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.     

噬菌体单链抗体导向溶栓剂的构建

Fibrin\|specific antibodies were isolated from a phage\|displayed single\|chain antibody(ScAb) library using affinity selection or panning.DNA shuffling was introduced to realign the specific antibody genes in order to mimic the antibody maturation in vivo. After cloning of shuffling products,a secondary library was constructed,from which the specific antibody against fibrin with higher activity was selected through panning and screening.The fibrin\|specific antibody gene and uPA functional domain gene fragment were joined together,then inserted into expression plasmid pET\|21a.The ScAb/uPA fusion protein was expressed with the induction of IPTG.In substrate S 2444 assay,the uPA activity of the chimera was about 3.1×10 3?IU/mg periplasmic protein of host E.coli .It also kept up the affinity to fibrin.     

Biopanning of HGV epitope from a phage displayed library

Three mouse monoclonal antibodies (mAb) specific to E2 antigen of human hepatitis G virus (HGV) were used to bio-panning of a phage displayed random peptide library of 15 amino acid residues. After 3 rounds of screening, the ratio of output to input increased to 1.1 x 10" and the false positive rate reduced to 0.2% , which means the enrichment was effective. At the third round of screening, 15 plage clones were selected for the use in binding and competitive inhibition tests. Thirteen of them could specifically react with the mAb M6. The inhibition rates of phage 10 clones out of 15 were over 60% . From the deduced insert sequence in the coat protein VIII, the core sequence NPLWP was found in 6 phage clones which are homologeous to the amino acids 301—305 of HGV E2.The sera from the mice immunized with the phage clone C2 containing motif sequence were found positive for anti-HGV. These indicate the possibility that NPLWP motif in the short peptide is the mimic of HGV E2 epitope that can be recognized by HGV mAb M6.     

Software Practicalization for Analysis of Wind-Induced Vibrations of Large Span Roof Structures

Wind loads are key considerations in the structural design of large-span structures since wind loads can be more important than earthquake loads, especially for large flexible structures. The analysis of wind loads on large span roof structures (LSRS) requires large amounts of calculations. Due to the combined effects of horizontal and vertical winds, the wind-induced vibrations of LSRS are analyzed in this paper with the frequency domain method as the first application of method for the analysis of the wind response of LSRS. A program is developed to analyze the wind-induced vibrations due to a combination of wind vibration modes. The program, which predicts the wind vibration coefficient and the wind pressure acting on the LSRS, interfaces with other finite element software to facilitate analysis of wind loads in the design of LSRS. The effectiveness and accuracy of the frequency domain method have been verified by numerical analyses of practical projects.     

A novel peptide,selected from phage display library of random peptides,can efficiently target into human breast cancer cell

To develop a targeting vector for breast cancer biotherapy, MDA-MB-231 cell, a human breast cancer cell line, was co-cultured with pC89 （9 aa） phage display library of random peptides. In multiple inde-pendent peptide-presenting phage screening trials, subtilisin was used as a protease to inactivate extra-cellular phages. The internalized phages were collected by cell lysising and amplified in E. coli XLI-Blue. Through five rounds of selection, the pepUde-presenting phages which could be internalized in MDA-MB-231 cells were isolated. A comparison was made between internalization capacities of peptide-presenting phages isolated from MDA-MB-231 cells and RGD-integrin binding phage by coculturing them with other human tumor cell lines and normal cells. The nucleoUde sequences of isolated peptide-presenting phages were then determined by DNA sequencing. To uncover whether phage coat protein or amino acid order was required for the character of the pepUde to MDA-MB-231 cells, three peptides were synthesized. They are CASPSGALRSC, ASPSGALRS and CGVIFDHSVPC （the shifted sequence of CASPSGALRSC）, and after coculturing them with different cell lines, their targeting capacities to MDA-MB-231 cells were detected. These data suggested that the internalization process was highly selective, and capable of capturing a specific peptide from parent peptide variants. Moreover, the targeting internalization event of pepUdes was an amino acid sequence dependent manner. The results demonstrated the feasibility of using phage display library of random peptides to develop new targeting system for intracellular delivery of macromolecules, and the peptide we obtained might be modified as a targeting vector for breast cancer gene therapy.     

