噬菌体展示技术系统发展进展

噬菌体展示技术(Phage display technology,PDT)是一种特殊的基因工程重组表达技术,噬菌体展示技术系统(Phage display system,PDS)是指包括经过遗传改造后的系列噬菌体、辅助噬菌体、宿主细菌等集成平台(含试剂盒)。文章从噬菌体分子遗传学及其基因(基因组)遗传工程改良角度,基于噬菌体M13、λ、T4和T7等4大类典型噬菌体展示技术系统的发展进展进行了综述。重点强调不同展示系统中的核心部件及其基因工程改造的分子遗传学原理、不同展示锚定位点的技术特征、相关试剂盒的研制状况及选择依据。     

T7 lytic phage-displayed peptide libraries exhibit less sequence bias than M13 filamentous phage-displayed peptide libraries

We investigated whether the T7 system of phage display could produce peptide libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and filamentous phage morphogenesis. Using a bioinformatics-assisted computational approach, collections of random peptide sequences obtained from a T7 12-mer library (X(12)) and a T7 7-mer disulfide-constrained library (CX(7)C) were analyzed and compared with peptide populations obtained from New England BioLabs' M13 Ph.D.-12 and Ph.D.-C7C libraries. Based on this analysis, peptide libraries constructed with the T7 system have fewer amino acid biases, increased peptide diversity, and more normal distributions of peptide net charge and hydropathy than the M13 libraries. The greater diversity of T7-displayed libraries provides a potential resource of novel binding peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7-displayed peptide libraries were screened for streptavidin- and neutravidin-binding phage. Novel binding motifs were identified for each protein.     

Citrulline-modified phage display: a novel high-throughput discovery approach for the identification of citrulline-containing ligands

Phage display is a high-throughput technology used to identify ligands for a given target. A drawback of the approach is the absence of PTMs in phage-displayed peptides. The applicability of phage display could be broadened considerably by the implementation of PTMs in this system. The aim of this study was to investigate the possible application of citrullination, a PTM of an arginine into a citrulline amino acid, in filamentous (M13) and lytic (T7) phage display. After in vitro citrullination of T7 and M13 phages, citrullination was confirmed and the infectivity of both citrullinated and non-citrullinated phage was compared by titer determination. We demonstrated the successful in vitro citrullination of T7 and M13 phage-displayed peptides. This in vitro modification did not affect the viability or infectivity of the T7 virions, a necessary prerequisite for the implementation of this approach in T7 phage display. For M13 phage, however, the infecting phage titer decreased five-fold upon citrullination, limiting the use of this modification in M13 phage display. In conclusion, in vitro citrullination can be applied in T7 phage display giving rise to a high-throughput and sensitive approach to identify citrulline-containing ligands by the use of the strengths of phage display technology.     

光学蛋白芯片技术在噬菌体M13KO7检测中的应用

将亲和素共价固定在表面改性后的硅片上,通过亲和素与生物素相互作用将生物素标记的噬菌体抗体GP3固定在亲和素膜层表面,当含有M13KO7噬菌体的样品经过抗体表面时,通过噬菌体与抗体之间的相互作用噬菌体就会被抗体捕获,生物学信号可以通过芯片上的膜层厚度变化表现出来,这种膜层厚度变化可以被椭偏生物传感器技术识别。结果表明,GP3抗体在芯片表面形成了饱和的抗体膜层,厚度为6.9nm;M13KO7噬菌体与芯片上固定的抗体会发生特异性相互作用,噬菌体被抗体捕获后形成的复合物膜层厚度为17.5nm,并且随着噬菌体浓度升高膜层厚度增加,检测含有M13KO7噬菌体的样品灵敏度为109pfu/mL。与其它研究病毒与抗体相互作用方法相比光学蛋白芯片技术具有简便快捷、无需标记待检样品和结果直观等优点,为研究病毒与其抗体相互作用以及疾病早期临床诊断提供了一个新的方法。     

Novel helper phage design: intergenic region affects the assembly of bacteriophages and the size of antibody libraries

