植物病毒载体系统研究进展

植物病毒基因组小,易于进行遗传操作而且感染过程简单,因而利用植物病毒载体表达外源基因在生物技术领域具有潜在的应用优势。本文主要介绍了抗原展示系统和多肽表达系统,并分别列举一些植物病毒作为表达载体的研究进展情况以及应用前景。     

噬菌体展示技术的发展及应用

噬菌体展示技术是一种用于筛选和改造功能性多肽的生物技术 ,编码多肽的ＤＮＡ片段与噬菌体表面蛋白的编码基因融合后 ,以融合蛋白的形式在噬菌体的表面表达出多肽序列。这是一种表型与基因型的统一。噬菌体展示技术最初是以Ｍ 13噬菌体为载体的 ,其宿主菌为大肠杆菌。以大肠杆菌为宿主的展示系统还有其他 ,如λ噬菌体和Ｔ4噬菌体等展示系统。还有利用真核细胞的病毒以及酵母菌作为展示系统的。这些展示系统各有各的优势 ,但最常用的仍是Ｍ 13噬菌体表达系统。最初的噬菌体展示系统是将外源肽或蛋白质与噬菌体外壳蛋白ＰⅢ或ＰⅧ的Ｎ末端融…     

噬菌体展示技术及其在寄生虫研究中的应用

噬菌体展示技术是将外源蛋白或多肽的编码基因或DNA序列插入到噬菌体外壳蛋白结构基因的适当位置,使外源基因随外壳蛋白的表达而表达,并随噬菌体的重新组装而展示到噬菌体表面的生物技术.在研究蛋白质识别或蛋白质与核酸相互作用的生物学过程、蛋白质定向改造、研制新型多肽药物、疫苗和抗体等多领域具有重要作用.就噬菌体展示技术基本原理及特点,以及噬菌体展示技术在寄生虫研究中的应用做一简要综述.     

细菌表面展示技术的应用研究进展

细菌表面展示系统是微生物表面展示系统的一个重要分支。由于呈现载体灵活多样,可根据不同的需要呈现蛋白或多肽等特点,细菌表面展示技术近年来得到了迅猛发展,在重组细菌疫苗、抗原表位分析、全细胞催化剂、全细胞吸附剂、多肽库筛选等多个领域得到广泛应用。本文就细菌表面展示技术的应用研究作一综述。     

噬菌体展示文库筛选技术的研究进展

噬菌体展示技术是将编码外源蛋白或多肽的基因片段定向插入到噬菌体的外壳蛋白基因区,使外源蛋白或多肽通过与噬菌体外壳蛋白融合而表达并展示于噬菌体表面,进而筛选表达特异蛋白或多肽的噬菌体,已发展成为生物学后基因组时代一个强有力的实验技术.噬菌体展示文库的筛选是其关键环节.为了提高筛选效率,许多研究者对传统的筛选技术进行了改进,如选择性感染噬菌体、迟延感染性噬菌体、以DNA为基础的筛选方法、亲合力捕获和反复筛选和封闭筛选法等,用于筛选的靶标也越来越具有多样性,使得这一技术有了更加广阔的发展前景.     

T4噬菌体表面展示技术的研究进展

噬菌体表面展示技术(phage display)是由Smith于1985年首先建立起来的一种新的生物技术[1],它能将表达的外源多肽或蛋白以融合蛋白的形式展示在噬菌体的表面,保持相对独立的空间构象和原有的生物活性[2].常用的噬菌体表面展示系统主要有丝状噬菌体、λ噬菌体及T4噬菌体展示系统等.虽然它们都具有噬菌体展示系统的优点,但对于丝状噬菌体来说,它不能展示那些难以分泌的肽和蛋白质,而且它的N端可融合外源多肽的容量有限,较大蛋白的融合会造成空间障碍,影响噬菌体的装配,使其失去感染力.而对于λ噬菌体,大分子蛋白的融合会抑制噬菌体的组装,使其生长受到影响,因此这两种噬菌体更适用于构建短肽库和cDNA表达文库[3],而不适于构建重组疫苗和表达分子量大具有完整结构域的蛋白质[4,5].     

