乳酸乳球菌组成型表面展示载体的构建及鉴定

目的探索融合有Usp45信号肽和3Lys M锚定序列的EGFP蛋白能否通过p32启动子组成型展示于乳酸乳球菌MG1363表面。方法融合PCR法分别将p32启动子与Usp45信号肽、3Lys M与egfp基因融合亚克隆至p MD-18T载体,鉴定正确后命名为18T-p Usp45、18T-3Lys M-egfp,分别将上述片段切下依次插入p MG36e构建p MG36e-p Usp45-3Lys M-egfp。将其转化入乳酸乳球菌MG1363,荧光显微镜观察及SDSPAGE、Western-blot检测。结果成功构建重组菌p MG36e-p Usp45-3Lys M-egfp/MG1363,荧光显微镜、SDSPAGE和Western-blot检测表明重组蛋白能在MG1363中组成型表达,且分泌至胞外并锚定在细胞壁上。结论成功地构建了组成型表面展示载体p MG36e-p Usp45-3Lys M-egfp,重组蛋白能分泌至胞外且展示在MG1363细胞壁上。     

带锚定基序3LysM的EGFP融合蛋白与乳酸乳球菌的体外混合展示

目的探索融合有锚定序列的EGFP蛋白,能否通过体外混合展示于乳酸乳球菌MG1363表面。方法采用融合PCR方法扩增带有锚定序列的egfp(3LysM-egfp),亚克隆至pMD18T载体,测序正确后,插入表达载体pET28a转化BL21(DE3)进行诱导表达、纯化,将纯化的3LysM-EGFP与乳酸乳球菌进行体外混合作用,荧光显微镜及Western-blot检测展示效果。结果成功构建表达重组菌pET28a-3LysM-egfp/BL21并诱导出可溶性3LysM-EGFP,纯化后蛋白纯度达到81.5%,荧光显微镜及Western-blot均检测到展示的目的蛋白。结论纯化后的3LysM-EGFP能在体外条件下展示在乳酸乳球菌表面。     

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。     

乳酸乳球菌表面表达Brazzein甜味蛋白系统的建立

参照甜味蛋白Brazzein基因序列,结合乳酸乳球菌的密码子偏嗜性的相关分析,对甜味蛋白Brazzein基因进行改造,将第29位天冬氨酸、31位的组氨酸和第41位的谷氨酸分别突变为赖氨酸、丙氨酸和赖氨酸,以期提高目的蛋白的甜度。采用重叠PCR的方法合成目的基因,目的片段亚克隆到pMD18-T载体上。经序列测定分析后,目的片段克隆入分泌表达载体pNZ8112,电转化乳酸乳球菌中,构建成表面展示表达甜味蛋白Brazzein的乳酸乳球菌表达系统。     

乳酸乳球菌作为基因工程受体菌研究进展

乳酸乳球菌(Lactococcus lactis)是一种"公认安全"的革兰氏阳性细菌,广泛存在于人、畜的肠道中并发挥许多重要的生理功能。由于它兼具安全性与益生性,近几年来研究者们开始关注用乳酸乳球菌作为受体菌来表达外源蛋白。随着生物技术的发展,人们对乳酸乳球菌基因表达及调控过程的认识不断深入并构建了一系列表达,成功地表达了许多外源蛋白,初步展示出良好的应用前景。主要对近年来国内外将乳酸乳球菌作为外源蛋白表达受体菌方面的研究进展做简要综述。     

乳酸乳球菌表面表达Brazzein甜味蛋白系统的建立北大核心CSCD

参照甜味蛋白Brazzein基因序列,结合乳酸乳球菌的密码子偏嗜性的相关分析,对甜味蛋白Brazzein基因进行改造,将第29位天冬氨酸、31位的组氨酸和第41位的谷氨酸分别突变为赖氨酸、丙氨酸和赖氨酸,以期提高目的蛋白的甜度。采用重叠PCR的方法合成目的基因,目的片段亚克隆到pMD18-T载体上。经序列测定分析后,目的片段克隆入分泌表达载体pNZ8112,电转化乳酸乳球菌中,构建成表面展示表达甜味蛋白Brazzein的乳酸乳球菌表达系统。     