Path Weight Complementary Convolutional Code for Type-Ⅱ Bit-Interleaved Coded Modulation Hybrid ARQ System

Bit-interleaved coded modulation (BICM) is suitable to bandwidth-efficient communication systems. Hybrid automatic repeat request (HARQ) can provide more reliability to high-speed wireless data transmission. A new path weight complementary convolutional (PWCC) code used in the type-Ⅱ BICM-HARQ system is proposed. The PWCC code is composed of the original code and the complimentary code. The path in trellis with large hamming weight of the complimentary code is designed to compensate for the path in trellis with small hamming weight of the original code. Hence, both of the original code and the complimentary code can achieve the performance of the good code criterion of corresponding code rate. The throughput efficiency of the BICM-HARQ system wit PWCC code is higher than repeat code system, a little higher than puncture code system in low signal-to-noise ratio (SNR) values and much higher than puncture code system, the same as repeat code system in high SNR values. These results are confirmed by the simulation.     

A New Thinking of the Objects Served Relationship Management in Complex System

Take a digital libraries＇ service system for example, Objects Served Relationship Management （OSRM） in complex systems is proposed firstly as a new concept, and its connotation is explained. The significances and constructions of OSRM are analyzed. Both the fundamental facts and the important natures that the things which are interested by Objects Served （OS） （e. g. publishers and readers） and the server （e. g. digital libraries are the servers of publishers and readers） will not be the same completely although there are a lot of common benefits between OS and servers, are indeed clarified. The valuable information,which should be used by OS and their server, is often hidden behind them. Thus, how to find, manage and control the relationship among OS and their servers is very necessary and important for the common benefits among all of them.（e. g. the three dimensions of OSRM in digital library system and its overall framwork are proposed. The different strategies to different cases in the digital library＇s multidimensional framework are analyzed.）     

AN INNOVATED REACTOR WITH VARYING DIAMETER AGITATOR AND ITS APPLICATIONS

The reactor is equipped with two different types of agitators: the diameter-variable agitator and the two-pitched-paddle agitator. The diameter of the former can be varied according to the requirement of the chemical reactions, thus making the concentration and temperature of the whole reaction system uniform throughout the reactor without any stagnant zone. It is shown that the apparatus has the preponderance over conventional agitator, especially when extremely rapid reactions are accompanied by large amount of reaction heat and when the viscosity of the reaction system varies in a wide range. The reactor is very effective when applied to the polycondensation reaction between paraphenylene diamine (PPDA) and terephthyl chloride (TPC). The inherent viscosity (η_(inh)) of the polymer comes up to more than 5. This innovative agitator has the advantage of simplicity in structure and flexibility in operation, etc.     

Path loss modelling and comparison based on the radio propagation measurement at 3.5GHz

Wideband IMT-Advanced mobile communication systems tend to operate in the high frequency bandsdue to a relatively large capacity available .Thus,Measurement and modelling methods of radio propaga-tion characteristics are proposed for the field test of Chinese 4th generation(4G)trial system.The mea-surement system is established for 3 .5GHz based on the sophisticated measurement instruments and thevirtual instrument technology.The characteristic parameters of radio propagation such as path loss(PL)expone...     

Reliable fuzzy control with domain guaranteed cost for fuzzy systems with actuator failures

The reliable fuzzy control with guaranteed cost for T-S fuzzy systems with actuator failure is proposed in this paper. The cost function is a quadratic function with failure input. When the initial state of such systems is known, a design method of the reliable fuzzy controller with reliable guaranteed cost is presented, and the formula of the guaranteed cost is established. When the initial state of such systems is unknown but belongs to a known bounded closed domain, a notion of the reliable domain guaranteed cost (RDGC) for such systems is proposed. For two classes of initial state domain, polygon domain and ellipsoid domain, some design methods for reliable fuzzy controllers with the RDGC are provided. The efficiency of our design methods is finally verified by numerical design and simulation on the Rossler chaotic system.     

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。     

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.     

小鼠噬菌体抗体库的构建和N-肽结合单链抗体的筛选

成功构建一库容量为1.7×10⁸的小鼠噬菌体抗体库,并从中淘选出对N-肽有结合活性的单链抗体。首先从未免疫小鼠的脾脏中提取总RNA,分离出mRNA,经逆转录合成其总cDNA。随后通过PCR分别扩增出抗体重链和轻链可变区V_H、V_L的基因片断,再经重组PCR将两片断由一编码(Gly₄Ser)₃十五肽的接头连在一起,并克隆到噬菌质粒pCANTAB 5E中,电击转化大肠杆菌TG1。经辅助噬菌体M13KO7超感染回收全部重组噬菌体,此即噬菌体抗体库。N-肽是一种新发现的神经肽,其N端与阿片肽高度同源,可与阿片肽受体相互作用。对其研究可能有助于了解成瘾机制和有临床应用价值。将生物素化的N-肽与抗体库相互作用,用偶联有链霉亲和素的磁珠富集与N-肽结合的重组噬菌体,经四轮淘选,得到亲和力较高的单链抗体。     

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.     

