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.     

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.     

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.     

抗口蹄疫病毒phage-scFv及可溶性scFv的构建

利用重组DNA技术在抗口蹄疫病毒单克隆抗体1C 7 VH基因和VL基因之间导入一段连接肽[(G ly4Ser)3],采用重叠延伸拼接法,经聚合酶链反应(PCR)扩增获得scF v基因。将scF v基因克隆至噬菌粒pCANTAB 5E载体,转化E.coli TG 1,构建噬菌体抗体文库。用M 13KO 7辅助噬菌体挽救及固相口蹄疫病毒(FMDV)抗原对噬菌体抗体文库的三轮“吸附-洗脱-扩增”的淘洗,筛选出scFv阳性克隆。将阳性克隆转化E.coli BH 2151,通过异丙基硫代-β-D-半乳糖苷(IPTG)诱导可溶性scFv蛋白的表达。酶联免疫吸附实验(EL ISA)检测表明:scFv克隆表达的phage-scFv及可溶性scFv与FMDV亲和力高,特异性强。     

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.     

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.     

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。     

Bispecific antibody and its clinical applications in cancer

Bispecific antibody (BsAb) usually consists of two different antigen-binding arms, by which it is capable of simultaneously binding to target cells and effector cells, and can directly mediate the killing of target cells by retargeting and activating effector cells. The development of BsAb research goes through three main stages: chemical cross- linking of murine-derived monoclonal antibody, hybrid hybridomas and engineered BsAb. Among them, engineered BsAb has more formats than the other two, such as diabody, ScdHLX, ScZip, ScCH3, ScFab and BsIgG, etc. Compared with former murine-derived BsAbs, engineered BsAb has lower immunogenicity and stronger penetrating capacity, and currently, some of them appear suitable for clinical application in yields and qualities. Up to now, several phase Ⅰand phase Ⅱ clinical studies of BsAb, for instance, some (Fab’)₂ and Diabodies, have been performed. Among those BsAbs, anti-CD3/anti-tumor BsAbs is most common, they not only can activate T cell and induce CD3AK cytotoxic activity in in vitro experiment, and inhibit the growth of tumor on tumor-bearing mouse by retargeting T cells to lyse tumor cells, but also offer great promise in the therapy of some malignancies in clinic, especially of some advanced cancers as well as elimination of minimal residual tumors, indicated by increasing the tumor/blood ratio of antibody in patients and improving the natural killer cell (NK) anti-tumor activity in tumor sites, and also presenting of an increase level in TNF-α,INF-γ, IL-6, IL-8, IL-10 and soluble CD25, etc. The responses are also shown via improving the quality of life and prolonging the survival of partial patients. The “Knobs into Holes” technology is a new strategy emerging during research on engineered BsAb, it is likely to be useful for heterodimerization and can improve the quantity, purity and stability of BsAb, it is also anticipated to increase the clinical potential of BsAb in the future.     

噬菌体筛选技术筛选与联萘酚(BINOL)相结合的抗体

利用噬菌体展示技术筛选只结合1种对应体的BINOL抗体,建立人类单链抗体(scFv)噬菌体文库--Griffin.1文库,以固相化抗原淘筛抗体库,ELISA鉴定噬菌体抗体.从经过4轮富集的次级抗体库中挑选到噬菌体克隆,用该克隆制备的单链抗体阳性克隆,经ELISA证实具有良好的抗原特异性.从人类scFv噬菌体文库中获得抗BINOL的特异性抗体.     

Generation of beta-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli

High-level expression of many eukaryotic genes has proved difficult to achieve even when a strong promoter and the ribosome binding sequence from highly expressed Escherichia coli genes have been placed in front of the coding sequences. To overcome this problem, many eukaryotic proteins have been efficiently produced as hybrids after fusion of their genes with a coding sequence of E. coli genes. However, such hybrid proteins are not suitable for functional studies or clinical use unless the authentic protein sequence can be released by specific cleavage. Here, we have inserted the sequence Ile-Glu-Gly-Arg between the 31 amino-terminal residues of lambda cII protein and Val 1 of human beta-globin, and produced this hybrid in high yield in E. coli. We then cleaved the hybrid specifically at the single arginine, using blood coagulation factor Xa and thus liberated the authentic beta-globin chain. As factor Xa is specific for the tetrapeptide Ile-Glu-Gly-Arg, which is rare in protein sequences, our expression/cleavage system is applicable to the efficient production of many eukaryotic proteins.     

