乳酸杆菌的基因工程

乳酸杆菌是一类广泛用于食品工业的革兰阳性杆菌,利用基因工程技术可对其性状进行改造。近20年中,人们构建了多种载体,并在乳酸酐菌中表达了多种蛋白,作为人体的正常菌群,乳酸杆菌已被用于肠道,阴道的微生态治疗,表达抗原的乳酸杆菌工程菌可经黏膜免疫诱导保护性免疫或免疫耐受,因此,深入研究和利用乳酸杆菌基因工程技术有重要意义。     

表达HIV单链抗体的重组乳酸杆菌的构建及表型分析

近年来,基于抗体药物的被动免疫策略在人类免疫缺陷病毒(HIV)预防和治疗中获得一定的成效,但仍需要定期补充抗体,才能维持体内长期有效的药物浓度,如何延长抗体药物的作用时间是亟待解决的问题。根据乳酸杆菌的食品安全性和黏膜定植稳定性,把HIV单链抗体连接到具有宿主同源启动子和信号肽的乳酸杆菌表达质粒pldhl0373中,电转化乳酸杆菌WCFS1感受态细胞,通过红霉素筛选出阳性单菌落,发酵培养后菌液经Western blot检测证明了单链抗体得到表达,并具有抗病毒能力。与野生型WCFS1菌株相比,重组菌体外生长能力未发生改变,发酵24h后菌液的CFU均可达到10~9/ml;单链抗体基因的插入不会明显改变重组菌黏膜细胞定植能力,每平方厘米单层上皮细胞可稳定定植10~5CFU的菌落。以宿主黏膜共生菌为媒介,在黏膜细胞表面实现抗病毒蛋白的长期稳定表达为预防HIV黏膜传播提供了一种新的思路。     

乳酸乳球菌作为黏膜免疫活载体疫苗传递抗原的研究进展

乳酸菌是人和动物肠道内的常见细菌,被公认为安全级(generally recognized as safe,GRAS)微生物。近年来,对于乳酸菌作为宿主菌表达外源蛋白或抗原的研究取得了一定进展。乳酸乳球菌(Lactococcus lactis)是乳酸菌的代表菌种,以其生长迅速、易于操作等优点成为表达外源抗原,作为黏膜免疫活载体疫苗的理想菌株。随着对乳酸乳球菌基因工程的研究逐渐深入,已发展了一系列组成型和诱导型乳酸乳球菌表达系统以及蛋白定位系统。破伤风毒素片段C、布氏杆菌L7/L12蛋白等多种病原微生物抗原已成功在乳酸乳球菌中表达,并已证明部分重组乳酸乳球菌作为黏膜免疫疫苗可以同时刺激局部黏膜免疫应答和系统免疫应答。目前,如何使活载体乳酸乳球菌以最佳方式向黏膜免疫系统提呈抗原继而诱导有效免疫反应是该领域的研究热点,也是巨大挑战。实现外源抗原在乳酸乳球菌中的准确定位及与细胞因子的共表达是未来研究的重要方向之一。乳酸乳球菌作为黏膜免疫活载体疫苗传递外源抗原具有广阔的应用前景。     

乳酸杆菌S-层蛋白的特性及功能性研究进展

文章综述了S-层蛋白的性质和功能,重点介绍了S-层蛋白对乳酸杆菌表面性质和黏附性的影响以及调节肠道功能的作用,包括减少病原菌引起的细胞凋亡、调节免疫细胞的活性、与SIGNR3相互作用参与肠道免疫反应、通过TLRS-My D88-NF-κB途径发挥生物学功能以及调控肠道黏膜相关蛋白表达。由此证明了S-层蛋白对于乳酸杆菌发挥免疫调节功能具有重要的作用。     

戈登链球菌的生物学特性及在黏膜疫苗的应用

戈登氏链球菌是一种非致病性革兰氏阳性黏膜共生菌, 参与组成人类口腔正常菌群。它具有特殊的生物学特性, 非常适合作为黏膜疫苗的载体。了解戈登氏链球菌的生物学特性, 常用表达体系及该菌在黏膜疫苗的应用情况, 将为其黏膜疫苗的进一步研制提供重要参考。     

戈登链球菌的生物学特性及在黏膜疫苗的应用

戈登氏链球菌是一种非致病性革兰氏阳性黏膜共生菌,参与组成人类口腔正常菌群.它具有特殊的生物学特性,非常适合作为黏膜疫苗的载体.了解戈登氏链球菌的生物学特性,常用表达体系及该菌在黏膜疫苗的应用情况,将为其黏膜疫苗的进一步研制提供重要参考.     