杆状病毒表面展示系统的研究进展

杆状病毒表面展示系统是近几年发展起来的一种新的真核展示系统 ,通过在病毒衣壳蛋白gp6 4插入外源肽、二者融合表达或与特异性的锚定部位结合 ,在病毒表面进行融合表达而筛选出目的活性肽或蛋白。可用来展示需糖基化、二硫键异构化等翻译后修饰才表现功能活性的复杂真核蛋白及构建多肽文库、抗体库等。本文简述了该技术的原理、研究进展、应用及发展前景等。可以预见 ,杆状病毒表面展示技术的发展必将对生命科学及相关领域的发展产生深远的影响     

MshB基因突变对维罗纳气单胞菌菌毛动力的影响

目的:通过探索野生型(菌毛表达)和MSHB基因突变型(菌毛不表达)维罗纳气单胞菌动力方面的差别,证明菌毛在细菌运动方面的重要功能。方法:首先培养细菌长至对数生长期,让后进行半固体培养基穿刺与平板挖沟培养实验,最后对阳性率进行T分析。结果:两者之间具有显著差别(P0.05)。结论:菌毛对维罗纳菌的动力具有重要作用。     

肺炎克雷伯菌菌毛的研究进展

肺炎克雷伯菌为条件致病菌,可引起肺炎、败血症等多种化脓性炎症,近年来肺炎克雷伯菌也成为医院内感染的主要致病菌之一。研究表明,菌毛作为细菌重要的毒力因子之一,在细菌黏附过程中起重要作用,细菌可借助于菌毛尖端黏附素黏附到宿主的组织器官,这是引起机体致病的首要条件。肺炎克雷伯菌菌毛包括Ⅰ型菌毛和Ⅲ型菌毛,绝大多数的肺炎克雷伯菌均可表达Ⅲ型菌毛,在医院感染的致病过程中起到关键作用。     

金黄色葡萄球菌蛋白A(Staphylococcal Protein A,SPA)分泌型融合表达系统

金黄色葡萄球菌蛋白Ａ（ＳｔａｐｈｙｌｏｃｏｃｃａｌＰｒｏｔｅｉｎＡ,ＳＰＡ）分泌型融合表达系统李节,邱平（南京大学生物化学系医药生物技术国家重点实验室,南京２１０００８）引言十几年前,我们制备蛋白质的方法还主要依靠从天然组织或细胞中提取,而现在人们可以通过基因工程方法,在细菌中生产大量有价值的活性蛋白和多肽。外源基因在细菌中表达的一个主要问题是基因产物如何折叠成天然构象。     

细菌表面呈现技术研究进展

自从首次描述外源蛋白在大肠杆菌表面呈现成功以来,细菌表面呈现技术得到了迅猛的发展,无论是革兰氏阴性菌还是革兰氏阳性菌都可用于异源蛋白的表面呈现,该技术被应用于微生物学、免疫学、分子生物学、疫苗学以及生物工程的多个领域的基础和应用研究。     

Display of proteins on bacteria

Display of heterologous proteins on the surface of microorganisms, enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and vaccinology. Gram-negative, Gram-positive bacteria, viruses and phages are all being investigated in such applications. This review will focus on the bacterial display systems and applications. Live bacterial vaccine delivery vehicles are being developed through the surface display of foreign antigens on the bacterial surfaces. In this field, 'second generation' vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals, through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. Certain bacteria have also been employed for display of various poly-peptide libraries for use as devices in in vitro selection applications. Through various selection principles, individual clones with desired properties can be selected from such libraries. This article explains the basic principles of the different bacterial display systems, and discusses current uses and possible future trends of these emerging technologies.     

Autodisplay: efficient bacterial surface display of recombinant proteins

To display a protein or peptide with a distinct function at the surface of a living bacterial cell is a challenging exercise with constantly increasing impact in many areas of biochemistry and biotechnology. Among other systems in Gram-negative bacteria, the Autodisplay system provides striking advantages when used to express a recombinant protein at the surface of Escherichia coli or related bacteria. The Autodisplay system has been developed on the basis of and by exploiting the natural secretion mechanism of the AIDA-I autotransporter protein. It offers the expression of more than 10⁵ recombinant molecules per single cell, permits the multimerization of subunits expressed from monomeric genes at the cell surface, and allows, after transport of an apoprotein to the cell surface, the incorporation of an inorganic prosthetic group without disturbing cell integrity or cell viability. Moreover, whole cells displaying recombinant proteins by Autodisplay can be subjected to high-throughput screening (HTS) methods such as ELISA or FACS, thus enabling the screening of surface display libraries and providing access to directed evolution of the recombinant protein displayed at the cell surface. In this review, the application of the Autodisplay system for the surface display of enzymes, enzyme inhibitors, epitopes, antigens, protein and peptide libraries is summarised and the perspectives of the system are discussed.     