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。     

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。     

F_(212-489)-3LysM融合蛋白与乳酸乳球菌的体外混合展示

探索C端融合有锚定序列3Lys M的呼吸道合胞病毒F截短蛋白(F212-489),能否通过体外混合展示于乳酸乳球菌MG1363表面。采用PCR方法扩增f212-489及3lys M基因,分别亚克隆至p MD18-T载体,在亚克隆载体中酶切回收上述两片段,插入p ET28a构成p ET28a-f212-489-3lys M,酶切鉴定并测序正确后转化BL21(DE3),经IPTG诱导获得的包涵体蛋白进行溶解、复性,将复性后的F212-489-3Lys M与乳酸乳球菌MG1363体外混合,通过全菌ELISA(enzyme linked immuno sorbent assay)检测融合蛋白吸附菌体的情况。成功构建重组菌p ET28-f212-489-3lys M/BL21(DE3),该菌诱导后F212-489-3Lys M主要以包涵体形式表达,F212-489-3Lys M与MG1363混合后,经全菌ELISA检测,F212-489-3Lys M组OD450远高于对照组,且差异非常显著(P0.01)。证明经大肠杆菌表达的重组蛋白F212-489-3Lys M经体外混合可展示于乳酸乳球菌MG1363表面。     

大肠埃希菌Mn-SOD在乳酸乳球菌中的表达

本文根据GenBank中报道的大肠埃希菌MG1655全基因组DNA序列中SOD的编码基因序列设计引物,PCR扩增大肠埃希菌锰超氧化物歧化酶(Mn-SOD)基因,并将其克隆入原核高效表达质粒载体pBV220中构建重组质粒pBV220-sod,并将其电转入乳酸乳球菌MG1363中获得了成功表达,为SOD发酵奶的研制奠定了基础。     

丝状真菌表面展示技术研究进展

丝状真菌表面展示技术是将表达的目的蛋白固定在丝状真菌细胞表面的一项新兴基因工程技术。丝状真菌具有极强的蛋白质分泌能力和良好的蛋白质翻译后加工能力,因而越来越多的丝状真菌表面展示技术得到开发和应用。本文就丝状真菌表面展示系统的研发和应用进展进行综述,并介绍与该系统构建密切相关的丝状真菌的细胞壁组成、锚定蛋白和遗传转化方法等技术。     

Cell surface display system for Lactococcus lactis: a novel development for oral vaccine

The food-grade Lactococcus lactis is a potential vector to be used as a live vehicle for the delivery of heterologous proteins for vaccine and pharmaceutical purposes. We constructed a plasmid vector pSVac that harbors a 255-bp single-repeat sequence of the cell wall-binding protein region of the AcmA protein. The recombinant plasmid was transformed into Escherichia coli and expression of the gene fragment was driven by the T7 promoter of the plasmid. SDS-PAGE showed the presence of the putative AcmA fragment and this was confirmed by Western blot analysis. The protein was isolated and purified using a His-tag affinity column. When mixed with a culture of L. lactis MG1363, ELISA and immunofluorescence assays showed that the cell wall-binding fragment was anchored onto the outer surface of the bacteria. This indicated that the AcmA repeat unit retained the active site for binding onto the cell wall surface of the L. lactis cells. Stability assays showed that the fusion proteins (AcmA/A1, AcmA/A3) were stably docked onto the surface for at least 5 days. The AcmA fragment was also shown to be able to strongly bind onto the cell surface of naturally occurring lactococcal strains and Lactobacillus and, with less strength, the cell surface of Bacillus sphericus. The new system designed for cell surface display of recombinant proteins on L. lactis was evaluated for the expression and display of A1 and A3 regions of the VP1 protein of enterovirus 71 (EV71). The A1 and A3 regions of the VP1 protein of EV71 were cloned upstream to the cell wall-binding domains of AcmA protein and successfully expressed as AcmA/A1 and AcmA/A3. Whole-cell ELISA showed the successful display of VP1 protein epitopes of EV71 on the surface of L. lactis. The success of the anchoring system developed in this study for docking the A1 and A3 epitopes of VP1 onto the surface of L. lactis cells opens up the possibilities of peptide and protein display for not only Lactococcus but also for other gram-positive bacteria. This novel way of displaying epitopes on the cell surface of L. lactis and other related organisms should be very useful in the delivery of vaccines and other useful proteins.     

