乳酸菌食品级基因表达系统

酸菌是一类重要工业菌株。最近,乳酸菌遗传学和分子生物学的研究取得长足进步,导致发展了乳酸菌食品级基因表达系统。通过介绍乳酸菌食品级基因表达系统的基本要求、食品级选择性标记、食品级诱导物及该系统的研究进展,展示了乳酸菌食品级基因表达系统的建立对研究乳酸菌的基因表达调控和它的深层次的开发利用所具有的重要意义。     

乳酸菌食品级载体选择标记

食品级载体不依靠抗生素抗性基因作为选择标记,因而更为安全。近年来,人们已构建了多种乳酸菌的食品级克隆载体。这些食品级克隆载体是在显性选择标记和互补选择标记的基础上建立的。最近提出了第三种新的方法。该就这三种食品级载体选择标记的功能、分子机制及应用前景作了简明的介绍。     

食品级乳酸菌表达系统研究进展

乳酸菌表达系统是近几年发展起来的食品级高效表达系统。乳酸菌具有益生菌特征,因此该表达系统与其他细菌表达系统相比有很多优点。介绍了糖诱导表达系统、噬菌体Φ31爆发式诱导的表达系统、乳链球菌素调控表达系统、温控表达系统等的研究进展,以及这些系统的应用前景。     

食品级高效诱导表达系统-NICE系统

乳酸菌NICE系统是在乳链菌肽诱导下由nisA启动子控制目的基因表达的,含nisR和nisK的两组分调节系统的高效诱导表达系统。由于NICE系统的诱导剂、宿主菌和载体都是食品级的,其应用前景十分广阔。     

乳酸菌食品级表达载体的研究与应用

乳酸菌是能够发酵糖类产生大量有机酸的革兰氏阳性菌的通称,在发酵食品中有着悠久的应用历史.乳酸菌通常被认为是安全菌株,这些微生物的基因工程操作在食品、医学等方面具有广阔的应用前景.表达载体是基因工程中常用的工具之一,大多数乳酸菌的表达载体通常以抗生素抗性基因作为选择标记,然而抗性基因具有潜在的转移性,因此需要开发食品级表...     

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。     

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。     

Nisin production of Lactococcus lactis N8 with hemin‐stimulated cell respiration in fed‐batch fermentation system

In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed‐batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed‐batch fermentation system with high fidelity (R² 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L^?1 h^?1, 3 μg mL^?1 and 40%, respectively. While 1711 IU mL^?1 nisin was produced by L. lactis N8 in control fed‐batch fermentation, 5410 IU mL^?1 nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed‐batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed‐batch fermentation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:678–685, 2015     

Inhibition of tyrosine-sensitive 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase by glyphosate in Candida maltosa

Abstract A gene block controlling sucrose-fermenting ability, nisin resistance and nisin production was found to be transmissible by a conjugation-like process. The 'pSN' (sucrose nisin) plasmid was transferred from 8 different nisin-producing donor strains into MG1614, a plasmid-free derivative of Streptococcus lactis 712. In the new host low yields of a plasmid of approx. 30 MDa were isolated but its authenticity as a pSN plasmid has not yet been established. Possibilities for increased nisin yield by genetic manipulation in S. lactis 712 must exist.     

16S rRNA和recA、groEL基因分类鉴定乳酸乳球菌乳酸亚种和乳脂亚种的比较

【目的】比较16S rRNA和recA、groEL基因部分序列用于乳酸乳球菌乳酸亚种和乳脂亚种分类鉴定的效果。【方法】对已鉴定的8株分离自传统发酵乳的乳酸乳球菌, 选取recA和groEL基因片段, 通过PCR扩增、测序, 将测序得到的序列比对后构建系统发育树, 并与16S rRNA基因序列分析技术进行比较。【结果】比较分析不同菌株16S rRNA和recA、groEL基因的亲缘关系, recA、groEL基因可以准确地完成乳酸乳球菌乳酸亚种和乳脂亚种的区分和鉴定。【结论】recA和groEL基因序列分析可以实现乳酸乳球菌乳酸亚种和乳脂亚种的区分, 因其具有快速、准确、稳定的特点, 可适合于乳酸乳球菌乳酸亚种和乳脂亚种间的快速分类鉴定。     

