双特异性单克隆抗体在治疗中的应用

双特异性抗体由于其独特的结构——两个不同抗原,引起研究机构广泛的重视。双特异性单克隆抗体有两个臂,研究者将治疗药物和针对疾病部位的特异目标分别放在两个臂上。治疗药物可以是药品、毒素、酶、DNA和放射性核素等。此外,双特异性单克隆抗体能使免疫效应细胞的细胞毒性作用于致病细胞或者诱导产生全身免疫应答。双特异性单克隆抗体从治疗来看,有很大的发展前景,这是由于:(1)最近重组DNA技术的重大突破;(2)人类基因组图谱工程的完成,提高了疾病的鉴别水平;(3)对人类免疫系统机制有了更好的了解。     

单个B细胞抗体制备技术研究进展

单克隆抗体在疾病预防、诊断和治疗方面具有重要意义，尤其在免疫机制研究方面具有突出贡献。随着单克隆抗体制备技术的发展，单个B细胞抗体制备技术成为新一代快速制备单克隆抗体的方法。该技术利用每个B细胞仅可产生一种特异性抗体的特性，直接筛选出分泌特异性抗体的B细胞，对其表达抗体重链和轻链的基因进行克隆和体外表达，从而获得特异性单克隆抗体。相较传统抗体制备技术，该技术具有快速、高效、产量高等优点，且表达的抗体具有天然构象，不仅可用于病原微生物相关抗原的抗体开发、病毒跨种传播机制等研究，还在抗肿瘤治疗、抗自身免疫病等方面发挥重要作用。本文主要对单个B细胞抗体制备过程和应用现状进行综述，以期为单克隆抗体制备技术的发展和完善提供参考，促进其更好地应用于相关领域。     

小分子化合物单克隆抗体的制备及其在动物医学领域的应用

农药、兽药、重金属、生物毒素等残留物质的痕量检测在医学诊断、食品安全、环境监测等领域均具有重要意义，其中基于抗原-抗体特异性识别的免疫分析技术具有特异性强、灵敏度高、稳定性好、操作简便快捷等优点，但如何获得小分子化合物的特异性抗体是研制免疫分析技术的关键。作者介绍了基于杂交瘤技术制备小分子化合物单克隆抗体的方法，分析了影响杂交瘤技术制备小分子化合物单克隆抗体的因素，然后综述了小分子化合物单克隆抗体在兽药残留、药物开发、环境保护等动物医学领域的应用，最后展望了小分子化合物单克隆抗体生产和应用的发展趋势，为小分子化合物单克隆抗体的制备与应用相关研究提供指导。     

针对银狐主要传染病血清学检测的免疫球蛋白单克隆抗体的制备

以饱和硫酸铵沉淀法和Agarose-Protein G亲和层析技术相结合从银狐血清中提纯免疫球蛋白(IgG),再经SDS-PAGE电泳鉴定,将纯化的免疫球蛋白(IgG)作为抗原,采用常规方法免疫BALB/c小鼠,3次免疫后取脾细胞和SP2/0骨髓瘤细胞相融合制备杂交瘤细胞,利用间接ELISA、Western blot等方法进行鉴定及阳性克隆的筛选,对杂交瘤细胞产生的单克隆抗体进行鉴定和活性测定。结果得到与银狐免疫球蛋白(IgG)发生特异性反应的3株单克隆抗体,分别命名为E2、1E3和2G10。Western blot结果表明,E2、1E3和2G10单克隆抗体能与银狐IgG蛋白特异性结合。经抗体亚型鉴定E2、1E3和2G10均为IgG1、Kappa链。生产提纯的腹水单克隆抗体经Western blot分析效价可达到1∶6 000。该研究利用抗银狐IgG单克隆抗体制备检测感染主要传染病的银狐血清抗体试纸条具有良好的特异性与灵敏性,可以作为检测银狐主要传染病的一种新方法。     

