抗弓形虫噬菌体单链抗体库的构建及筛选

采用弓形虫可溶性抗原攻击的小鼠,取小鼠脾脏从中提取细胞总RNA,通过RT-PCR扩增鼠源抗体VH和VL基因,并采用重叠PCR (SOE-PCR)方法构建ScFv基因,将其克隆入噬粒载体pCANTAB5E中,转化于感受态大肠杆菌TG1,通过辅助噬菌体M13K07援救构建噬菌体单链抗体库.从20个噬菌体克隆中筛选到15个具...     

抗PAF单链抗体的基因构建及蛋白质3D模建

构建抗PAF单链抗体的基因并通过计算机模建分析几种不同连接类型的单链抗体空间结构差异。采用重叠延伸PCR方法扩增了VH和VL基因,形成抗PAF的VH-Linker-VL(ScFv)基因产物,并经菌落PCR和测序分析鉴定,通过Internet对其3D结构进行了模建与分析,研究结果表明：PCR产物电泳可见一条与目的基因大小一致的片段,经菌落PCR和测序分析鉴定,抗PAF单链抗体的基因构建成功;VH和VL在单链抗体中的顺序对单链抗体3D的空间结构有影响。     

具有GPX活性单链抗体的制备及其抗氧化效应

为实现单链抗体的可溶性表达, 制备具有谷胱甘肽过氧化物酶（GPX）活力的单链抗体, 在单链抗体表达载体pTMF-2F3上去除原2F3基因N端非必需的18个氨基酸, 采用新的表达载体pRose质粒, 在2F3的N端引入13个氨基酸的前导肽, 转入BL21（lys S）中, 使其分泌到大肠杆菌的周质腔中得以表达, 获得可溶性表达产物. 产物经过分离纯化、 Wester n Blot印迹和硒化测活确定具有GPX活性的鼠单链抗体, 其GPX活力为2 530 U/μmol. 以脂质过氧化、 细胞存活率和细胞膜完整性为指标的抗氧化实验研究表明, 具有GPX活性的单链抗体对大鼠乳鼠表皮细胞有抗紫外线损伤的作用, 是膜脂质过氧化的有效抑制剂.     

Construction of Large Human Single—chain Antibody Phage Display Library

A large human naive single chain antibody (scFv) library is constructed from 60 healthy donors via phage display technique.During the period,some methods are employed to optimize the diversity,such as multi donors,different annealing temperature,half-nest PCR.and assembly by two-way fusion PCR.In this study,78 electroporations resulted in 1010 library,diversity of which is assayed by enzyme fingerprint.The efficiency and diversity are all better than other researches.     

等规聚苯乙烯单链单晶的结构和耐辐照性能

利用电子衍射（ED）和高分辨电子显微（HREM）技术，研究等规聚苯乙烯（i－PS）单链单晶的结构．纳米级的单链单晶具有很强的耐电子辐照性能．按照i－PS的晶胞能对ED图中的衍射环和HREM像中的晶格条纹进行晶面指标化，但发现低指数衍射缺失．由于单链单晶的尺寸很小，电子辐照所产生的次级电子可以逸出晶体，使辐照损伤大大减小．另外，单链单晶存在着较少的低指数晶面，未能产生足够的衍射强度，使低指数晶面的衍射缺失．单链单晶对电子辐照稳定，在室温下，可得到高分辨晶格条纹象，这为研究高分子晶体的结构开辟了新的实验方法．     

EL-4荷瘤鼠单链噬菌体抗体库的构建

目的:构建一个EL-4荷瘤鼠的单链噬菌体抗体库,为筛选高特异性和高亲和力的单链抗体做准备.方法:于C57小鼠腋区接种EL-4细胞,待肿瘤长大后,从脾细胞中提取总RNA,RT-PCR技术扩增小鼠抗体重、轻链可变区基因(VH、VL),用Linker奖VH和VL基因连成单链抗体可变区片段(scFv).双酶切后(Not Ⅰ、Sfi Ⅰ)与预备好的pCANTAB5E噬粒载体连接,转化入感受态TG1,构建EL-4荷瘤鼠的单链噬茵体抗体库.随机抽取转化后的20个克隆,用以检测外源DNA的转入情况.结果:PCR扩增出的VH约有340bp和VL约有320bp,scFv的长度约有750bp.转化后的TG1约有1.67×107个茵落,随机挑取20个克隆,双酶切显示五分之一的茵落转化了外源DNA片段,有效库容为3.34×106.结论:成功构建了EL-4荷瘤鼠的单链噬茵体抗体库.     

