乳酸乳球菌两组分食品级NICE系统载体的构建

乳酸菌食品级NICE系统是目前较理想的可控制蛋白质生产的食品级诱导表达系统。本实验构建的食品级表达载体pRNA48含α-aga食品级选择标记、θ型复制子和nisA启动子，与宿主菌L．hctis NZ9000共同构成乳酸乳球菌食品级NICE系统，可用于目的基因在乳酸乳球菌中高效诱导表达。     

乳酸乳球菌NICE系统食品级载体pRNB48的构建

乳酸菌食品级NICE系统是目前较理想的可控制蛋白质生产的食品级诱导表达系统。实验构建的食品级表达载体pRNB48含α-aga食品级选择标记、θ型复制子和nisA启动子,与宿主菌L.lactisNZ9000共同构成乳酸乳球菌食品级NICE系统,可用于目的基因在乳酸乳球菌中高效诱导表达。     

cA-GFP融合蛋白在大肠杆菌中的高效功能性表达

为了实现乳酸乳球菌锚定蛋白cA在大肠杆菌中可溶表达以及直观检测其生物学活性,以乳酸乳球菌MG1363为模板,扩增N-乙酰葡萄糖胺糖苷酶AcmA基因,将其C末端序列与GFP基因融合,连接至大肠杆菌表达载体pET28a,构建重组质粒pET28a-cA-GFP,转化至大肠杆菌表达菌株BL21(DE3),通过低温诱导表达重组蛋白cA-GFP。工程菌超声破碎后的上清液与经热酸处理的乳酸乳球菌常温孵育,经SDS-PAGE和荧光显微镜检测。结果表明,工程菌可以表达分子量约53ku的目的融合蛋白质,与预期大小相符。cA-GFP通过锚定蛋白cA的引导回向锚定,成功将GFP展示在乳酸乳球菌表面,目的蛋白cA-GFP在乳酸乳球菌表面展示量为121mg/g干重菌体。GFP锚定至乳酸乳球菌后于4℃保存,连续6d测定其荧光强度,荧光强度仍可达82.2%,证明其稳定性较好。成功获得了具有生物学功能的cA-GFP可溶性蛋白,为进一步展示功能性外源蛋白奠定了坚实的基础。     

利用淀粉的酿酒酵母基因工程菌研究进展

酿酒酵母是发酵工业的重要微生物，主要用于酒精、啤酒和面包工业。在酿酒酵母中已表达的淀粉酶基因包括α-淀粉酶基因和糖化酶基因。外源淀粉水解酶能在酵母中高效表达并分泌，其主要因素在于：在构建载体时组入强启动子；酿酒酵母中带有合适的酵母受体系统；具有外源淀粉酶蛋白的分泌信号；外源淀粉酶基因的遗传稳定性和生长介质。构建分解淀粉酿酒酵母已取得相当大的进展，但在构建的多数菌株利用糊精及淀粉的能力仍然有限，而且降解速率较慢。构建直接分解和利用淀粉的酵母工程菌，可简化工艺、节省能源和酶制剂、降低成本，有重要的应用前景。(孙悟)     

甜味蛋白Brazzein在乳酸乳球菌的表面表达及鉴定

为了提高甜味蛋白Brazzein的产量及甜度,将第29位的天冬氨酸、第31位的组氨酸和第41位的谷氨酸突变为赖氨酸、丙氨酸、赖氨酸,然后结合乳酸乳球菌密码子偏嗜性,对甜味蛋白编码序列进行优化。将优化的Brazzein基因克隆入表面表达载体p NZ8112,构建甜味蛋白Brazzein的乳酸乳球菌表面表达系统。通过SDSPAGE、免疫印迹及免疫荧光等方法验证甜味蛋白Brazzein成功表达于乳酸乳球菌表面,这为甜味蛋白Brazzein的商品化开发奠定基础。     