乙肝表面抗体和抗人红细胞单克隆抗体的F(ab′)_2偶联与鉴定

制备人红细胞非凝集型单克隆抗体,形成双功能抗体试剂,用于临床诊断。用人O型红细胞膜蛋白作为抗原免疫Balb/c小鼠,采用淋巴细胞杂交瘤技术进行细胞融合,制备非凝集型单克隆抗体,通过化学偶联法将非凝集性单克隆抗体和乙肝表面抗体的F(ab′)2片段偶联,制备出双功能抗体,用直接凝集法检测乙肝表面抗原。获得5株分泌非凝集型杂交瘤细胞,利用戊二醛一步法制备出双功能抗体,形成全血凝集检测试剂,并进行了初步鉴定。     

禽腺病毒血清4型中国流行株fiber2蛋白单克隆抗体制备与鉴定

旨在制备禽腺病毒血清4型(fowl adenovirus serotype 4,FAdV-4)中国流行株的fiber2蛋白的特异性单克隆抗体。以纯化的FAdV-4中国流行株CH/HNJZ/2015免疫BALB/c小鼠，取小鼠脾脏与SP2/0细胞融合，采用基于fiber2蛋白的间接ELISA筛选阳性杂交瘤细胞，经过5次亚克隆，获得2株分泌抗fiber2蛋白抗体的杂交瘤细胞株，分别命名为2C3和7B4。以制备的2C3和7B4腹水进行亚型鉴定、反应原性、特异性以及中和活性测定。结果显示，成功制备了抗FAdV-4 fiber2蛋白的特异性单克隆抗体，获得的单克隆抗体均为IgM亚型，可特异性识别FAdV-4及其fiber2蛋白，为FAdV-4的免疫检测和fiber2蛋白功能研究提供了研究材料。     

猪塞尼卡谷病毒单克隆抗体的制备及鉴定

[目的] 纯化猪塞尼卡谷病毒（Seneca Valley virus，SVV）SVV-CH-HB2016毒株，并制备其结构蛋白VP1、VP2和VP3的单克隆抗体。[方法] 以蔗糖密度梯度离心法纯化的SVV-CH-HB2016病毒颗粒作为抗原，免疫BALB/c小鼠，取脾细胞与骨髓瘤细胞（SP2/0）进行细胞融合。通过间接免疫荧光试验（IFA）结合间接ELISA筛选阳性细胞株，制备能特异性分泌针对结构蛋白的杂交瘤细胞株。采用Western blotting和IFA方法分别检测单克隆抗体与重组表达蛋白及天然结构蛋白的反应性，并对单克隆抗体的病毒中和保护效果进行测定。利用空斑试验和实时荧光定量PCR方法探究中和性单克隆抗体对SVV-CH-HB2016毒株吸附过程的影响，最后用抗体相加试验来分析14株单克隆抗体的抗原表位。[结果] 在蔗糖密度梯度为5%~45%（W/V）时获得了纯度较好、浓度较高的SVV-CH-HB2016毒株结构蛋白，免疫小鼠血清抗体效价均达到了1：12 800，成功制备了17株能稳定分泌特异性单克隆抗体的杂交瘤细胞株。经验证14株单克隆抗体能与重组结构蛋白发生Western blotting反应，17株单克隆抗体能与病毒发生IFA作用；2G6、4A3和4C11 3株单克隆抗体对SVV-CH-HB2016毒株感染的BHK-21细胞具有明显中和保护作用，也能有效抑制SVV-CH-HB2016毒株对293T细胞的吸附。经分析发现，除1F5与2E1、4B8与4F11外，其余10株单克隆抗体分别针对不同抗原表位。[结论] 本研究初步建立了SVV的纯化方法，制备了17株特异性针对SVV-CH-HB2016毒株的单克隆抗体，为后期进一步开展SVV全病毒灭活疫苗的研发、ELISA检测方法的建立及保护性抗原表位的鉴定奠定了基础。     

犬细小病毒单克隆抗体的制备及其在胶体金免疫层析试纸研发中的应用

为制备犬细小病毒胶体金免疫层析试纸,将F81细胞中分离的CPV免疫Balb/c小鼠,取其脾细胞与骨髓瘤细胞融合,建立间接ELISA方法筛选分泌CPV单克隆抗体的杂交瘤细胞,获得1株能稳定分泌CPV单克隆抗体的杂交瘤细胞株,命名为3E2。3E2的亚类为IgG1型,腹水抗体效价为1.8×105,交叉试验表明,3E2可与CPV特异性结合,与其他犬常见病毒无交叉反应。用3E2单克隆抗体制备的检测CPV的胶体金免疫层析试纸条特异性良好,为诊断和研究CPV奠定了基础。     

