Construction and characterization of a 9-mer phage display pVIII-library with regulated peptide density

The construction of a new phagemid vector for display of peptides on the pVIII major coat protein of filamentous bacteriophage is described, in which expression of pVIII-peptide fusions was placed under the control of the arabinose-inducible P_BAD promoter. The new phagemid showed excellent capacity for the regulation of peptide expression, as judged by enzyme-linked immunosorbent assay (ELISA) and electron microscopy of immunogold-labeled FLAG peptides displayed on phages. Regulation of the density of peptide fusions displayed on phages may offer advantages in the search for new peptide ligands due to the possibility of regulating the stringency of binding, reducing selection based on avidity effects during biopanning. Furthermore, the peptide expression in the absence of inducer was effectively shut off, minimizing growth bias of individual clones. A 9-mer phage display library prepared using the constructed phagemid was generated by insertion of randomly synthesized oligonucleotides close to the N-terminal of the pVIII protein. The library comprised a total of 9.4 × 10⁹ unique transformants, and was confirmed to show high diversity. The functional utility of the library was confirmed by the successful affinity selection of peptides binding to matrix metalloproteinase-9 (MMP-9). The majority of selected peptides shared the consensus motif R(D/N)XXG(M/L)(V/I)XQ, not previously selected during biopanning against MMP-9.     

人白细胞介素6cDNA克隆的分离与鉴定

我们从重组的人α干扰素处理的单层HeLa细胞常规提取Poly(A)~+RNA作为逆转录合成cDNA第一链之模板,用引物-适配接头法在噬菌质粒pTz19R中构建cDNA文库。以~(32)p-标记的480bpIL-6cDNA片段作探针进行菌落原位及狭缝印迹杂交,筛选出6个阳性克隆。其中两个克隆并用限制酶切分析及DNA序列测定做进一步鉴定。结果证明,一个克隆的cDNA片段长1.3kb,含有人白介素6全长编码区;另一个的cDNA插入片段为0.9kb,缺乏信号肽及成熟IL-6N端30个残基的编码序列。     

基于噬菌体展示技术抗黄曲霉毒素B1单链抗体的筛选及其蛋白结构分析

黄曲霉毒素B1(aflatoxin B1,AFB1)是一种毒性强、污染广的真菌毒素,建立高效、准确、快速的AFB1检测方法具有重要意义。利用噬菌粒-辅助噬菌体展示系统构建单链抗体(single chain variable fragment,scFv)文库,应用"淘选-洗脱"的策略,是筛选高亲和配体的常用方法之一。同时结合同源建模和分子对接等计算机辅助手段,分析得到抗体与抗原的结合位点与关键氨基酸,为在基因水平改造抗体提供基础。从AFB1-BSA免疫小鼠的脾细胞内扩增重链可变区和轻链可变区,将组合成的scFv片段插入噬菌粒pCANTAB5e中,构建了噬菌体展示单链抗体文库,以不同浓度的AFB1-OVA作为抗原,从文库中筛选到一株亲和力较好的抗AFB1单链抗体scFv,其亲和常数为8×10~5L/mol;根据同源建模和分子对接发现,与抗原AFB1结合时,scFv中Tyr33、Ser52和Tyr102起关键作用,分别以π-π共轭键、氢键和范德华力与AFB1结合。     

应用抗病毒抗体表位筛选的多肽文库的构建

1985年,Sndth在《科学》杂志上提出构建"融合噬菌体",即在这种丝状病毒的包膜蛋白上表达融合蛋白[1]．1990年,Scott和Smith山利用这种表面表达载体建立了随机多肽文库,可以很方便地筛选出抗体及其他功能蛋白的强结合配基[2]．我们建立了十二肽的随机多肽库,将从中筛选出抗HIV-gP160抗体的抗独特型多肽．1材料和方法1．1质粒、菌株和培养墓噬菌粒成h血四、大肠杆菌XLI-Blue和辅助噬菌体VCSM13由军事医学科学院微生物流行病研究所王海涛教授惠赠．SB液体培养基：30gtmptone,20g酵母膏,10gMops溶于IL去离子水中,调pH7．0．枝…     