TGEV和PEDV融合S蛋白在乳酸乳球菌食品级细胞壁锚定高效诱导表达与免疫原性分析

近年来猪传染性胃肠炎和猪流行性腹泻在我国广泛流行,给养殖业带来了巨大的经济损失。目前对此两种疾病的防治手段还停留在使用常规疫苗。现有的疫苗免疫效果不够理想,急需寻求一种安全高效的防治手段。本文首先构建了猪传染性胃肠炎病毒(TGEV)和猪流行性腹泻病毒(PEDV)融合S基因,以乳酸乳球菌食品级细胞壁锚定高效诱导表达载体p RNV48为基础,构建了TGEV和PEDV融合S基因的表达质粒p RNV48-TPs。将得到的重组质粒用电穿孔方法转入乳酸乳球菌NZ9000感受态细胞中,获得了重组菌乳酸乳球菌Z9000/p RNV48-TPs。再利用nisin对重组菌进行诱导,提取细胞壁蛋白后采用聚丙烯酰胺凝胶电泳(SDS-PAGE)分析目的蛋白的表达情况,然后分别对兔子进行注射免疫和口服免疫,并用Western Blot分析。通过乳酸乳球菌食品级细胞壁锚定高效诱导表达载体,诱导表达TGEV与PEDV融合S蛋白产生中和抗体,以期探讨口服疫苗的免疫反应机理,为由TGEV与PEDV研发的安全、高效的疫苗奠定良好的基础。     

乳源血管紧张素转移酶抑制肽在乳酸乳球菌中的表达

在乳酸乳球菌中表达乳源血管紧张素转移酶抑制肽。选取了4种不同的来源于牛酪蛋白的血管紧张素转移酶(ACE)抑制肽,为了确保能够在人体消化液作用下正常发挥它们的ACE抑制活性,4种短肽以串联多肽(TP)的形式进行表达,并在各短肽单体间引入了人体内主要消化酶的酶切位点。根据TP的氨基酸序列和乳酸乳球菌的偏爱密码子,人工合成TP基因。然后将TP基因和绿色荧光蛋白(GFP)基因串联于载体pSEC-E7,从而构建了pSEC-TP:GFP质粒,实现了2种蛋白在乳酸乳球菌中的共表达。经电击转化,将该重组质粒转入乳酸乳球菌NZ9000中,获得重组菌株NZ9000(pSEC-TP:GFP)。用Nisin诱导TP:GFP蛋白表达。RT-PCR、激光共聚焦扫描显微镜和SDS-PAGE鉴定表达产物。RT-PCR结果表明,TP:GFP蛋白在RNA水平表达成功;SDS-PAGE表明目标产物是35 ku的条带。在乳酸乳球菌中实现了乳源血管紧张素转移酶抑制肽的表达。     

乳酸片球菌素PA-1在大肠杆菌中的表达与纯化

为了实现该细菌素的外源表达,本实验首先利用聚合酶链式反应从乳酸片球菌PAF中扩增出乳酸片球菌素PA-1的结构和免疫基因,然后克隆到表达载体pGEX-6p-1,构建了N端含有GST-His-DDDDK标签的重组质粒pGEX/his-pedAB,然后转化进入大肠杆菌Rosetta（DE3）感受态细胞,经异丙基硫代半乳糖苷诱导,重组乳酸片球菌素PA-1在大肠杆菌胞内成功表达。表达的融合蛋白先经过镍亲合层析柱纯化,然后注入谷胱甘肽S-转移酶亲和色谱柱用肠激酶处理,释放出成熟的乳酸片球菌素PA-1。利用高效液相色谱和质谱技术检测乳酸片球菌素PA-1纯度。以单核细胞增生李斯特氏菌CMCC54004为指示菌,利用琼脂扩散法检验乳酸片球菌素PA-1活性。结果表明,携带GST-His-DDDDK标签的融合蛋白无活性,标签切除后其抑菌活性恢复,且其纯度达90%以上。     

基于CRISPR/Cas9系统对乳酸乳球菌进行绿色荧光蛋白标记

乳酸乳球菌是治疗剂在体内运送的良好载体,研究其在体内的真实运送情况需对其进行标记。该实验利用CRISPR/Cas9系统对乳酸乳球菌(Lactococcus lactis)NZ9000进行增强型绿色荧光蛋白(enhanced green fluorescent protein, eGFP)标记,用于研究乳酸乳球菌在体内的运送,评价其作为益生菌的功能。基于该实验室已构建的乳酸乳球菌CRISPR/Cas9编辑质粒pLL25构建重组质粒pYSH,其上携带eGFP及同源臂,电转入乳酸乳球菌NZ9000感受态细胞中,将基因组中的乳酸脱氢酶基因(ldh)替换为绿色荧光蛋白基因,从而使Lactococcus lactis NZ9000获得标记,表达绿色荧光蛋白。对绿色荧光标记的Lactococcus lactis NZ9000突变株,酶标仪定量分析eGFP的表达强度。荧光强度定量分析结果表明,在乳酸乳球菌不同生长阶段,eGFP基因均能稳定表达。     