双特异性抗体的研制方法及其在奶牛乳腺炎治疗中的应用

双特异性抗体具有两个不同的抗原结合位点,能分别与靶细胞和免疫细胞结合,从而引导免疫细胞定向杀灭靶细胞。双特异性抗体的研制经历了鼠源性单克隆抗体的化学偶联,双杂交瘤和基因工程3个发展阶段,基因工程双特异性抗体与以往鼠源性双特异性抗体相比具有较低的免疫原性和较好的组织穿透能力。knobs into holes技术是基因工程双特异性抗体研制过程中出现的一种新技术,该技术能引导异源二聚体的配对,提高双特异性抗体的产量和质量。目前已有一些Bs(Fab)2和diabody等类型双特异性抗体进入了临床试验阶段,其中以抗CD3/抗肿瘤双特异性抗体最为常见。抗中性粒细胞/金黄色葡萄球菌的试验结果表明,双特异性抗体对中性粒细胞体外抗金黄色葡萄球菌具有明显的导向作用,且对中性粒细胞的免疫功能具有诱导性。     

IL-17单克隆抗体的制备与鉴定

为制备山羊IL-17单克隆抗体,诱导重组菌rIL-17-pET32a-TransB(DE3)表达山羊IL-17重组蛋白(rIL-17),纯化后免疫BALB/c小鼠,取免疫合格的小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,采用间接ELISA方法筛选阳性克隆细胞株。对获得的杂交瘤细胞进行染色体组型分析,并对其分泌的IL-17单克隆抗体进行抗体特异性、抗体类别等分析。结果显示,成功获得了纯化的山羊IL-17原核表达产物;筛选到1株能特异性分泌山羊IL-17单克隆抗体的杂交瘤细胞株-B6,其分泌的单克隆抗体亚型为IgG1,抗体轻链为κ。     

Comparison of intramuscular and footpad subcutaneous immunization with DNA vaccine encoding HSV-gD2 in mice

Herpes simplex virus type 2 is the most common infectious agent in humans that causes genital herpes disease and vaccination is a desirable method to prevent herpes infections. An effective therapeutic vaccine will need to elicit virus-specific immune responses. The route of immunization has important role in immune responses. In this study, DNA vaccine encoding glycoprotein D of herpes simplex virus type 2 (HSV-gD2) was prepared and injected via intramuscular and footpad routes to determine the optimal method of delivery for immune stimulation. The control manipulation of immune response by concerning route of administration is highly appreciated issue by researches. Although DNA vaccine containing HSV-gD2 is effective in both intramuscular and footpad injection routes, the latter could induce significantly higher cellular responses against HSV-2.     

Proteomic analysis of soluble protein extract of adult Toxocara cati

Toxocara cati is a cat roundworm and the causative agent of toxocariasis as a cosmopolitan zoonotic disease. As no information has been reported so far, identification of T. cati proteins can be useful for the development of new diagnostic strategies. This study was conducted to identify the major proteins in the adult T. cati tegument using bi-dimensional electrophoresis (2-DE) and shotgun proteomics. A total proteins were identified, among them the metabolic enzymes were the largest group, including: Enolase, triose phosphate isomerase, fructose-bisphosphate aldolase, aldehyde dehydrogenase. The other important protein groups recognized in T. cati, belong to the HSP-family, the structure and motor proteins, such as actin. The role of these proteins have been implicated in parasite–host interactions and modulating cellular immune response, immune regulation in evasion mechanisms of the host immune response. Characterizing T. cati adult proteins play a key role not only in host-parasite interactions, but also in the discovery of drug targets, subunit vaccines against toxocariasis, immunodiagnostic kits for toxocariasis and the identification of novel immuno-modulators that can form the next generation of therapeutic possibilities for inflammatory diseases.     

