乳酸乳球菌载体疫苗

乳酸乳球菌是一种在食品工业中广泛应用的安全级微生物,应用基因工程手段能使乳酸乳球菌表达多种病毒、细菌、寄生虫的外源蛋白。乳酸乳球菌可经粘膜途径免疫,能有效递呈抗原,诱导外源蛋白的特异性免疫应答,并能同时诱导粘膜免疫与全身免疫,因此可作为潜在的疫苗载体。本文对乳酸乳球菌载体疫苗的优势、应用以及疫苗设计时需要考虑的问题进行了概述。     

乳酸乳球菌食品级表达载体的研究进展

乳酸乳球菌（L.lactis）是乳球菌属中最重要和最典型的一个种,在食品工业中应用广泛,被公认为安全的（generally regards as safe,GRAS）食品级微生物。以乳酸乳球菌作为宿主菌,构建表达载体用来表达异源蛋白和酶,逐渐成为食品工业、生物制药和疫苗研究的热点。近年来,乳酸乳球菌的分子微生物学研究取得了重大进展,这为表达载体的构建奠定了基础,一些具有不同用途的乳酸乳球菌基因表达载体已经构建,用来表达抗原蛋白、细胞因子和生物酶等。其中,以来源于食品级微生物的DNA片段构建的食品级表达载体引起人们的关注。     

鼠疫抗原LcrV在乳酸乳球菌中的表达与鉴定

目的以乳酸乳球菌(Lactococcus lactis)为载体,将鼠疫抗原LcrV基因导入乳酸乳球菌内,构建重组肠道微生态菌株,作为黏膜免疫疫苗的先期探索和尝试。方法采用酸诱导P170启动子,乳酸乳球菌本身的SP310mut2信号肽,将鼠疫杆菌LcrV抗原结构基因克隆到质粒pAM J397上,电转化感受态Lactococcus lactis PSM565。结果经重组子PCR鉴定,SDS-PAGE检测,W estern-b lot鉴定,在Lactococcus lactisPSM565/pAM J397-V培养基上清中获得了38 kD的鼠疫抗原LcrV蛋白。结论在乳酸乳球菌中成功表达了鼠疫V抗原,为下一步鼠疫黏膜疫苗的研制打下基础。     

乳酸菌载体pMG36e的应用现状

乳酸乳球菌通用表达质粒pMG36e是一个经典的人工构建的组成型表达载体,是以乳酸乳球菌乳脂亚种蛋白酶基因的转录和翻译信号为基础构建而成的。它包含一个强启动子,能够在多种细菌中表达外源蛋白。已用于研究细菌素作用机制,乳酸菌基因工程菌株的改造以及口服疫苗的开发等,应用领域十分广泛,已成为乳酸菌基因工程研究的重要工具质粒之一。本文主要从载体构成、基因表达与食品级载体改造等三方面的应用对其进行综述,旨在为该质粒今后研究提供资料。     

基于冰核蛋白的乳酸菌表面展示系统的构建

[目的]验证来源于丁香假单胞菌的冰核蛋白在乳酸乳球菌表面展示外源蛋白的可能性.[方法]以绿色荧光蛋白(Green Fluorescence Protein,GFP)基因gfp为报告基因,以冰核蛋白基因的N末端和NC端作为展示单元,构建乳酸菌表面展示载体pHZ101和pHZ102,并转化大肠杆菌(Escherichia coli JM109和乳酸乳球菌(Lactococcus lactis)MG1363.[结果]荧光显微镜观察显示重组大肠杆菌和乳酸乳球菌均能检测到绿色荧光.Western blot结果表明GFP蛋白在重组大肠杆菌和乳酸乳球菌中均得到表达,并且INPN-GFP蛋白多数滞留于乳酸乳球菌细胞质内,而INPNC-GFP蛋白则大部分定位于乳酸乳球菌的细胞膜上.[结论]以上结果表明丁香假单胞菌的冰核蛋白能引导外源蛋白定位于乳酸乳球菌的细胞膜上,为乳酸菌表面展示系统的构建提供了新的方向.     