基于CRISPR/Cas9系统对乳酸乳球菌进行绿色荧光蛋白标记

乳酸乳球菌是治疗剂在体内运送的良好载体,研究其在体内的真实运送情况需对其进行标记。该实验利用CRISPR/Cas9系统对乳酸乳球菌(Lactococcus lactis)NZ9000进行增强型绿色荧光蛋白(enhanced green fluorescent protein, eGFP)标记,用于研究乳酸乳球菌在体内的运送,评价其作为益生菌的功能。基于该实验室已构建的乳酸乳球菌CRISPR/Cas9编辑质粒pLL25构建重组质粒pYSH,其上携带eGFP及同源臂,电转入乳酸乳球菌NZ9000感受态细胞中,将基因组中的乳酸脱氢酶基因(ldh)替换为绿色荧光蛋白基因,从而使Lactococcus lactis NZ9000获得标记,表达绿色荧光蛋白。对绿色荧光标记的Lactococcus lactis NZ9000突变株,酶标仪定量分析eGFP的表达强度。荧光强度定量分析结果表明,在乳酸乳球菌不同生长阶段,eGFP基因均能稳定表达。     

A food-grade expression/secretion vector for Lactococcus lactis that uses an alpha-galactosidase gene as a selection marker

A new food-grade expression/secretion vector for lactococci, pFMN30, was developed using an alpha-galactosidase gene (melA) of Lactobacillus plantarum as a selection marker. The 4.9-kb pFMN30 is a derivative of the lactococcal vector pMG36e containing a broad-host-range replicon of pWV01. In Lactococcus lactis, transformants carrying the vector were easily detectable by the appearance of a blue colony on a X-alpha-gal-containing medium and also by the growth on a medium containing melibiose as a sole carbon source. The expression/secretion vector was equipped with the controllable and strong nisA promoter. In addition, usp45 signal peptide was inserted for the efficient secretion of a foreign protein outside cells. The vector pFMN30 was used for the expression and secretion of alpha-amylase as a reporter gene, lacking a signal sequence derived from Bacillus licheniformis in L. lactis. These results show that the food-grade expression/secretion vector constructed in the present study could be used for the production of foreign proteins in L. lactis for the production food materials and also for the medicinal purposes.     

乳酸乳球菌乳脂亚种MG1363中盐诱导启动子的克隆

为了从乳酸菌中筛选和克隆启动子,实验利用缺失T7启动子的质粒载体PRSET/LacZ直接在大肠杆菌(E.coli)DH5α中分离乳酸乳球菌乳脂亚种(Lactococcus lactis subsp.cremoris)MG1363的基因启动子片段,获得了10多个具有抗氨苄和盐诱导出蓝斑的重组子。反复筛选并对其中一个抗性最高的重组子PRSET-osm进行序列测定和同源性分析发现,所克隆的基因启动子片段来自乳酸乳球菌乳脂亚种MG1363的基因组,并具有原核启动子的保守序列(Pribnow框和Sextama框)。对启动子osm进行进一步序列分析和鉴定发现,其在大肠杆菌BL21中启动LacZ基因的表达,确定osm为盐诱导启动子。     

Genetically modified Lactococcus lactis: novel tools for drug delivery

The dairy bacterium Lactococcus lactis can be genetically engineered to secrete bioactive cytokines. This enabled the development of an entirely novel drug delivery system: topical and active delivery of therapeutic proteins by genetically modified micro-organisms. In mice, intestinal inflammation can be successfully treated with interleukin-10- (IL-10) or trefoil factor-secreting L. lactis . In human application, biological containment is guaranteed by exchange of the gene encoding thymidylate synthase for IL-10. The recombinant strain is now absolutely dependent on exogenous thymidine. Thymidine starvation will irreversibly lead to 'thymidine-less death' induction. This accomplishment has opened applications in human medicine.     