IL-17细胞因子家族调节黏膜免疫的研究进展

皮肤和黏膜上皮细胞既是机体的物理屏障,又是机体防御微生物的第一道防线。上皮细胞与白细胞、树突细胞(DCs)等之间的相互作用,是机体产生适应性免疫反应的重要因素。IL-17细胞因子家族是最近新发现的具有强大的促炎症作用的细胞因子,它们在机体的固有和适应性免疫中发挥着重要作用,IL-17A、IL-17C和IL-17F能够直接作用于组织的上皮细胞,诱导各种免疫反应来对抗病原体,且能够促进组织的修复。IL-17E最基本的作用是作用于白细胞和诱导Ⅱ型免疫,这在其对抗寄生虫的作用中是非常关键的,此外,IL-17E还可以反向调节白细胞对IL-17A和IL-17F的产生;而对于IL-17B和IL-17D的研究则相对较少一些。     

Issues and consequences of using nutrition to modulate the avian immune response

Combatting antimicrobial-resistant bacteria is a high priority for both public health and agriculture in the United States and globally. Antibiotic use in animals and humans is considered a major risk factor in the development of antibiotic-resistant bacteria in animals, humans, and the environment. A worldwide emphasis on research into the development of new alternatives to antibiotics is ongoing. Alternatives to antibiotics are broadly defined as any substance that can be substituted for therapeutic drugs, which are increasingly becoming ineffective against pathogenic bacteria, viruses, or parasites. One promising approach involves host-directed immunomodulatory therapies, whereby natural mechanisms in the host are exploited to enhance therapeutic benefit. The objective is to initiate or enhance protective antimicrobial immunity while limiting inflammation-induced tissue injury. The interaction between poultry nutrition, nutrients, and dietary factors and the bird’s immune response has long been known. However, the interplay between nutritional processes and the immune system is incompletely understood. Specifically, precise cellular and molecular immune responses invoked by feed components and the role of the gut barrier and microbiota on the interaction between the immune system and nutrition remains to be fully elucidated. Further, this review will point out some of the areas that have been either neglected when studying the effects of nutrition and nutrients on immune function (the effect of the gut microbiome) or the non-intentional effects of over-feeding various nutrients on the avian immune response (feed-induced inflammation and meta-inflammation).     

Specific immunity in mice to heartwater

Mice develop a specific immune response following infection with the mice strains of heartwater. In the case of the Kümm strain the agent can persist in some tissues for up to 365 days. Transfer of spleen cells from immune mice confers protection against homologous challenge in recipient mice showing that cell mediated immunity is important. A comparison with immune mechanisms occurring in other Rickettsia is discussed.     

Induction of respiratory immune responses in the chicken; implications for development of mucosal avian influenza virus vaccines

The risk and the size of an outbreak of avian influenza virus (AIV) could be restricted by vaccination of poultry. A vaccine used for rapid intervention during an AIV outbreak should be safe, highly effective after a single administration and suitable for mass application. In the case of AIV, aerosol vaccination using live virus is not desirable because of its zoonotic potential and because of the risk for virus reassortment. The rational design of novel mucosal-inactivated vaccines against AIV requires a comprehensive knowledge of the structure and function of the lung-associated immune system in birds in order to target vaccines appropriately and to design efficient mucosal adjuvants. This review addresses our current understanding of the induction of respiratory immune responses in the chicken. Furthermore, possible mucosal vaccination strategies for AIV are highlighted.     

猪肺炎支原体P46基因的原核表达与间接ELISA方法的建立

猪肺炎支原体是猪喘气病的病原体,本研究选择猪肺炎支原体P46膜蛋白基因亲水区序列进行克隆,并将其内3个编码Trp的TGA突变成TGG,然后再克隆到pET28a(+)载体中,在大肠杆菌BL21 (DE3)细胞内实现了高效表达,表达产物相对分子质量约为31 ku,约占菌体总蛋白35％,表达形式为包涵体,通过Western blotting 证明表达产物与猪肺炎支原体高免血清具有很好的反应原性和特异性.将大肠杆菌表达的猪肺炎支原体P46重组蛋白经过洗涤、过柱纯化后,作为间接ELISA包被抗原用于检测猪血清中猪肺炎支原体抗体,通过对各参数和试剂的优化建立了rP46-ELISA方法,获得了较好的效果,通过与现有ELISA检测方法的比较,结果表明二者间具有较高的符合率.