Targeting of fluorescent Lactococcus lactis to colorectal cancer cells through surface display of tumour-antigen binding proteins

Development of targeted treatment for colorectal cancer is crucial to avoid side effects. To harness the possibilities offered by microbiome engineering, we prepared safe multifunctional cancer cell-targeting bacteria Lactococcus lactis. They displayed, on their surface, binding proteins for cancer-associated transmembrane receptors epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2) and co-expressed an infrared fluorescent protein for imaging. Binding of engineered L. lactis to tumour antigens EpCAM and HER2 was confirmed and characterised in vitro using soluble receptors. The proof-of-principle of targeting was demonstrated on human cell lines HEK293, HT-29 and Caco-2 with fluorescent microscopy and flow cytometry. The highest L. lactis adhesion was seen for the HEK293 cells with the overexpressed tumour antigens, where colocalisation with their tumour antigens was seen for 39% and 67% of EpCAM-targeting and HER2-targeting bacteria, respectively. On the other hand, no binding was observed to HEK293 cells without tumour antigens, confirming the selectivity of the engineered L. lactis. Apart from cell targeting in static conditions, targeting ability of engineered L. lactis was also shown in conditions of constant flow of bacterial suspension over the HEK293 cells. Successful targeting by engineered L. lactis support the future use of these bacteria in biopharmaceutical delivery for the treatment of colorectal cancer.     

Identification of candidate carrier proteins for surface display on Lactococcus lactis by theoretical and experimental analyses of the surface proteome

Lactococcus lactis is a lactic acid bacterium of proven safety for use in human oral applications. For this purpose, surface display of recombinant proteins is important, and new approaches for it are being sought. Analysis of the bacterial surface proteome is essential in identifying new candidate carrier proteins for surface display. We have made two different predictions of surface-associated proteins of L. lactis MG1363 by using Augur and LocateP software, which yielded 666 and 648 proteins, respectively. Surface proteins of L. lactis NZ9000, a derivative of MG1363, were identified by using a proteomics approach. The surface proteins were cleaved from intact bacteria, and the resulting peptides were identified by mass spectrometry. The latter approach yielded 80 proteins, 34 of which were not predicted by either software. Of the 80 proteins, 7 were selected for further study. These were cloned in frame with a C-terminal hexahistidine tag and overexpressed in L. lactis NZ9000 using nisin-controlled expression. Proteins of correct molecular weight carrying a hexahistidine tag were detected. Their surface localization was confirmed with flow cytometry. Basic membrane protein A (BmpA) was exposed at the highest level. To test BmpA as a candidate carrier protein, the hexahistidine tag was replaced by the B domain of staphylococcal protein A in the genetic construct. The B domain was displayed on the surface with BmpA as a carrier. The advantage of covalent BmpA binding was demonstrated. BmpA was thus shown to be a suitable candidate for a carrier protein in lactococcal surface display.     

枯草芽孢杆菌表面展示技术用于黏膜疫苗的研究进展

枯草杆菌全名枯草芽孢杆菌(Bacillus subtilis),因其优秀的益生特性及芽孢良好的抗逆性而备受研究者青睐,由于芽孢的特殊结构及独特的生理特性,是酶和免疫原等外源蛋白的理想锚定点。采用枯草杆菌进行芽孢表面展示被认为是表达高活性和高稳定性的外源蛋白的方法之一。本文主要对枯草杆菌芽孢表面展示抗原蛋白以生产黏膜疫苗的策略和应用前景进行综述。     

Cell-surface binding domains from Clostridium cellulovorans can be used for surface display of cellulosomal scaffoldins in Lactococcus lactis

