(variabledomainofheavychainofheavy chainantibody ,VHH)。研究发现,VHH抗体具有易表达、可溶性好、稳定性强等优点。另外,骆驼的重链抗体与人VH3家族抗体同源,对人VH3家族抗体的重链可变区进行类似VHH的特征性改造,可以使这些抗体在保持亲和力、特异性不变或者变化很小的情况下,优化抗体的其它性质。已有的研究表明VHH抗体作为一种小型化的基因工程抗体在基础研究、药物开发等领域有广阔的应用前景。     

骆驼来源单域抗体在免疫治疗中的研究进展

近几年多种单克隆抗体药物已成功上市并用于治疗人类多种疾病,但因单克隆抗体的复杂结构和高昂的生产成本,限制了其在临床上的广泛应用。随着生物技术的进步,使抗体朝着小型化的基因工程单域抗体发展。20世纪末,在骆驼科动物(camelidae)和护士鲨(nurse shark)体内发现一种天然缺失轻链的重链抗体(heavy chain antibody,HCAb),这种特殊抗体的抗原结合位点仅由单一结构域构成。该结构域在骆驼中称为VHH,经基因重组修饰易于进行工程表达,产物即称为VHH单域抗体,具有分子量小,可溶性好,稳定性高及组织穿透力强等优势,在调节免疫功能方面的应用前景更加广阔。就目前国际上对骆驼来源的VHH单域抗体在免疫治疗应用研究的进展情况进行综述,为基因工程单域抗体改造提供新思路。     

基因工程抗体中噬茵体抗体库技术的应用

通过基因工程可以大规模地制备能与人相容的单克隆抗体或片段.其中,噬茵体抗体库技术可以模拟体内抗体产生和成熟过程,不经细胞杂交,甚至不经免疫制备针对任何抗原的单克隆抗体.就基因工程抗体及噬茵体抗体库技术的发展与应用作一概述.     

绵羊肌肉生长抑制素MSTN原核表达及其纳米抗体文库的构建与鉴定

克隆绵羊肌肉生长抑制素（myostatin,MSTN）基因并在大肠杆菌中诱导表达,纯化重组蛋白免疫健康的双峰驼（Bactrian camel）,分离其外周血淋巴细胞提取总RNA,利用RT-PCR扩增骆驼重链抗体IgG2、IgG3的可变区（VHH）基因片段,将VHH片段与pCANTAB5E连接后电转入大肠杆菌TG1构建纳米抗体文库。结果显示,纳米抗体文库容量为9.5×10⁵,挑取部分克隆进行测序分析,所获得的纳米抗体文库具有良好的多态性,为进一步筛选绵羊MSTN的高特异性纳米抗体片段奠定了基础。     

不同给药途径的治疗性纳米抗体研究进展

骆驼科及鲨鱼科动物血清中天然存在的纳米抗体具有不同于传统单克隆抗体的独特结构和分子量,这为抗体药物开发提供了全新的思路。纳米抗体较小的分子量和优异的稳定性使其在给药方面具有更大的灵活性,可以在一定程度上克服传统单克隆抗体在给药途径方面存在的局限性。同时,较小的分子量使纳米抗体具有双重药代动力学特征,既有优异的组织渗透性,又表现出快速的血液清除。重点介绍纳米抗体的药物代谢动力学特征和进一步改善药代动力学的方法,综述不同给药途径的纳米抗体药物研究进展,对其治疗特定疾病的可行性、安全性以及治疗效果进行分析,以期为纳米抗体药物研发中给药途径的选择提供参考。     

新疆双峰驼天然单域抗体库的构建及初步鉴定

旨在构建新疆双峰驼天然单域抗体库,及从中快速筛选VHH抗体.采用两种不同的抗原(溶菌酶和cAb-HEWL23)用天然文库进行筛选,成功筛选到了相应的抗体,并融溶菌酶筛选的VHH抗体(A3-1,A4-1和A10-1)进行表达和初步的ELISA检测.结果显示,A3-1和A10-1具有结合溶菌酶的能力.该方法简单方便、省时省力,可以快速从天然单城抗体库中筛选VHH抗体.     