Genetic selection of lambda phage clones from a gene clonotheque

Summary A genetic procedure for selection of specific clones, by homologous recombination between clones from a gene clonotheque and sequences cloned into a plasmid, was developed. Resulting clones are isolated in transduction experiments by plating infected Escherichia coli cells under conditions selecting for the antibiotic resistance marker carried by the plasmid. The feasibility of the method was demonstrated in a model test system as well as by isolation of -interferon-specific sequences from the human gene clonotheque.     

Drop-out phagemid vector for switching from phage displayed affinity reagents to expression formats

Affinity reagents that are generated by phage display are typically subcloned into an expression vector for further biochemical characterization. This insert transfer process is time consuming and laborious especially if many inserts are to be subcloned. To simplify the transfer process, we have constructed a “drop-out” phagemid vector that can be rapidly converted to an expression vector by a simple restriction enzyme digestion with MfeI (to “drop-out” the gene III coding sequence), which generates alkaline phosphatase (AP) fusions of the affinity reagents on religation. Subsequently, restriction digestion with AscI drops out the AP coding region and religation generates affinity reagents with a C-terminal six-histidine tag. To validate the usefulness of this vector, four different human single chain Fragments of variable regions (scFv) were tested, three of which show specific binding to three zebrafish (Danio rerio) proteins, namely suppression of tumorigenicity 13, recoverin, and Ppib and the fourth binds to human Lactoferrin protein. For each of the constructs tested, the gene III and AP drop-out efficiency was between 90% and 100%. This vector is especially useful in speeding up the downstream screening of affinity reagents and bypassing the time-consuming subcloning experiments.     

Genetic systems development in the clostridia

Abstract: This review describes recent developments in the genetic manipulation of the solventogenic clostridia, Clostridium acetobutylicum and C. beijerinckii . It is to be noted that our laboratory stock of C. acetobutylicum ATCC 824, which was obtained from the American Type Culture Collection, has recently been re-identified as C. beijerinckii NCIMB 8052 based on DNA similarity studies using the S1 nuclease method (personal communication, Dr. Jiann-Shin Chen, Virginia Polytechnic Institute and State University). Reference to our laboratory 824 culture has been changed to C. beijerinckii NCIMB 8052 throughout this paper in order to be consistent with this finding. The focus of this review specifically involves the characterization of an M13-like genetic system for the clostridia based on the pCAK1 phagemid, as well as preliminary work on development of a plasmid-based vector based on the indigenous pDM11 plasmid recovered from C. acetobutylicum NCIB 6443. The construction of a C. beijerinckii strain with amplified endoglucanase activity was achieved by inserting the engB gene from C. cellulovorans into C. beijerinckii . The successful expression of a heterologous engB gene from C. cellulovorans in C. beijerinckii NCIMB 8052 has important industrial significance for the eventual utilization of cellulose by this acetone-butanol-ethanol fermentation microorganism.     

辅助噬菌体在噬菌体展示中的应用及研究进展

噬菌体展示技术是一种将外源肽或蛋白质与特定噬菌体衣壳蛋白相融合,展示于噬菌体表面来构建蛋白质或多肽文库,并从中筛选目的蛋白、多肽或抗体的基因工程高新技术。噬菌粒/辅助噬菌体系统是最常用的噬菌体展示系统,此系统中辅助噬菌体对噬菌粒的复制和组装发挥着至关重要的作用。本文结合当今该领域的最新研究动态,概述了噬菌粒和辅助噬菌体双基因组系统,着重介绍了不同辅助噬菌体的特点及其突变机制,并对其应用前景进行了展望,以期为该技术的进一步完善提供一定的借鉴作用。