Abstract Phagemid vectors for display of proteins/peptides on the surface of filamentous phage utilize a plasmid genome carrying the phage origin of replication, along with the gene fused to a fragment of gene III. Generation of phage particles displaying the fusion protein also requires superinfection of the host bacterium with a helper virus. We describe here the construction of a new gene III mutant of M13 KO7 bacteriophage and compare its ability to act as helper phage with two mutants derived from Fd tet (fKN 16 and fCA 55). Furthermore, we investigate their capability to act as helper phages in SAP selection, where non-infectious helper phage, expressing antibody fragments but not protein 3, can still infect by first reacting with a soluble antigen-protein 3 fusion protein. Gene III mutants were found to be non-infectious, and high titers of infective particles were obtained only when the helper phage was grown in cells harbouring a gene III-containing plasmid. An amplification of the phage titer of 10⁶× was achieved in M13-derived phages, when used for the selection of specific antibody fragments.     

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA^- strain were G:C → T:A transversions, occurring within the sequence which in recA⁺ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.     

利用重组噬菌体诱导表达的大肠杆菌表达系统

将编码噬菌体T7RNA聚合酶的基因克隆至噬菌体M13mpl8RFDNA中,置于lac启动子的控制之下,得到了可表达T7 RNA聚合酶的重组噬菌体M13HEP。利用该噬菌体感染含T7启动子表达质粒的宿主菌以提供T7RNA聚合酶,可以诱导T7启动子控制下的外源基因的表达。该噬茵体诱导表达系统已成功地表达了多种外源基因,特别是一些表达产物对宿主菌有毒性的基因。同时,通过细菌接合将F',因子从大脑杆菌XL1-blue转至大肠杆菌HMS174,构建了新的大脑杆菌菌株HMSl74F,,使得T7表达质粒构建、表达及单链制备可以在同一菌株中完成,得到了一个完整的T7表达系统。     

High-density functional display of proteins on bacteriophage lambda

We designed a bacteriophage lambda system to display peptides and proteins fused at the C terminus of the head protein gpD of phage lambda. DNA encoding the foreign peptide/protein was first inserted at the 3' end of a DNA segment encoding gpD under the control of the lac promoter in a plasmid vector (donor plasmid), which also carried lox P(wt) and lox P(511) recombination sequences. Cre-expressing cells were transformed with this plasmid and subsequently infected with a recipient lambda phage that carried a stuffer DNA segment flanked by lox P(wt) and lox P(511) sites. Recombination occurred in vivo at the lox sites and Amp(r) cointegrates were formed. The cointegrates produced recombinant phage that displayed foreign protein fused at the C terminus of gpD. The system was optimised by cloning DNA encoding different length fragments of HIV-1 p24 (amino acid residues 1-72, 1-156 and 1-231) and the display was compared with that obtained with M13 phage. The display on lambda phage was at least 100-fold higher than on M13 phage for all the fragments with no degradation of displayed products. The high-density display on lambda phage was superior to that on M13 phage and resulted in selective enrichment of epitope-bearing clones from gene-fragment libraries. Single-chain antibodies were displayed in functional form on phage lambda, strongly suggesting that correct disulphide bond formation takes place during display.This lambda phage display system, which avoids direct cloning into lambda DNA and in vitro packaging, achieved cloning efficiencies comparable to those obtained with any plasmid system. The high-density display of foreign proteins on bacteriophage lambda should be extremely useful in studying low-affinity protein-protein interactions more efficiently compared to the M13 phage-based system.     