猪细小病毒VP2与大肠杆菌不耐热肠毒素B亚单位在干酪乳杆菌表面共表达

将分别编码猪细小病毒(PPV)主要免疫保护性抗原VP2蛋白与大肠杆菌不耐热肠毒素B亚单位(LTB)基因插入乳酸杆菌细胞表面表达载体pPG中, 成功构建了重组表达载体pPG-VP2-LTB, 将其电转化干酪乳杆菌Lactobacillus casei 393, 获得了表达猪细小病毒VP2-LTB融合蛋白的重组乳酸菌表达系统, 经2%乳糖诱导, SDS-PAGE和Western-blot检测表明, 有大小约78 kD的蛋白得到了表达, 具有与天然病毒蛋白一样的抗原特异性, 全细胞ELISA结果表明, LTB同     

人干扰素α-2b基因在德氏乳杆菌的表达与鉴定

目的:试图用乳酸杆菌在局部分泌干扰素,以达到预防和治疗病毒感染.方法:以pBLUE/IFN α-2b为模板,采用PCR技术将IFNα-2b基因扩增然后插入乳酸菌表达载体pSClllAE,将重组质粒电转德氏乳杆菌DM8909,获得重组菌株.在含乳糖的MRS培养基培养并诱导表达,表达产物分泌到菌液上清.结果:分别用PCR和ELISA鉴定目的基因和表达产物,利用WISH-VSV系统采用细胞病变抑制法,测定表达产物IFN α-2b的抗病毒活性,平均效价为1.41×105IU/ml.结论:本研究为以乳酸杆菌为载体在局部产生治疗性药物奠定了基础.     

新型大肠杆菌高效表达载体pHsh的构建与应用

在现代生物学和生物技术研究中,通过基因的重组表达获得目标蛋白是一种应用最广泛的方法。因其培养简单、操作方便、遗传背景清楚、克隆表达系统成熟完善,大肠杆菌表达系统通常是人们表达重组蛋白的首选,而表达载体在重组蛋白的生产中起决定作用。pHsh及其衍生质粒是近年发展起来的新型大肠杆菌表达载体,其调控外源基因表达的原理不同于所有其他表达系统,并且具有表达水平高、成本低廉等特点。介绍大肠杆菌表达系统的组成和常用表达载体,并对由pHsh系列载体组成的Hsh表达体系的构建策略、表达调控机制及其使用方法进行综述。Hsh表达体系的建立和发展有望从一个不同的角度帮助解决基因的重组表达中常见的表达水平低、诱导剂成本高、包涵体形成等问题。     

纳米抗体异源表达的研究进展

羊驼体内存在天然缺失轻链的重链抗体(HcAb),其单域抗原结合片段也称为VHH或纳米抗体(nanobody,Nb),是目前已知的最小抗原结合片段。与传统抗体相比,纳米抗体具有分子量小(12~15kDa)、稳定性好和免疫原性低等特点,这些特点使得VHH在基础研究、诊断及治疗上具有极大的应用价值,目前已有多种纳米抗体进入了临床研究阶段。综述了近年来VHH在革兰氏阴性菌(大肠杆菌)、革兰氏阳性菌(芽孢杆菌和乳酸杆菌)、酵母、丝状真菌、昆虫细胞、哺乳动物细胞和植物细胞中异源表达的研究现状,包括表达系统、宿主、载体特点、载体构建方式及产量等;从分子水平、表达水平和理性设计三个层面探讨了纳米抗体产量提高的策略,以期为纳米抗体研究者提供借鉴和思路。     

Expression of the protective antigen of Bacillus anthracis by Lactobacillus casei: towards the development of an oral vaccine against anthrax

Bacillus anthracis is the causative organism of the disease anthrax. The ability of the organism to form resistant spores and infect via the aerosol route has led to it being considered as a potential biological warfare agent. The current available human vaccines are far from ideal, they are expensive to produce, require repeated doses and may invoke transient side-effects in some individuals. There is also evidence to suggest that they may not give full protection against all strains of B. anthracis. A new generation of anthrax vaccine is therefore needed. The use of Lactobacillus as a vector for expression of heterologous proteins from pathogens supplies us with a safe system, which can be given orally. Lactobacilli are commensals of the gut, generally regarded as safe and have intrinsic adjuvanticity. Oral vaccines may stimulate the mucosol immune system to produce local IgA responses in addition to systemic responses. These vectors are delivered at the mucosal surface, the site where the infection actually occurs and where the first line of defence lies. The gene encoding the protective antigen (PA) of B. anthracis, an immunogenic non-toxic component of the two toxins produced, is being cloned into different homologous vectors and subsequently transformed to various Lactobacillus strains. High intracellular expression levels for the PA in Lact. casei were achieved. Mucosal antigen presentation and humoral and cellular immune responses following immunization with transformants expressing PA in various ways (intracellular, surface-anchored and extracellular) are being studied.     

Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge

Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade 1 PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed.     

Prospects for the genetic manipulation of lactobacilli

Abstract Efforts are underway in a number of laboratories around the world to develop methods for the improvement of Lactobacillus strains by recombinant DNA techniques. Research has centered on: (i) characterization and construction of chimeric shuttle vectors based on endogenous Lactobacillus plasmids which are capable of replicating in lactobacilli; (ii) molecular cloning of genes and operons from lactobacilli encoding important pathways such as the lactose : phospho enol pyruvate phosphotransferase system, phosphogalactoside-β- d -galactohydrolase, and β-galactosidase; and (iii) methods for introduction of genes in vivo and in vitro through conjugation, transfection and transformation. The lack of natural gene exchange systems has prompted research efforts to devise a protoplast transformation system. Initial early successes in gene cloning, vector development, transfection, conjugation, protoplast fusion and, recently, transformation, have laid the ground-work for rapid development of gene exchange systems for the genus.     

Transgene Regulation Using the Tetracycline-Inducible TetR-KRAB System after AAV-Mediated Gene Transfer in Rodents and Nonhuman Primates

Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Krüppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive transactivators with the potential to not trigger the host immune system are still needed.     

乳酸菌食品级nisin控制的基因表达系统NICE

乳酸菌安全应用于人们的生产和生活已有上千年的历史,是一种食品级的微生物。在过去二十年里,其生理及遗传学特性已被彻底研究。由于其遗传可行且操作简单,乳酸菌除了其传统应用外已被广泛用于表达异源基因,在食品、农业及医药工程领域具有重要的应用前景。人们已开发了一系列乳酸菌食品级基因表达系统。本文主要介绍了乳酸菌,重点是其模式菌Lactococcus lactis最常见的食品级诱导表达系统--nisin控制的基因表达系统NIC E及其食品级诱导物nisin、食品级的宿主及表达载体系统,以及NICE系统在表达异源基因方面的应用。     

Development of an inducible gene expression system for Lactobacillus sakei

AIM: To develop an inducible gene expression system for Lactobacillus sakei, based on the regulatory system of sakacin A production. METHODS AND RESULTS: A Lactobacillus/Escherichia coli shuttle vector; pKRV3, was constructed including the signal transducing system genes of the bacteriocin sakacin A. The gusA gene fused to PsapA promoter, cloned in this vector allowed for inducible beta-glucuronidase expression in L. sakei and L. plantarum following the addition of the sakacin A inducing peptide. PsapA appeared to be a strong and tightly controlled promoter when compared with known promoters. CONCLUSION: The pKRV3 system can be used as an inducible gene expression system in lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel, inducible gene expression system has been developed for lactic acid bacteria relevant in food fermentations.     

A vaccine strategy utilizing a combination of three different chimeric vectors which share specific vaccine antigens

A large number of recombinant of viral and bacterial systems have been engineered as vectors to express foreign genes for vaccination and/or gene therapy. A common problem is the immune response to the vector itself. The presence of anti-vector immune responses may preclude sufficient 'priming' or immunogenicity if pre-existing immune responses are present, or they may impair optimal 'boosting' upon repeated immunization or delivery with the same vector. To circumvent this problem we developed a strategy using different chimeric vectors which share only the expression of common specific antigens desired for immunization. This approach not only has the advantage of avoiding increased anti-vector responses, but allows the use of combinations of vectors which could subsequently present the same or related antigen differently to the immune system as well as at alternative sites to induce the optimal type of immunity against the pathogen of interest.     

A vaccine strategy utilizing a combination of three different chimeric vectors which share specific vaccine antigens

A large number of recombinant of viral and bacterial systems have been engineered as vectors to express foreign genes for vaccination and/or gene therapy. A common problem is the immune response to the vector itself. The presence of anti-vector immune responses may preclude sufficient 'priming' or immunogenicity if pre-existing immune responses are present, or they may impair optimal 'boosting' upon repeated immunization or delivery with the same vector. To circumvent this problem we developed a strategy using different chimeric vectors which share only the expression of common specific antigens desired for immunization. This approach not only has the advantage of avoiding increased anti-vector responses, but allows the use of combinations of vectors which could subsequently present the same or related antigen differently to the immune system as well as at alternative sites to induce the optimal type of immunity against the pathogen of interest.     

Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology

Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.