应用噬菌体随机肽库研究抗溶藻弧菌及其外膜蛋白单抗识别的特异性表位

用噬菌体随机七肽库筛选制备的抗溶藻弧菌及其外膜蛋白的纯化单抗，经3轮淘洗，获得较高程度富集的噬菌体，效价较高，第3轮淘洗比第1轮淘洗的产量高出近百倍。同时获得的阳性噬菌体经ELISA检测能与Va抗原发生强的阳性反应，确证了淘洗筛选的结果。     

Effects of interlinker sequences on the biological properties of bispecific single-chain antibodies

Single-chain bispecific antibody (scBsAb) is one of the promising genetic engineering antibody formats for clinical application. But the effects of interlinker sequenees on the biological properties of bispecific single-chain antibodies have not been studied in detail. Three interlinker sequences were designed and synthesized, and denominatedas Fc, HSA, 205C‘, respectively. Universal vectors with these different interlinker sequences for scBsAb expression in E.coli were constructed. A model scBsAb based on a reshaped single-chain antibody (scFv) against human CD3 and a scFv directed against human ovarian carcinoma were generated and expressed in E. coli. The results of SDS-PAGE and Western blot showed that the different interlinker sequences did not affect the expression level of scBsAb. However, as demonstrated by ELISA and pharmacokinetics studies performed in mice, scBsAbs with different interlinker sequenees had difference in the antigen-binding activities and terminal haw-life time (T1/2β) in vivo, the interlinker HSA could remarkably prolong the retention time of scBsAb in blood. These results indicated that the peptide sequence of intertinker could affect important biological properties of scBsAb, such as antigen-binding properties and stability in vivo. So, selection of an appropriate interlinker sequence is very important for scBsAb construction. Optimal interlinker can bring scBsAb biological properties more suitable for clinical application.     

噬菌体展示禽流感病毒抗原变异性基因片段文库的构建

构建T7噬菌体展示禽流感病毒抗原变异性基因片段文库. 首先， 从Gene Bank中查找筛选禽流感病毒抗原变异性基因, 将其截短、 修饰、 简并后得到禽流感病毒抗原变异性基因微阵列. 其次， 将合成的禽流感病毒抗原变异性基因片段文库扩增、 酶切, 链接到双酶切后的T7噬菌体载体基因上, 构成重组噬菌体DNA. 最后， 重组噬菌体DNA经体外包装和扩增, 得到T7噬菌体展示文库, 并进行T7噬菌体展示文库滴度、 重组率和免疫活性测定. 实验结果表明, 从Gene Bank中查找、 筛选、 剪切和修饰共获得96 258条序列构建T7噬菌体展示文库, 原始文库滴度为3.6×107个菌落/mL, 重组率大于90%. 用禽流感病毒H5N1抗体进行捕获, 经聚合酶链式反应(PCR)鉴定, 得到理想目的条带, 证明噬菌体表面展示蛋白具有抗原活性, 可用于禽流感病毒感染患者的快速检测及抗原表位筛选.     

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.     

噬菌体展示禽流感病毒抗原变异性基因片段文库的构建

构建T7噬菌体展示禽流感病毒抗原变异性基因片段文库. 首先, 从Gene Bank中查找筛选禽流感病毒抗原变异性基因, 将其截短、 修饰、 简并后得到禽流感病毒抗原变异性基因微阵列. 其次, 将合成的禽流感病毒抗原变异性基因片段文库扩增、 酶切, 链接到双酶切后的T7噬菌体载体基因上, 构成重组噬菌体DNA. 最后, 重组噬菌体DNA经体外包装和扩增, 得到T7噬菌体展示文库, 并进行T7噬菌体展示文库滴度、 重组率和免疫活性测定. 实验结果表明, 从Gene Bank中查找、 筛选、 剪切和修饰共获得96 258条序列构建T7噬菌体展示文库, 原始文库滴度为3.6×107个菌落/mL, 重组率大于90%. 用禽流感病毒H5N1抗体进行捕获, 经聚合酶链式反应(PCR)鉴定, 得到理想目的条带, 证明噬菌体表面展示蛋白具有抗原活性, 可用于禽流感病毒感染患者的快速检测及抗原表位筛选.