快速全血凝集试验在中国不同人群HBsAg检测中的应用

为了评价本实验室建立的快速全血凝集法检测HBsAg的效果,检测了河北省不同人群534份全血样品,得到了满意结果.来自不同人群的369份样品(其中62份来自乙肝患者,180份来自献血者,127份来自正常人群),经快速全血凝集法和国家认证的市售ELISA试剂盒平行检测后,总符合率为96.7%,灵敏度为90.99%,特异性为98.7%.乙肝患者、献血者和正常人群中阳性率分别为52.6%,4.3%和8.9%.实验结果说明快速全血凝集法不仅简单快速、不需要特殊设备,而且具有高的敏感性和特异性,因此它可以广泛应用于基层卫生机构和临床HBsAg的检测,尤其适用于病人的床边诊断.     

黄麻秸秆及有机肥对滨海盐土生物性质的影响

为改善滨海盐土质量,通过黄麻秸秆还田及施用有机肥对盐土的改良试验,研究不同施用量的秸秆与有机肥配施分别对土壤酶活性、微生物的影响。结果表明:以3 600 kg/hm2秸秆施用量分别与有机肥施用量为2 400、3 600 kg/hm2配施时对土壤生物性质改良效应较为明显,土壤脲酶分别比对照增加53%和98%、蔗糖酶增加700%和825%、过氧化氢酶增加49%和65%、磷酸酶增加99%和161%、土壤微生物数量增加了5.8、9.5倍,其中,真菌增幅较大,两种方式下分别增加55.1、90.8倍。说明秸秆与有机肥配施能明显提高滨海盐土酶活性,增加土壤微生物数量,增强土壤生物学活性,从而提高土壤肥力。     

抗体药物的植物反应器生产

大量的重组抗体被用于疾病的预防、诊断和治疗.目前,这些重组抗体绝大多数是利用鼠源细胞生产的,它的缺点是有潜在的隐患和价格昂贵.然而,植物作为反应器生产抗体药物既具有安全性又可大规模廉价生产的特点,具有广阔的前景.     

不同免疫佐剂对大肠杆菌疫苗免疫效果的影响

将大肠杆菌经培养后制成疫苗,分别加入矿物油佐剂、氢氧化铝佐剂和不加任何免疫佐剂,按一定免疫程序分别注射实验兔,取兔血清进行凝集试验测其抗体效价。结果表明：加矿物油佐剂苗的抗体效价最高（1：256）,不加佐剂苗的抗体效价次之（1：128）,加入氢氧化铝佐剂苗的抗体效价最低（1：64）。     

HBscFv毕赤酵母工程菌的中试发酵及产物纯化

中试规模生产可溶性重组人抗HBsAg单链抗体 (HBscFv) ,分泌表达HBscFv的巴氏毕赤酵母 (P .pastoris)工程菌在 30L发酵罐中进行补料分批培养 .上清中的HBscFv以离子交换及免疫亲和层析两步法进行纯化 .发现酵母工程菌经 7d补料分批培养后 ,6 0 0nm波长下的光密度值 (OD6 0 0 )达到 334,目的蛋白表达量为 2 6 0mg/L .纯化后 ,可获得纯度为95 %的HBscFv ,产量为 171mg/L .活性测定结果表明 ,HBscFv制品的比活性为 (2 5 2±0 17) μg- 1,与大肠杆菌来源的HBscFv无明显差异 .     

抗HBsAg单链抗体的等电点测定和结构模拟

应用实验生物学和计算生物学方法对重组人源抗HBsAg单链抗体(HBscFv)的部分理化性质和空间结构进行分析．首先应用蛋白质分析软件包Antheprot预测HBscFv等电点，然后等电聚焦测定HBscFv等电点，在此基础上，用神经网络法预测HBscFv二级结构，然后用同源建模的方法，获得HBscFv三级结构．结果发现，HBscFv等电点理论值为8．51，实验值为7．3～8．1；PHDsec结果表明，HBscFv是一种全β蛋白质；三级结构建模表明，HBscFv的VH结构域由9个片层组成，VL结构域由8个片层组成；由于单链抗体的特殊性，三级结构建模无法给出VH与VL结构域之间进一步折叠后形成的结构。     

Effects of biological pre-treatment of pine chips on the beating performance of Kraft pulp