T7家族噬菌体感染宿主菌的起始——吸附和穿入

噬菌体和细菌互相作用的研究,是分子生物学的重要内容,在细菌感染性疾病的治疗等方面有重要的应用价值。噬菌体的感染从噬菌体吸附于宿主菌表面并将核酸注入开始。介绍了T7家族代表株T7噬菌体在吸附和穿入阶段需要的结构和具体过程,并简要综述了T7家族3个亚群的特征。     

抗乙型肝炎病毒表面抗原T7噬菌体抗体库的构建

构建T7噬菌体单链抗体(scFv)库筛选抗乙型肝炎病毒表面抗原抗体.从抗-HBs阳性患者外周血淋巴细胞中提取总RNA,反转录合成cDNA第1条链,PCR分别扩增抗体重链可变区基因(VH)和轻链可变区基因(VL),经重叠延伸拼接(SOE)PCR组成scFv基因,并将其与T7噬菌体载体的2个臂相连接.体外包装后,在宿主菌BLT5403中,扩增重组噬菌体抗体库.以乙型肝炎病毒表面抗原进行4轮“吸附-洗脱-扩增”的筛选,酶免疫实验检测抗体活性.所建抗体库库容为1.53×107,扩增后初级库滴度为2.42×1010pfu/mL.以乙型肝炎病毒表面抗原筛选后抗体出现特异性富集,经酶免疫实验鉴定,得到2株与HBsAg抗原特异结合的噬菌体抗体,成功构建了抗HBsAg蛋白T7噬菌体抗体库.     

High efficiency transduction of single strand plasmid DNA into enteric bacteria

Summary This report demonstrates high efficiency transduction of enteric bacteria using single strand plasmids packaged in M13 phage capsids. Transformation by plasmid DNA is usually a very inefficient process in many enteric bacteria other than Escherichia coli K12. Plasmids carrying an M13 origin of replication can be replicated and packaged when cells carrying such plasmids are infected with M13 or a derivative helper phage. By introducing an F plasmid into E. coli, Serratia marcescens, Citrobacter freundii, and Enterobacter aerogenes, these species can now be infected at high efficiency with M13 phage and with packaged single strand plasmids, yielding an efficient method to introduce cloned DNA fragments into these bacteria. The titer of colony forming units in a lysate was essentially equivalent in all the bacteria, demonstrating an equal efficiency of transduction of these other enteric bacteria compared to E. coli.     

Temperature- and solvent-tolerant mutants of filamentous bacteriophage helper M13 KO7

Mutants of helper phage M13 KO7 were isolated whose survival was up to 300-fold greater than the wild type after 30 min at 95°C, and had increased solvent tolerance (up to 200-fold in 75% v/v N,N-dimethylformamide). These mutants retained their ability to package pIII-fusion display phagemids with normal efficiency and therefore will be useful in the isolation of stable protein variants by phage display.     

T7噬菌体转运真核表达载体入胞表达平台的构建

[目的]构建携带锚定序列的真核表达载体,研究T7噬菌体识别、包裹和转运真核表达载体进入细胞实现蛋白表达的可行性,为DNA疫苗研发建立新的技术平台.[方法]本研究通过重叠延伸PCR方法获得候选锚定序列并插入真核表达载体;建立荧光定量PCR方法比较T7噬菌体识别、包裹真核表达载体的效率;激光共聚焦显微镜观察T7噬菌体转运真...     

噬菌体抗体库的构建及抗乳腺癌细胞单链抗体的筛选

构建抗人乳腺癌细胞MCF 7的噬菌体单链抗体库 ,从中筛选MCF 7细胞特异性单链抗体。用MCF-7细胞免疫BALB C小鼠 ,取脾脏 ,提取总RNA ,用RT-PCR技术扩增小鼠抗体重链 (V_H)和轻链 (V_L)可变区基因 ,经重叠PCR(SOE-PCR) ,在体外将V_H和V_{L连接成单链抗体 (scFv)基因 ,并克隆到噬菌粒载体pCANTAB5E中 ,电转化至大肠杆菌TG1,经辅助噬菌体超感染 ,构建噬菌体单链抗体库。从该抗体库中筛选特异性识别MCF-7细胞的噬菌体单链抗体 ,将表面展示单链抗体的单克隆噬菌体转化大肠杆菌TOP10进行可溶性表达。成功地构建了库容为12×10⁶ 的抗MCF-7乳腺癌细胞的单链抗体库 ,初步筛选到了与MCF 7细胞特异性结合的scFv,Westernblot检测表明 ,在大肠杆菌TOP10中实现了单链抗体可溶性表达}     