Salmonella enteritidis fimbriae displaying a heterologous epitope reveal a uniquely flexible structure and assembly mechanism

Two distinct Salmonella fimbrins, AgfA and SefA, comprising thin aggregative fimbriae SEF17 and SEF14, respectively, were each genetically engineered to carry PT3, an alpha-helical 16-amino acid Leishmania T-cell epitope derived from the metalloprotease gp63. To identify regions within AgfA and SefA fimbrins amenable to replacement with this epitope, PCR-generated chimeric fimbrin genes were constructed and used to replace the native chromosomal agfA and sefA genes in Salmonella enteritidis. Immunoblot analysis using anti-SEF17 and anti-PT3 sera demonstrated that all ten AgfA chimeric fimbrin proteins were expressed by S. enteritidis under normal growth conditions. Immunoelectron microscopy confirmed that eight of the AgfA::PT3 proteins were effectively assembled into cell surface-exposed fimbriae. The PT3 replacements in AgfA altered Congo red (CR) binding, cell-cell adhesion and cell surface properties of S. enteritidis to varying degrees. However, these chimeric fimbriae were still highly stable, being resistant to proteinase K digestion and requiring harsh formic acid treatment for depolymerization. In marked contrast to AgfA, none of the chimeric SefA proteins were expressed or assembled into fimbriae. Since each PT3 replacement constituted over 10% of the AgfA amino acid sequence and all ten replacements collectively represented greater than 75% of the entire AgfA primary sequence, the ability of AgfA to accept large sequence substitutions and still assemble into fibers is unique among fimbriae and other structural proteins. This structural flexibility may be related to the novel fivefold repeating sequence of AgfA and its recently proposed structure Proper formation of chimeric fimbrial fibers suggests an unusual assembly mechanism for thin aggregative fimbriae which tolerates aberrant structures. This study opens a range of possibilities for Salmonella thin aggregative fimbriae as a carrier of heterologous epitopes and as an experimental model for studies of protein structure.     

A two-plasmid Escherichia coli system for expression of Dr adhesins

This paper presents a very efficient expression system for production of Dr adhesins. The system consists of two plasmids. One is the pACYCpBAD-DraC-C-His, which contains the draC gene under the control of the arabinose promoter (pBAD), encoding the DraC usher. The second is the pET30b-syg-DraBE, which contains the draB and draE genes under the control of the T7lac promoter, encoding the DraB chaperone and the DraE adhesin, respectively. Those plasmids have different origin of replication and can therefore coexist in one cell. Since different promoters are present, the protein expression can be controlled. The Dr adhesion expression system constructed opens up a lot of possibilities, and could be very useful in experiments focusing on understanding the biogenesis of Gram-negative bacteria adhesins. For this purpose we showed that the AfaE-III adhesin (98.1% identity between the DraE and the AfaE-III adhesins, with three divergent amino acids within the sequences) was able to pass through the DraC channel in the Escherichia coli BL21(DE3) strain. Immunoblotting analysis and immunofluorescence microscopy showed the presence of AfaE-III on the bacterial cell surface. In addition, the system described can be useful for displaying the immune-relevant sectors of foreign proteins on the bacterial cell. The heterologous epitope sequence of the HSV1 glycoprotein D was inserted into the draE gene in place of the N-terminal region of surface exposed domain 2. Chimeric proteins were exposed on the bacterial surface as evidenced by immunoblotting and immunofluorescence microscopy. The effective display of peptide segments on Dr fimbriae expressed at the bacterial cell surface, can be used for the development of a fimbrial vaccine.     

Type 1 fimbriae of Escherichia coli as carriers of heterologous antigenic sequences

A strategy has been designed for the construction of recombinant bacterial strains which eventually may become useful as live vaccines and which may also be relevant for the preparation of conventional vaccines. The approach used is the fusion of small antigenic peptide sequences into specific segments of a protein whose location on the bacterial surface ensures that the recombinant organism is able to present the inserted antigen to the host (animal or human) infected by the bacterium. The chosen surface protein is a naturally occurring polymer of Escherichia coli, viz., type 1 fimbriae. The results obtained show that fusion of such foreign sequences into selected points of the structural protein of the fimbriae results in the production of functionally normal type 1 fimbriae. Furthermore, hybrid fimbriae carrying such small epitope sequences can be recognized by antibodies directed against the foreign parent protein. This observation is an important prerequisite for the eventual design of useful vaccines. The analysis of the fimbrial protein and its potential as a carrier of foreign peptides from hepatitis B surface antigen, foot-and-mouth disease virus and poliovirus indicated that there may be several positions in the protein which may turn out to be relevant for this purpose and be important fusion sites.     