猪传染性胃肠炎病毒S蛋白在乳酸菌中的表达

目的：构建猪传染性胃肠炎病毒S蛋白的细胞内表达重组乳酸乳球菌,确定其最佳表达条件,为重组乳酸菌作为口服疫苗防治猪传染性胃肠炎奠定基础。方法：根据猪传染性胃肠炎病毒纤突（S）蛋白的全基因序列及表达载体质粒的基因融合特点,设计一对引物,进行PCR,获得含有TGEV S基因4个主要抗原位点的约2000bp目的片段,将其与表达载体质粒pNZ8048进行连接,通过电转化进入宿主菌乳酸乳球菌NZ9000细胞内,在乳链菌肽（Nisin）的诱导下进行表达,确定最佳表达条件;并通过SDS-PAGE进行检测和Western-blot分析表达蛋白活性。结果：成功获得了TGEV S蛋白在乳酸乳球菌细胞内的表达并且表达的蛋白具有TGE全病毒的抗原性。确定了乳酸乳球菌表达TGEV S蛋白的最佳表达条件为在以1ng/ml的乳链杆菌肽nisin诱导下,诱导后3h,重组蛋白表达效率达最高,重组蛋白约占菌体总蛋白含量的8．7％。结论：在乳酸乳球菌细胞内表达的重组TGEV S蛋白获得了理想表达,为进一步研制开发防治TGE口服疫苗提供物质基础。     

海藻酸钙微囊对乳酸乳球菌在胃肠道环境中的保护作用

乳酸菌是机体内一类重要的益生菌,因其益生功能和安全性,在食品行业和医疗保健领域有着广泛的应用,此外将乳酸菌作为口服疫苗载体或药物传递载体也是目前的研究热点之一。乳酸菌到达肠道的活菌数是影响其功能有效性的一个重要因素,需考虑为其提供一定的防护来抵御胃酸等恶劣环境。通过化学法制备能稳定表达Gfp的乳酸乳球菌海藻酸钙微囊,以Gfp作为活菌标记,检测了海藻酸钙微囊对乳酸乳球菌的保护作用。体外实验结果显示,酸处理30、60、90、120 min后,海藻酸钙微囊包裹使乳酸乳球菌的存活率分别提高了1 370、525、235和105倍。动物体内实验也表明,在灌胃2 h后,海藻酸钙微囊包裹使乳酸乳球菌在肠道内的活菌数增加90多倍。上述结果说明海藻酸钙微囊对乳酸乳球菌在胃肠道环境中具有明显的保护作用,为今后乳酸乳球菌口服制剂的研究及开发提供重要的参考依据。     

乳酸乳球菌表达系统的发展现状与前景展望

乳酸乳球菌作为全球公认安全的微生物,具有多种益生作用,常被用作基因工程宿主菌.在过去的二十年中,乳酸乳球菌作为载体在递呈病毒、细菌抗原等方面得到了广泛的应用,并且在不同领域发挥着重要作用.本文以乳链菌肽控制的表达(nisin-controlled expression,NICE)系统为例,介绍了基于乳酸乳球菌的表达系统...     

作为宿主系统的几株乳酸菌的表型特征

目的：研究乳酸菌载体－宿主系统。方法：采用涂片染色和形态特征观察,用鉴别生化实验如过氧化氢酶实验,碳水化合物发酵产酸实验,精氨酸水解实验及抗生素抗性实验等对含有pMG36e质粒的乳酸菌MG1363,乳球菌IL1403和乳杆菌ATCC4356进行研究鉴定。结果：乳酸乳球菌乳脂亚种MG1363,乳酸乳球菌乳酸亚种IL1403含有质粒pMG36e的MG1363致及嗜酸乳杆菌ATCC4356其表型特征分别与伯杰氏手册中相应细菌特征一致,质粒pMG36e含有红霉素抗性基因,结论：此乳酸菌宿主一载体系统可用载体来源的红霉素抗性进行筛选,用于外源基因在乳酸菌中克隆和表达的研究。     