The Calabrien pine (Pinus brutia) wood chips prior to kraft pulping were biologically pre-treated with selected white-rot fungi (Ceriporiopsis subvermispora), which was recorded to be preferentially attacking the lignin component of the wood. The effects of this treatment on beating performance and physical strength of resultant papers were studied in detail. Bio-treated samples showed comparable and, in most cases, higher physico-mechanical properties than those obtained from untreated controls. Under the same beating conditions the bio-treated kraft pulp was noted to have the lower SR° indicating a lower degree of external fibrillation. The paper made from bio-treated kraft pulp has a higher density, tensile property, air permeability and swellability. Furthermore, remarkable energy savings up to 33 % were observed when beating bio-treated kraft pulp. This study contributes to a better understanding of the mechanisms taking place during bio-treatment and the modification processes of cell wall components.     

基于生物序列模式提取技术的邮件过滤算法

为了解决垃圾邮件过滤问题,考虑到中文垃圾邮件的特点和过滤系统的效率要求,应用生物信息化技术中模式提取算法TEIRESIAS的原理,设计了基于生物序列模式提取技术的垃圾邮件过滤算法BioMatrix,并实现了基于此算法的中英文邮件过滤系统.过滤系统由数量控制过滤提供垃圾邮件训练集,通过提取其中的特征模式对邮件进行分类,可以识别出约94.2%的垃圾邮件,误过滤率约0.04%.与Bayes过滤算法对比的实验结果表明,将生物序列模式提取技术应用于邮件过滤具有较好的研究和实用价值.     

Design and optimization of a linker for fusion protein construction

Bivalent, bispecific single-chain antibody fusion protein construction appears to be a promising tool for tumor therapy. One of its drawbacks is that the function and activity of the fusion proteins have varied affinity and/or anti-tumor activity compared with the original molecules from which they are derived. A more optimized linker for fusion proteins would confer more favorable biological activities upon bispecific single-chain antibodies. Thus, it is critical to design and optimize an inter-peptide linker. With different functional domains and optimized linkers, fusion proteins provide a solid base for targeted immune therapy for malignancies. In this paper, we review the inter-peptide linker studies and the design of an optimized linker using genetic algorithms. The spatial structure of the fusion protein can be predicted by using genetic and bioinformatics research. Based on current research, the future focus will be on different correlation models to perform simulations of spatial structures and drug molecule design.     

A Co—expression System Based on Phage and Phagemid to Select Cognate Antibody—antigen Pairs in vivo

A modified selectively-infective phage(SIP) is developed to facilitate the selection of interacting antibody-antigen paris from a large single-chan antibody(scFv)library in vivo.The system is constructed with a modified helper phage M13KO7 and phagemid pCANTAB 5E.The antigen fused to the C-terminal of N1-N2 domain and the scFv to the N-terminal of CT domain of the gIIIp of filamentous phage are encoded on the phage and phagemid vectors respectively.The phages produced by co-transformants restore infectivity via interaction between antigen and antibody fusions in the cell periplasm.In a model system,the scFv fragment of the anti-hemagglutinin 17/9 antibody and its corresponding antigen are detected in the presence of a 10^5 fold excess of a non-interacting cotrol paris,which demonstrates this system to be very sensitive and facile to screen a large single-chain antibody library.     

Isolation by phage display and characterization of a single-chain antibody specific for O6-methyldeoxyguanosine

New approaches of making single chain Fv antibodies against O⁶-methyl-2′-deoxyguanosine (O⁶MdG) have been demonstrated by using the phage antibody display system. Using O⁶MdG as an antigen, 21 positive clones were identified by ELISA from this library, one of which, designated H3, specifically binds to O⁶MdG with high affinity. The H3 scFv antibody has an affinity constant (K_aff) of 5.94×10O¹¹(mol/L)^-1. H3 scFv has been successfully used to detect O⁶MdG in DNA hydrolyses from yeast or E. coli cells treated with a DNA methylating agent. To our knowledge, this is the first report of the selection of a specific scFv against DNA adducts. The results demonstrate the potential applications of the phage display technology for the detection of DNA lesions caused by mutagens and carcinogens.     

鸡卵黄抗体的研究与应用

免疫母鸡的卵黄内舍有抗体这一发现，推动了卵黄抗体制备技术的发展．研究发现卵黄抗体更优于哺乳动物所产生的抗体，而且减少了对动物的伤害．分析了卵黄抗体的生物学特性、作用机理以及用途.