Tuning of electronic properties of one-dimensional cyano-bridged Cu-Ni, Cu-Pd, and Cu-Pt bimetallic assemblies by stereochemistry of ligands

Preparations, XPS and electronic spectroscopy, and magnetism of seven new one-dimensional cyano-bridged coordination polymers, chiral [Cu(RR-chxn)₂][Pd(CN)₄] · 2H₂O (1), [Cu(trans-chxn)₂][M(CN)₄] · 2H₂O (2, 4, and 6 for M = Pd, Ni, and Pt), and [Cu(cis-chxn)₂][M(CN)₄] · 2H₂O (3, 5, and 7 for M = Pd, Ni, and Pt) (RR-chxn = cyclohexane-(1R,2R)-diamine, trans-chxn = racemic trans-cyclohexane-(1,2)-diamine, and cis-chxn = racemic cis-cyclohexane-(1,2)-diamine) have been reported in view of tuning of their electronic properties by stereochemistry of chxn ligands and metal-substitution. Comparison of Cu 2p_1/2 and 2p_3/2 peaks of XPS and broad d-d bands around 18 000 cm⁻¹ of electronic spectra are described systematically for 1-7. Variable-temperature magnetic measurement shows that complexes 1-7 indicate weak antiferromagnetic interactions via cyano-bridges. Because of semi-coordination coupled with pseudo Jahn-Teller elongation and electrostatic interaction for 1, the axial Cu-N coordination bond distances of 2.330(7) and 3.092(8) Å are considerably longer than those of equatorial ones in the range from 2.016(6) to 2.030(6) Å. The former bond distances of 1 are intermediate values among the related Ni (2.324(6) and 3.120(8) Å) and Pt (2.34(1) and 3.09(1) Å) complexes.     

Land,livestock, and livelihoods: Changing dynamics of gender,caste, and ethnicity in a Nepalese Village

Over the past 10 years, Ghusel VDC, Lalitpur District has moved from primarily subsistence agriculture into the wider cash economy aided by the Small Farmers' Development Program (SFDP), which provides credit to farmers mainly for the purchase of buffalo for milk production, and by the National Dairy Corporation, which supports local dairy cooperatives. Analysis reveals that buffalo-keeping and milk sales are increasing the well-being of many households, while at the same time creating new inequalities in gender roles and responsibilities, greater inequities between Brahmin and Tamang residents in Ghusel, and placing pressures on the ecosystem for increased supplies of fodder and fuelwood. Evidence suggests that there is critical, need for attention to the social, and particularly gender-based, implications of maintaining livestock for milk sales and to the ecological underpinnings of this livelihood system.     

Structure and function of S-adenosylhomocysteine hydrolase

In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1′R, 2′S, 3′R)-9-(2′,3′-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17° rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.     

Chiral Separation of G‐type Chemical Warfare Nerve Agents via Analytical Supercritical Fluid Chromatography

Chemical warfare nerve agents (CWNAs) are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Undirected synthesis of G‐type CWNAs produces stereoisomers of tabun, sarin, soman, and cyclosarin (GA, GB, GD, and GF, respectively). Analytical‐scale methods were developed using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer for the separation, quantitation, and isolation of individual stereoisomers of GA, GB, GD, and GF. Screening various chiral stationary phases (CSPs) for the capacity to provide full baseline separation of the CWNAs revealed that a Regis WhelkO1 (SS) column was capable of separating the enantiomers of GA, GB, and GF, with elution of the P(+) enantiomer preceding elution of the corresponding P(–) enantiomer; two WhelkO1 (SS) columns had to be connected in series to achieve complete baseline resolution. The four diastereomers of GD were also resolved using two tandem WhelkO1 (SS) columns, with complete baseline separation of the two P(+) epimers. A single WhelkO1 (RR) column with inverse stereochemistry resulted in baseline separation of the GD P(–) epimers. The analytical methods described can be scaled to allow isolation of individual stereoisomers to assist in screening and development of countermeasures to organophosphorus nerve agents. Chirality 26:817–824, 2014. © 2014 The Authors. Chirality published by John Wiley Periodicals, Inc.     

Influence of Fluoride on Rat Kidney Antioxidant System: Effects of Methionine and Vitamin E

The aim of the study has been to determine and compare the influence upon the kidney antioxidative system, exercised by administration of vitamin E, and vitamin E in combination with methionine, under conditions of oxidative stress induced by sodium fluoride. The experiment was carried out on Wistar FL rats (adult males) that, for 35 days, were administered water, NaF, NaF with vitamin E, or vitamin E with methionine (doses: 10 mg NaF/kg of body mass/24 h, 3 mg vitamin E per 10 μl per rat for 24 h, 2 mg methionine per rat for 24 h). The influence of administered sodium fluoride and antioxidants upon the antioxidative system in kidney was examined by analyzing the concentration of malondialdehyde (MDA) and the activity of the most important antioxidative enzymes (SOD, total and both its isoenzymes, GPX, GST, GR, and CAT). The studies carried out confirmed the disadvantageous effect of the administered dose of NaF upon the antixodiative system in rats (increase in the concentration MDA, decrease activity of all antioxidative enzymes). The administration of vitamin E increased the activity of studied enzymes with the exception of glutathione reductase GR; it also reduced the procesess of lipid peroxidation. It has been found that combined doses of vitamin E and methionine were most effective in inhibiting lipid peroxidation processes. The results confirmed the antioxidative properties of methionine.     