乳酸乳球菌表面展示表达系统的构建及其鉴定

目的:旨在构建一个将抗原靶向于乳酸乳球菌细胞表面的表达系统。方法:运用PCR技术从金黄色葡萄球菌基因组中克隆出蛋白A(SPA)C-末端544个碱基对的锚定域序列(Spax)。通过酶切、连接将Spax构建入分泌型质粒pAMJ399形成携有整合外源基因位点BglⅡ的pAMJ400质粒。将报告蛋白—绿色荧光蛋白的基因(Gfp)插入载体pAMJ400的整合位点产生模式质粒pAMJ401并电转化其于乳酸乳球菌MG1363。绘制转化子MG1363(pAMJ401)生长曲线确认诱导期。调节pH值(6.0~6.5)诱导转化子并在荧光显微镜下观察杂合蛋白(GFP:SPAX)的表达情况。结果:在395nm的蓝色激发光下,诱导后的细菌发出较明亮的绿色荧光,而未诱导的细菌几乎不产生荧光。结论:成功地构建了乳酸乳球菌表面展示表达系统,此系统可以作为口服活菌疫菌研究的可行性操作平台。     

Heterologous protein production and delivery systems for Lactococcus lactis

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as live vehicles for the production and delivery of heterologous proteins of vaccinal, medical or technological interest has therefore been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium). A promising application of L. lactis is its use as an antigen delivery vehicle, for the development of live mucosal vaccines. The expression of heterologous proteins and antigens as well as the various delivery systems developed in L. lactis, and its use as an oral vaccine carrier are discussed.     

Lactococcus lactis as a live vector: heterologous protein production and DNA delivery systems

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as mucosal delivery vehicles for vaccinal, medical or technological use has been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins and for plasmid DNA delivery to eukaryotic cells. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium) and more recently to efficiently transfer DNA to eukaryotic cells. A promising application of L. lactis is its use for the development of live mucosal vaccines. Here, we have reviewed the expression of heterologous protein and the various delivery systems developed for L. lactis, as well as its use as an oral vaccine carrier.     

Development of Vaccine Delivery Vehicles Based on Lactic Acid Bacteria

Live recombinant bacteria represent attractive antigen delivery systems able to induce both mucosal and systemic immune responses against heterologous antigens. The first live recombinant bacterial vectors developed were derived from attenuated pathogenic microorganisms. In addition to the difficulties often encountered in the construction of stable attenuated mutants of pathogenic organisms, attenuated pathogens may retain a residual virulence level that renders them unsuitable for the vaccination of partially immunocompetent individuals such as infants, the elderly or immunocompromised patients. As an alternative to this strategy, non-pathogenic food-grade lactic acid bacteria (LAB) maybe used as live antigen carriers. This article reviews LAB vaccines constructed using antigens other than tetanus toxin fragment C, against bacterial, viral, and parasitic infective agents, for which protection studies have been performed. The antigens utilized for the development of LAB vaccines are briefly described, along with the efficiency of these systems in protection studies. Moreover, the key factors affecting the performance of these systems are highlighted.     

Lactococcus lactis as an adjuvant and delivery vehicle of antigens against pneumococcal respiratory infections

Most studies of Lactococcus lactis as delivery vehicles of pneumococcal antigens are focused on the effectiveness of mucosal recombinant vaccines against Streptococcus pneumoniae in animal models. At present, there are three types of pneumococcal vaccines: capsular polysaccharide pneumococcal vaccines (PPV), protein-polysaccharide conjugate pneumococcal vaccines (PCV) and protein-based pneumococcal vaccines (PBPV). Only PPV and PCV have been licensed. These vaccines, however, do not represent a definitive solution. Novel, safe and inexpensive vaccines are necessary, especially in developing countries. Probiotic microorganisms such as lactic acid bacteria (LAB) are an interesting alternative for their use as vehicles in pneumococcal vaccines due to their GRAS (Generally Recognized As Safe) status. Thus, the adjuvanticity of Lactococcus lactis by itself represents added value over the use of other bacteria, a question dealt with in this review. In addition, the expression of different pneumococcal antigens as well as the use of oral and nasal mucosal routes of administration of lactococcal vaccines is considered. The advantages of nasal live vaccines are evident; nonetheless, oral vaccines can be a good alternative when the adequate dose is used. Another point addressed here is the use of live versus inactivated vaccines. In this sense, few researchers have focused on inactivated strains to be used as vaccines against pneumoccoccus. The immunogenicity of live vaccines is better than the one afforded by inactivated ones; however, the probiotic-inactivated vaccine combination has improved this matter considerably. The progress made so far in the protective immune response induced by recombinant vaccines, the successful trials in animal models and the safety considerations of their application in humans suggest that the use of recombinant vaccines represents a good short-term option in the control of pneumococcal diseases.     