苯丙氨酸脱氨酶在乳酸乳球菌NICE系统的高效表达及其实验动物研究

为构建苯丙氨酸脱氨酶的一种更新更高表达的基因工程菌,取得治疗高苯丙氨酸血症大鼠的更佳疗效。将欧芹PAL cDNA重组到质粒pNZ8048中,构建翻译融合载体和转录融合载体,分别转化乳酸乳球菌NZ9000。对两种高效表达菌株的酶活性差异进行比较,观察Nisin诱导的动态进程,并利用新鲜培养的p(NZ8048-PAL)₁/NZ9000工程菌进行了高苯丙氨酸血症大鼠(称PKU动物模型)的治疗实验。结果表明：（1）翻译融合型菌株具有更高的酶活性;（2）当Nisin的诱导时间达6h时, 产物肉桂酸生成量基本达到峰值;（3）给高苯丙氨酸血症大鼠灌喂新鲜培养的p(NZ8048-PAL)₁/NZ9000工程菌（翻译融合型）, 观察到该工程菌能显著降低模型动物血中Phe浓度,从而取得了更好的疗效。     

利用PCR技术克隆乳链菌肽前体基因

乳链菌肽(Nisin)是由核糖体合成,经翻译后修饰、加工而含有稀有氨基酸的一种小肽分子。它对许多革兰氏阳性菌,包括利斯特氏菌属、梭菌属和芽孢杆菌属这些腐败苗和病原菌有强烈抑制作用。 已被50多个国家和地区广泛应用于乳制品、罐头食品、高蛋白食品及乙醇饮料的防腐保鲜,是越来越 受到人们重视的一种无毒的天然食品防腐剂⑴。对乳链菌肽分子结构与功能及生物合成遗传控制的研 究不仅有应用价值,而且有其理论意义。本实验室以产乳链菌肽的乳酸乳酸球菌ATCC 11454总DNA 为模板,利用PCR技术成功地扩增了乳链菌肽前体基因,并对其核苷酸序列进行了测定。     

抗艰难梭菌毒素纳米抗体在乳酸乳球菌中重组表达及其功能分析

本研究旨在构建表达抗艰难梭菌毒素的纳米抗体重组乳酸乳球菌。所述的抗体为免疫羊驼后从外周血中分离的天然缺失轻链的纳米抗体（分别命名为E3、AA6）。构建重组质粒pNZ8148- E3-AA6,转化乳酸乳球菌NZ9000后诱导表达,SDS-PAGE及Western blot检测到32 kD的目的蛋白。利用镍柱亲和层析法纯化重组蛋白E3-AA6,BCA试剂盒测定其浓度。通过细胞变圆实验以及CCK8实验鉴定其功能,E3-AA6能够抑制艰难梭菌毒素TcdB的毒性。本实验成功构建了重组表达载体pNZ8148- E3-AA6,通过NICE系统实现了二价抗体的表达,为治疗艰难梭菌引发的感染提供了新的治疗途径。     

Heterologous expression of azurin from Pseudomonas aeruginosa in food-grade Lactococcus lactis

Abstract

In this study, azurin, a bacteriocin with anticancer property, was produced by food-grade Lactococcus lactis using the Nisin Controlled Gene Expression (NICE) System. In addition, the antibacterial and cytotoxic properties of recombinant azurin in the culture supernatant were also investigated. Azurin gene from Pseudomonas aeruginosa was cloned into the pNZ8149 vector and the resulting recombinant DNA was transformed into food grade L. lactis NZ3900. The expression of azurin protein was induced by the optimum concentration of nisin for 3?h. Inhibition zones for Escherichia coli and Bacillus cereus were observed at 5.0 and 10?mg/mL concentrations of lyophilized supernatants containing azurin, but no inhibition zone at azurin-free lyophilized supernatants. When HUVEC, HT29, HCT116, and MCF7 cell lines were treated with lyophilized culture supernatants with azurin or without azurin, cell viability decreased with increasing concentrations of the supernatant. Furthermore, the supernatants containing azurin showed more anti-proliferative effect than the azurin-free supernatants. This work provides a practicable method to produce recombinant azurin in the food grade L. lactis strain. As a result, the recombinant L. lactis strain, producing azurin, can be used in the investigation of food biopreservatives and in the development of a therapeutic probiotic.     

多肽抗生素apidaecin基因在乳酸乳球菌中的融合表达

利用乳链菌肽(nisin)诱导表达系统,以泛素(ubiquitin)融合蛋白的形式在乳酸乳球菌(Lactococcus lactis)中表达了多肽抗生素apidaecin。利用TricineSDSPAGE和Western blotting均可在诱导后的宿主菌中检测到特异蛋白带。表达产物的最高产量可达宿主菌可溶性蛋白的7.2%左右。在体外用泛素特异性蛋白酶UBPI从融合蛋白中切除泛素后,产物具有明显的抗菌活性。