脊髓灰质炎病毒C3表位和C—myc＋肽表位在细菌表面的表达

为了进一步验证载体pCSB136和pCSX72作为表面呈现载体的可行性,化学合成脊髓灰质炎病毒C3表位和C-myc十肽有位的基因片段,并插入到上述载体的相应位点间,采用全菌PCR筛选到正向插入的重组子,全细胞ELISA和电镜观察显示,重组蛋白以杂和菌毛的形式得到了表达,并保持CS3和外源位的抗原性,表明脊髓灰质炎病毒C3表位和C-myc十肽捕位在细菌表面得到了表达,证实载体pCSB136和pCSX72可用于外源表位的呈现,为构建基因工程活苗疫苗奠定了基础。     

细菌表面展示技术研究新进展

细菌表面展示是将靶标蛋白质表达于细菌表面以更好地实现其功能的一种技术,它在重组细菌疫苗、生物燃料电池、全细胞催化剂和生物修复等多个领域均有广泛的应用.随着相关技术的发展,表面展示系统的各种性能被不断地改良,同时新的表面展示系统也陆续被开发和应用,使该技术得到持续的丰富和发展.本文重点关注近年研究得较多的细菌表面展示系统,主要对各类细菌表面展示系统的开发、改造和修饰,以及该技术在生物修复和生物传感器方面的应用作一综述.     

Anaplasma marginale major surface protein 1a directs cell surface display of tick BM95 immunogenic peptides on Escherichia coli

The surface display of heterologous proteins on live Escherichia coli using anchoring motifs from outer membranes proteins has impacted on many areas of biochemistry, molecular biology and biotechnology. The Anaplasma marginale major surface protein 1a (MSP1a) contains N-terminal surface-exposed repeated peptides (28-289 amino acids) that are involved in pathogen interaction with host cell receptors and is surface-displayed when the recombinant protein is expressed in E. coli. Therefore, it was predicted that MSP1a would surface display on E. coli peptides inserted in the N-terminal repeats region of the protein. The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that a recombinant protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region is displayed on the E. coli surface and is recognized by anti-BM86 and anti-MSP1a antibodies. This system provides a novel approach to the surface display of heterologous antigenic proteins on live E. coli and suggests the possibility to use the recombinant bacteria for immunization studies against cattle tick infestations.     

Expression of functional Porphyromonas gingivalis fimbrillin polypeptide domains on the surface of Streptococcus gordonii.

Genetically engineering bacteria to express surface proteins which can antagonize the colonization of other microorganisms is a promising strategy for altering bacterial environments. The fimbriae of Porphyromonas gingivalis play an important role in the pathogenesis of periodontal diseases. A structural subunit of the P. gingivalis fimbriae, fimbrillin, has been shown to be an important virulence factor, which likely promotes adherence of the bacterium to saliva-coated oral surfaces and induces host responses. Immunization of gnotobiotic rats with synthetic peptides based on the predicted amino acid sequence of fimbrillin has also been shown to elicit a specific immune response and protection against P. gingivalis-associated periodontal destruction. In this study we engineered the human oral commensal organism Streptococcus gordonii to surface express subdomains of the fimbrillin polypeptide fused to the anchor region of streptococcal M6 protein. The resulting recombinant S. gordonii strains expressing P. gingivalis fimbrillin bound saliva-coated hydroxyapatite in a concentration-dependent manner and inhibited binding of P. gingivalis to saliva-coated hydroxyapatite. Moreover, the recombinant S. gordonii strains were capable of eliciting a P. gingivalis fimbrillin-specific immune response in rabbits. These results show that functional and immunologically reactive P. gingivalis fimbrillin polypeptides can be expressed on the surface of S. gordonii. The recombinant fimbrillin-expressing S. gordonii strains may provide an effective vaccine or a vehicle for replacement therapy against P. gingivalis. These experiments demonstrated the feasibility of expressing biologically active agents (antigens or adhesin molecules) by genetically engineered streptococci. Such genetically engineered organisms can be utilized to modulate the microenvironment of the oral cavity.