TMV重组外壳蛋白Sc1754CP的原核表达和多克隆抗体制备

烟草花叶病毒（tobacco mosaic virus,TMV）重组外壳蛋白（coat protein,CP）Sc1754CP是一种能够激发敏感烟草产生过敏性反应（hypersensitive reaction, HR）的蛋白, 但是这种蛋白无法通过植物病毒的大量扩增获得.为了获得足够量的上述蛋白,并对这种蛋白在植物中激发的HR反应进行深入研究,作者在大肠杆菌中大量表达Sc1754CP蛋白,并且获得了Sc1754CP高度专一性的多克隆抗体.上述研究为进一步深入研究Sc1754CP及其激发的新的过敏性反应机制奠定了基础.     

5＇端序列对hGM－CSF在大肠杆菌中表达水平的影响

设计并构建了三种起始密码子ＡＴＧ上下游不同序列的人ＧＭ－ＣＳＦ原核表达质粒，对其核苷酸序列进行分析验证后，使它们分别在大肠杆菌中表达了人ＧＭ－ＣＳＦ．结果表明：ｐＬＧＭ３对ＧＭ－ＣＳＦ的表达量比ｐＬＧＭ１高约６倍，比ｐＬＧＭ２高约３倍．     

半模正合列的性质

采用文献[1]中半模的正合列、短正合列的定义,将环上模的正合列的一些性质推广到半模范畴。     

人溶菌酶N端与Exendin-4嵌合蛋白的基因克隆及原核表达

目的:克隆嵌合多肽人溶菌酶N端-Exendin-4基因并进行原核表达和纯化.方法:通过重组PCR技术将人溶菌酶N端74个氨基酸的基因序列与Exendin-4多肽基因序列相连接,其间引入一段由凝血酶和二肽基肽酶识别位点组成的连接序列.以嵌合基因hLYZ(N74)-Ex4与质粒pET-32a(+)构建原核表达体,转化大肠杆菌BL21(DE3)并诱导表达.表达蛋白经Ni-NTA亲和层析纯化、Western blotting鉴定;透析复性后,以肠激酶切割并回收目的多肽.结果:重组质粒pET-32a/hLYZ(N74)-Ex4构建正确,目的蛋白主要以包涵体形式存在,37℃诱导4h、IPTG浓度为0.6 mmol/L时表达量最高,约占菌体蛋白总量的30%.Western blotting检测显示重组蛋白为单一清晰条带.重组蛋白经肠激酶切割后,回收得到高纯度的嵌合多肽.结论:成功构建hLYZ(N74)-Ex4嵌合基因的原核表达质粒,高效原核表达并获得高纯度目的蛋白.     

Research advances in genetic engineering of plantibody

The combination of engineering antibody and plant biotechnology creates the plantibody. It has been reported that the engineering antibodies expressed in plant, no matter whether they are intact or small-molecular antibodies, keep their antigen-binding specificity, which means they are functional. This trait makes plantibody notable. Now the researches are focused mainly on the following three aspects: (i) Therapeutic antibody in clinic. It is expected to offer the opportunity of large-scale antibody procluction in agriculture systems, for the purpose of decreasing the cost greatly. Moreover, the property of multiple sexual hybridization between plants can be used to produce bior multi-functional antibodies and create new antibody medicines. (ii) Plant physiology. Engineering antibodies against plant hormones or some active materials can block their activities in plant, so the trait of plantibody could be used to study the growth and development of plant. (iii) Plant disease-resistance. The gene of antibody against plant virus can be transferred into plant to defend the intrusion of virus in order to cure the disease.     