乳链菌肽的基因工程研究进展

乳链菌肽是某些Lactococcus lactis和Streptococcus lactis产生的小肽。它对许多革兰氏阳性菌及厌氧菌具有强烈抑制作用,是人们广为应用的一种天然食品防腐剂,但其低产量和高成本而制约着Nisin商业应用。很多研究者尝试通过基因工程方法生产乳链菌肽从而提高其产量。论文综述了Nisin的分子结构、合成及分泌机制、基因工程应用等。     

Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris

Pichia pastoris expression system has been widely used in recombinant protein production. So far the majority of heterologous proteins are expressed by methanol inducible promoter P_AOX1 and constitutive promoter P_GAP. The use of other promoters is rather limited. Here we selected 16 potentially efficient and regulatory promoter candidates based on the RNA‐seq and RNA folding free energy ΔG data. GFP and recombinant amylase were inserted after these promoters to reveal their strength and efficiency under different carbon sources and culture scales. Two novel promoters were successfully identified and could possibly be applied in recombinant protein expression: the methanol‐inducible promoter P₀₅₄₇ and the constitutive promoter P₀₄₇₂.     

Extracellular Expression of a Recombinant Ganoderma lucidium Immunomodulatory Protein by Food-Grade Lactococcus lactis System

Lactococcus lactis is a food-grade microorganism of major commercial importance in food industry. For commercial application of genetically modified L. lactis, a food-grade expression system is strongly recommended. In this study, two food-grade selection markers, nisin immunity gene nisI and nisin resistance gene nsr, were evaluated as dominant markers for the L. lactis food-grade expression system. By using an efficient P_SlpAl promoter fused to a signal peptide from subtilisin YaB (SP_YAB), a functional recombinant Ganoderma lucidium immunomodulatory protein rLZ8 was expressed extracellularly in L. lactis. Replacing the antibiotic marker gene into the proper food-grade selection marker nsr gene, the rLZ8 was expressed extracellularly in the food-grade L. lactis system. This study provides a rationale basis for a food-grade system to express functional peptides extracellularly as an important tool for oral administration of genetically modified L. lactis.     

Bacteriophage resistance in Lactococcus lactis ssp. lactis using antisense ribonucleic acid.

Antisense RNA against a conserved bacteriophage gene when expressed in a Lactococcus lactis ssp. lactis strain renders it resistant to bacteriophage infection. Two open reading frames have been identified in a L. lactis ssp. lactis bacteriophage that are conserved in a majority of isolates. They code for an 18-kDa (designated GP18C) protein and a 24-kDa (GP24C) protein, respectively, which are arranged along with previously identified open reading frames in a tandem motif similar to other bacteriophages. The presence of gp18C and gp24C in a number of bacteriophage isolates was confirmed by polymerase chain reaction using primers specific for these regions. Plasmids bearing various fragments of gp18C, gp24C, or both were constructed such that the respective open reading frames were positioned in the antisense direction relative to the Lactococcus lactis ssp. cremoris Wg2 promoter, p59. These antisense RNA-producing vectors inhibited the efficiency of plaquing of L. lactis ssp. lactis bacteriophage phi 7-9 up to 50%; the resulting plaques were extremely small and irregular in shape. The replication of the bacteriophage was severely inhibited, and the total number decreased over the first 3 h during infection in strains expressing antisense RNA compared with the host strain alone, in which the bacteriophage number increased 10(4)-fold.     

乳酸克鲁维酵母作为微生物细胞工厂的应用研究进展

乳酸克鲁维酵母（Kluyveromyces lactis）是一种典型的非常规酵母,在微生物基础研究和应用研究方面都有着非常重要的用途。该酵母具有食品安全级别高、蛋白分泌能力强、整合表达能力高效及大规模发酵能力优异等特点,因此,工业应用前景较为广阔。目前,乳酸克鲁维酵母作为蛋白表达系统已在食品和医药等行业中得到了广泛应用。近年来,国内外生物技术领域的科研人员以乳酸克鲁维酵母作为底盘细胞,利用合成生物学技术已经成功构建出了能够生产各种生化产品的微生物细胞工厂,该技术展示出了极大的发展潜力。本文主要对乳酸克鲁维酵母的菌种特点、合成生物学元件、遗传操作工具、基因编辑策略进行介绍,并综述乳酸克鲁维酵母作为细胞工厂的应用研究进展,可以为今后利用合成生物学方法在乳酸克鲁维酵母底盘中构建生产各种高附加值产品的高效微生物细胞工厂提供理论指导。