Engineering microbial strains combining efficient lignocellulose metabolization and high-value chemical production is a cutting-edge strategy towards cost-sustainable 2^nd generation biorefining. Here, protein components of the Clostridium cellulovorans cellulosome were introduced in Lactococcus lactis IL1403, one of the most efficient lactic acid producers but unable to directly ferment cellulose. Cellulosomes are protein complexes with high cellulose depolymerization activity whose synergistic action is supported by scaffolding protein(s) (i.e., scaffoldins). Scaffoldins are involved in bringing enzymes close to each other and often anchor the cellulosome to the cell surface. In this study, three synthetic scaffoldins were engineered by using domains derived from the main scaffoldin CbpA and the Endoglucanase E (EngE) of the C. cellulovorans cellulosome. Special focus was on CbpA X2 and EngE S-layer homology (SLH) domains possibly involved in cell-surface anchoring. The recombinant scaffoldins were successfully introduced in and secreted by L. lactis. Among them, only that carrying the three EngE SLH modules was able to bind to the L. lactis surface although these domains lack the conserved TRAE motif thought to mediate binding with secondary cell wall polysaccharides. The synthetic scaffoldins engineered in this study could serve for assembly of secreted or surface-displayed designer cellulosomes in L. lactis.     

Surface display of respiratory syncytial virus glycoproteins in Lactococcus lactis NZ9000

Aims: A system for displaying heterologous respiratory syncytial virus (RSV) glycoproteins on the surface of Lactococcus lactis NZ9000 was developed. Methods and Results: Fusion of the USP45 signal peptide and the cA (C terminus of the peptidoglycan‐binding) domains of AcmA, a major autolysin from L. lactis, to the N‐ and C‐terminal of the target proteins, respectively, was carried out. The target protein was the major immunogenic domain of either the F (40·17‐kDa) or G (11·49‐kDa) glycoprotein domains of the RSV. Whole‐cell ELISA readings obtained after 24 h of induction showed an increase in protein expression as the cA domain repeats increased, for the G glycoprotein of RSV. On the other hand, the F glycoprotein indicated decreasing expression levels as the number of cA domain repeats increased. The difference in the expression levels of the F and G domains may be attributed to the different sizes of the antigenic domains. Conclusions: The size and properties of the target proteins are vital in determining the amount of antigenic domains being displayed on the surface of live cells. Significance and Impact of the Study: The system demonstrated here can aid in the utilization of the generally regarded as safe (GRAS) bacteria L. lactis, as a vaccine delivery vehicle to surface display the antigenic proteins of RSV.     

利用乳酸乳球菌AcmA表面展示β-1,3-1,4-葡聚糖酶

采用PCR扩增乳酸乳球(Lactococcus lactis)MBl91菌株的全长肽聚糖水解酶基因acmA,通过C-末端融合构建了与绿色荧光基因gfp的融合基因acmA-gfp,再连接于表达载体pMG36k上后得到可组成型表达AcmA-GFP融合蛋白的重组质粒pMB137,然后将该质粒电转化导入到乳酸乳球菌AS1.2829中获得重组菌MB137.经SDS-PAGE检测.重组菌MB137可表达预期的分子量约74 kD的蛋白质.Western blotting、细胞分级分离组分的荧光活性测定和特异GFP 二抗标记的流式细胞仪检测证实GFP被成功锚定在重组茵细胞表面,被锚定蛋白约占总表达融合蛋白的35%.进一步通过从枯草芽胞杆菌BF7658基因组中扩增去信号肽序列的β-1,3-1,4葡聚糖酶基因gls,来取代pMB137中的gfp,得到携带融合基因acmA-gls的重组质粒pMB138,经导入到乳酸乳球茵AS1.2829后得到重组菌MB138,其全细胞β-1,3-1,4-葡聚糖水解酶的活性约为12 U/mL茵液,明显高于对照茵株.     

Divergence of Genomic Sequences between Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris

Relatedness between Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis was assessed by Southern hybridization analysis, with cloned chromosomal genes as probes. The results indicate that strains of the two subspecies form two distinct groups and that the DNA sequence divergence between L. lactis subsp. lactis and L. lactis subsp. cremoris is estimated to be between 20 and 30%. The previously used phenotypic criteria do not fully discriminate between the groups; therefore, we propose a new classification which is based on DNA homology. In agreement with this revised classification, the L. lactis subsp. lactis and L. lactis subsp. cremoris strains from our collection have distinct phage sensitivities.