Reorientation of Microfibrils and Microtubules at the Outer Epidermal Wall of Maize Coleoptiles During Auxin-Mediated Growth

Auxin-mediated elongation growth of isolated subapical coleoptile segments of maize (Zea mays L.) is controlled by the extensibility of the outer cell wall of the outer epidermis (Kutschera et al., 1987). Here we investigate the hypothesis that auxin controls the extensibility of this wall by changing the orientation of newly deposited microfibrils through a corresponding change in the orientation of cortical microtubules. On the basis of electron micrographs it is shown that cessation of growth after removal of the endogenous source of auxin is correlated with a relative increase of longitudinally orientated microfibrils and microtubules at the inner wall surface. Conversely, reinduction of growth by exogenous auxin is correlated with a relative increase of transversely orientated microfibrils and microtubules at the inner wall surface. These changes can be detected 30–60 min after the removal and addition of auxin, respectively. The functional significance of directional changes of newly desposited wall microfibrils for the control of elongation growth is discussed.     

Gamma-glutamylcysteine protects ergothioneine-deficient Mycobacterium tuberculosis mutants against oxidative and nitrosative stress

Mycobacterium tuberculosis (M.tb.), the causative agent of tuberculosis (TB), cannot synthesize GSH, but synthesizes two major low molecular weight thiols namely mycothiol (MSH) and ergothioneine (ERG). Gamma-glutamylcysteine (GGC), an intermediate in GSH synthesis, has been implicated in the protection of lactic acid bacteria from oxidative stress in the absence of GSH. In mycobacteria, GGC is an intermediate in ERG biosynthesis, and its formation is catalysed by EgtA (GshA). GGC is subsequently used by EgtB in the formation of hercynine-sulphoxide-GGC. In this study, M.tb. mutants harbouring unmarked, in-frame deletions in each of the fives genes involved in ERG biosynthesis (egtA, egtB, egtC, egtD and egtE) or a marked deletion of the mshA gene (required for MSH biosynthesis) were generated. Liquid chromatography tandem mass spectrometry analyses (LC-MS) revealed that the production of GGC was elevated in the MSH-deficient and the ERG-deficient mutants. The ERG-deficient ΔegtB mutant which accumulated GGC was more resistant to oxidative and nitrosative stress than the ERG-deficient, GGC-deficient ΔegtA mutant. This implicates GGC in the detoxification of reactive oxygen and nitrogen species in M.tb.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

八种脑-肠肽侧脑室内注射对大鼠基础胃酸分泌的影响

用乌拉坦麻醉大鼠作急性实验,采用连续灌流胃并收集流出液的方法,观察向侧脑室内注射微量脑-肠肽对大鼠基础胃酸分泌的影响。实验结果如下:(1)雨蛙肽、八肽胆囊收缩素、促甲状腺素释放激素及四肽胃泌素均使总酸排出量增加;(2)生长抑素、胰多肽、P 物质、胰高血糖素则使总酸排出量减少;(3)上述肽类用侧脑室注射的剂量作肌肉注射,除四肽胃泌素也产生明显的刺激胃酸分泌作用外,对胃酸分泌均无明显影响。以上结果提示,脑内的一些肽类可能以神经递质或调制物的方式,参与中枢对胃酸分泌的调节。     

A chromium-tolerant plant growing in Cr-contaminated land

In this paper, a kind of Typhaceae plant species, Typha angustifolia L, with a high tolerance to Cr is described. Experiments were carried out to examine its ability to tolerant Cr and its physiological response. The results showed that there was no difference in growth, plant height, and biomass response to external Cr (VI) between the plants exposed to 100 microM Cr (VI) and control (0 microM), while increasing Cr levels to 200-800 microM induced a significant decrease in plant height and biomass, but no significant injury was detected, even for the plants exposed to 800 microM Cr. Chromium induced significant increases in superoxide dismutase (SOD) and peroxidase (POD) activities. Meanwhile, a significantly positive correlation was found between Cr and Mn or Cu in leaves and roots, respectively. The Cr tolerance of the plant appeared to be associated with the enhancement of SOD and POD activities and the improvement in uptake and translocation of the essential microelements.