Lactic acid bacteria as live vaccines

Mucosal routes for vaccine delivery offer several advantages over systemic inoculation from both immunological and practical points of view. The development of efficient mucosal vaccines therefore represents a top prority in modern vaccinology. One way to deliver protective antigens at the mucosal surfaces is to use live bacterial vectors. Until recently most of these were derived from attenuated pathogenic microorganisms. As an alternative to this strategy, non-pathogenic food grade bacteria such as lactic acid bacteria (LAB) are being tested for their efficacy as live antigen carriers. The LABVAC european research network is presently comparing the vaccine potential of Lactococcus lactis, Streptococcus gordonii and Lactobacillus spp. To date, it has been shown that systemic and mucosal antigen-specific immune responses can be elicited in mice through the nasal route using the three LAB systems under study. Data on successful oral and vaginal immunisations are also accumulating for L. lactis and S. gordonii, respectively. Moreover, the immune responses can be potentiated by co-expression of interleukins. Future areas of research include improvement of local immunisation efficiency, analysis of in vivo antigen production, unravelling of the Lactobacillus colonisation mechanisms and construction of biologically contained strains.     

Current prophylactic and therapeutic uses of a recombinant Lactococcus lactis strain secreting biologically active interleukin-12

The noninvasive and food-grade Gram-positive bacterium Lactococcus lactis is well adapted to deliver medical proteins to the mucosal immune system. In the last decade, the potential of live recombinant lactococci to deliver such proteins to the mucosal immune system has been investigated. This approach offers several advantages over the traditional systemic injection, such as easy administration and the ability to elicit both systemic and mucosal immune responses. This paper reviews the current research and advances made with recombinant L. lactis as live vector for the in situ delivery of biologically active interleukin-12, a potent pleiotropic cytokine with adjuvant properties when co-delivered with vaccinal antigens, at mucosal surfaces. Three well-illustrated examples demonstrate the high potential of interleukin-12-secreting lactococci strains for future prophylactic and therapeutic uses.     

Genome analysis of lactic acid bacteria in food fermentations and biotechnological applications

Lactic acid bacteria are an important group of microorganisms, several of which are used in fermented food processes. Lactococcus lactis is a non-pathogenic, non-invasive and non-colonising gram-positive lactic acid bacterium, the genome sequence of which has been established. A great deal is known about the genetics, vectors, gene expression systems and protein secretion apparatus of this bacterium. Recently, recombinant strains of L. lactis have been developed that might provide in vivo delivery of cytokines and specific antigens across mucosal surfaces to the immune system of animals.     

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。     

Cytoplasmic and extracellular expression of pharmaceutical-grade mycobacterial 65-kDa heat shock protein in Lactococcus lactis

Lactic acid bacteria (LAB) are an attractive and safe alternative for the expression of heterologous proteins, as they are nonpathogenic and endotoxin-free organisms. Lactococcus lactis, the LAB model organism, has been extensively employed in the biotechnology field for large-scale production of heterologous proteins, and its use as a "cell factory" has been widely studied. We have been particularly interested in the use of L. lactis for production of heat shock proteins (HSPs), which reportedly play important roles in the initiation of innate and adaptive immune responses. However, this activity has been questioned, as LPS contamination appears to be responsible for most, if not all, immunostimulatory activity of HSPs. In order to study the effect of pure HSPs on the immune system, we constructed recombinant L. lactis strains able to produce and properly address the Mycobacterium leprae 65-kDa HSP (Hsp65) to the cytoplasm or to the extracellular medium, using a xylose-induced expression system. Approximately 7 mg/L recombinant Hsp65 was secreted. Degradation products related to lactococcal HtrA activity were not observed, and the Limulus amebocyte lysate assay demonstrated that the amount of LPS in the recombinant Hsp65 preparations was 10-100 times lower than the permitted levels established by the U.S. Food and Drug Administration. These new L. lactis strains will allow investigation of the effects of M. leprae Hsp65 without the interference of LPS; consequently, they have potential for a variety of biotechnological, medical and therapeutic applications.