拟南芥AtERF1蛋白的原核可溶性表达及多克隆抗体的制备

转录因子AtERF1属于ERF/AP2家族的B3亚家族,参与植物的防卫反应.根据密码子简并原理,用基因全合成的方法合成了满足大肠杆菌密码子嗜好的编码拟南芥AtERF1蛋白的碱基序列,并将其克隆到原核表达载体pET-29b.将表达载体AtERF1-pET-29b转入大肠杆菌BL21(DE3),用IPTG成功诱导表达了分子量约30kD的AtERF1蛋白,该蛋白主要以可溶性蛋白的形式存在.以该蛋白为抗原免疫家兔,制备出的多克隆抗体经免疫双扩散测定效价达1∶16,Western blot检测表明该抗血清与AtERF1蛋白识别良好.AtERF1抗体的制备为进一步从生化水平上研究AtERF1的功能奠定了良好基础.     

PMEPA1蛋白的原核表达、纯化及其抗体制备

为了从蛋白水平上检测前列腺跨膜蛋白PMEPA1(prostate transmembrane protain,androgen induced 1)在细胞内的表达和功能,构建了原核表达重组质粒pTrcHisA-PMEPA1和pGEX4T-2-PMEPA1,转化大肠杆菌BL21-RIPL后,IPTG诱导蛋白表达,分别用Ni-NTA和谷胱甘肽琼脂糖凝胶层析柱纯化蛋白。用His-PMEPA1免疫兔子制备抗体,并用GST-PMEPA1对抗体进行纯化。免疫印迹检测显示抗体能够特异识别细胞中内源PMEPA1蛋白。     

A putative second cell-derived oncogene of the avian leukaemia retrovirus E26

The acute avian leukaemia retroviruses AMV and E26 both induce myeloblastosis in vivo and transform myeloblasts in vitro. Both viruses contain the oncogene v-myb first described for AMV. Unlike AMV, E26 has the additional capacity to induce erythroblastosis in vivo and to transform erythroblasts. Previous analyses indicated that the genome of E26 also contained nucleotide sequences distinct from v-myb and unrelated to viral replicative genes. Using a molecularly cloned E26 provirus, we have now identified a novel nucleotide sequence designated v-ets (for E-twenty-six specific) of approximately 1.5 kilobase pairs (kbp) located next to v-myb. v-ets possesses all the structural characteristics of a putative new oncogene: it has a conserved cellular counterpart c-ets which is transcribed in some normal chicken cells as a major 7.5-kb polyadenylated RNA. Although our results now await elucidation of their biological significance, we propose that v-ets could be a new oncogene accounting for the additional transforming properties of E26, or potentiating the transforming properties of the v-myb oncogene.     

沙门氏菌rDNA ISR序列测定及特征分析

以细菌ｒＤＮＡＩＳＲ通用经物对鼠伤寒等８种沙门氏菌进行了ＰＣＲ分析，表明在沙门氏菌属的不同菌株中都存在１个比大肠杆菌的Ｌ－ＩＳＲ长几十年碱基对的Ｌ－ＩＳＲ。进一步测定和分析鼠伤寒，纽波特和田纳西３种沙门氏菌Ｌ－ＩＳＲ的全序列，揭示出不同血清型的沙门氏菌具有相似的ｔＲＮＡ基因的间隔区和特征性核苷酸序列。这些特征序列可以作为沙门氏各的分子标志，用于沙门氏菌的分子检验和系统学鉴定。     

Analysis of components of conserved “backbone sequences”among genomes of Shigella spp. strains

Difference in the genomic compositions of prokaryotes is the basis of the diversity in their biological characters. However, besides their flora-or strain-specific genes,those floras with closer relationship in the evolution also have conserved “backbone sequences”, which reveal the marks of their origin and evolution, and these “backbone sequences”are just the basis of their elementary living abilities and common biological properties. Shigella is very closely related to E. coli in the origin and evolution, and may turn out to belong to the same genus. In this study, a microarray containing E. coli K-12 whole genome and Sf301 specific ORFs is used to investigate the genomic components of four Shigella strains. The results indicate that 16%-22% K-12 ORFs sequences are not detected in the genome of Shigella strains while the genome of Shigella contains at least 2800 conserved ORFs, which compose the common “backbone sequences”.Advanced analysis indicated that the “backbone sequences”are the essential components in maintaining the normal physiological activities of intestinal bacteria. Furthermore,only 20% Sf301-specific ORFs exist in other strains simultaneously, which demonstrate the great genome heterogeneity and the genetic diversity among the strains.