Screening recombinant phage M13 plaques with RNA probes; a one-step procedure which identifies clones containing either of the complementary DNA strands

We describe a method for detecting specific DNA sequences cloned in M13 phage vectors, based on the procedure of Woo (in Wu, R., Methods in Enzymology, Vol. 68, Academic Press, New York, 1979, pp. 389-395). M13 plaques are adsorbed to a nitrocellulose filter that has been pre-saturated with bacteria. The filter is incubated on an agar plate to amplify the phage; the DNA is alkali-denatured and then hybridized with a radioactive RNA probe. Unlike standard procedures, this method detects and distinguishes M13 plaques containing phage particles which harbor either the coding or non-coding (RNA-like) DNA strand, when single-stranded RNA is used as probe. We have optimized this procedure with M13 clones containing mouse histidine tRNA gene sequences and have used it to determine the sequence of both strands of a mouse glycine tRNA gene.     

Identification of C10 biotinylated camptothecin (CPT-10-B) binding peptides using T7 phage display screen on a QCM device

A peptide sequence that can bind to camptothecin (CPT), a natural cytotoxic compound, was screened for using a T7 phage display system combined with a cuvette type quartz crystal microbalance (QCM) device. In this screen, after only 10 min of monitoring of the interaction between injected T7 phage pool with immobilized C10 biotinylated CPT (CPT-10-B) on a gold electrode surface, six different kinds of phage (A–F) were identified as judged by the size of PCR product on agarose gel electrophoresis. Injection of each single phage (A–E) pool individually caused a frequency decrease, suggesting interaction with the immobilized CPT-10-B. In addition, the peptide sequence displayed on phages A–C is consistent with chemical and biological studies of the interaction of CPTs with topoisomerase I (TopI), human E prostanoid receptor third cytoplasmic polypeptide, and a series of esterases. The efficacy of T7 phage display screening for small molecules on QCM devices, target discovery from primary peptide sequence, and application of this strategy to various drug-like small molecules are discussed.     

Bovine oocyte development following different oocyte maturation and sperm capacitation procedures

Various procedures have been reported for successful in vitro maturation and in vitro fertilization (IVM/IVF) of bovine follicular oocytes. Direct comparisons of these different recommended procedures have been rare. In this research, involving a total of 5,128 oocytes, a series of experiments were conducted to compare oocyte maturation, fertilization, and development in vitro with 2 maturation systems (with or without added hormones) and 3 types of sperm treatment procedures. Oocytes were collected from ovarian antral follicles (2–7 mm in diameter) within 3 hr after slaughter of cows or heifers. Those with intact or at least 4 layers of cumulus cells were selected for IVM/IVF. Oocytes were incubated for 22 hr in either Medium 199 with 7.5% fetal calf serum (M199 + FCS) alone or M199 + FCS with added hormones (M199 + FCS + H; oFSH 0.5 μg/ml, oLH 5.0 μg/ml, and E₂ 1.0 μg/ml) at 39°C in 5% CO₂ and 95% air. For IVF, frozen-thawed sperm were treated with either 0.1 μM calcium ionophore A23187 (A23187) for 1 min, or 10 or 100 μg/ml heparin (H10 or H100) for 15 min. Our results demonstrated the following: (1) both M199 + FCS and M199 + FCS + H supported maturation development to the metaphase II stage (90–95%, P > 0.05); (2) when oocytes were matured in M199 + FCS without added hormones, A23187 sperm treatment was superior to H10 or H100 treatment for fertilization and blastocyst development of the inseminated oocytes (P < 0.05); (3) when oocytes were matured in M199 + FCS + H, A23187 treated sperm again produced a higher fertilization rate than the H10 group (P < 0.05), but the development to the blastocyst stage was similar among all 3 sperm treatment groups (P > 0.05); (4) direct comparison of the 2 maturation systems with A23187 treated sperm resulted in no difference in all criteria measured; however, (5) when compared retrospectively, beneficial effects of added hormones are evident for blastocyst development (but not for fertilization) when sperm were treated with heparin procedures. © 1993 Wiley-Liss, Inc.