人铜锌超氧化物歧化酶基因的克隆和在乳酸乳球菌中的表达

采用RT-PCR技术从人肝总RNA中分离扩增了0.45kb的人铜锌超氧化物歧化酶(Cu/Zn SOD)基因的cDNA序列,首先克隆至大肠杆菌表达质粒pET23b,进行了序列测定和超高表达。将Cu/Zn SOD cDNA亚克隆至乳酸乳球菌表达载体pMG36e,用电穿孔法将重组质粒pMG36esod转化到乳酸乳球菌中,获得Cu/Zn SOD的组成型表达,其表达量约占乳酸乳球菌可溶性蛋白的5%以上,活性染色表明该工程菌表达的Cu/Zn SOD具有较好的酶活性。     

Tfh 细胞在NOD小鼠糖尿病发病过程中的作用机制的研究

目的:研究滤泡辅助性T细胞(Follicular Helper T cell,Tfh)在非肥胖性糖尿病小鼠(Non-obese Diabetic mice,NOD)发病过程中的作用机制。方法:实验动物NOD小鼠按血糖值分为胰岛炎组(血糖浓度≤9 mmol/L)及糖尿病组(血糖浓度≥20 mmol/L)。ELISA法检测各组中糖尿病自身抗体谷氨酸脱羧酶抗体(65-kda glutamate decarboxylase antibody,GAD65Ab)、抗胰岛素自身抗体(Insulin autoantibody,IAA)表达水平,Western blot检测B细胞型淋巴瘤6蛋白(B-cell lymphoma 6 protein,Bcl-6)及可诱导共刺激分子(Inducible costimulatory molecule,ICOS)表达,流式细胞仪检测各组外周血及脾脏Tfh细胞水平。结果:糖尿病组NOD鼠自身抗体GAD65Ab(1.21±0.23 nmol/L)、IAA(0.96±0.12 nmol/L)浓度较胰岛炎组(0.32±0.09 nmol/L,0.25±0.06 nmol/L)均有明显升高;糖尿病组NOD鼠Bcl-6及ICOS表达较胰岛炎组NOD鼠有明显升高,外周血和脾脏Tfh细胞水平糖尿病组NOD鼠(24.55%)较胰岛炎组NOD鼠(4.27%)升高明显。结论:NOD小鼠自发糖尿病与自身抗体浓度升高相关,Tfh细胞可能参与NOD鼠糖尿病发生及发展过程。     

鼠李糖乳杆菌D-(+)-乳酸脱氢酶基因的克隆及在大肠杆菌中的表达

利用PCR的方法从鼠李糖乳杆菌基因组DNA中扩增到D-(+)-乳酸脱氢酶基因(ldhD),并连接到载体pSE380上,构建表达质粒pSE-ldhD,将重组质粒pSE-ldhD转化大肠杆菌BL21(DE3),重组菌株经IPTG诱导表达,SDS-PAGE电泳分析表明ldhD在大肠杆菌中实现了表达,表达产物的分子量约为37kD。同时采用紫外分光光度法测定D-乳酸脱氢酶的酶活,测得重组菌株的D-乳酸脱氢酶活力为5.4U/mL,最适反应温度为35℃,最适pH为5.6。     

硬脂酰-辅酶A脱氢酶基因在食品级乳酸菌中的表达

为了实现硬脂酰-辅酶A脱氢酶1编码基因在乳酸乳球菌中的表达,采用PCR技术扩增获得人类scd1的编码序列。Nco I和Xba I双酶切后定向插入到食品级表达载体pNZ8149中,构建表达载体pNZ8149-scd1。电转化乳酸乳球菌NZ3900,经菌落PCR和测序鉴定scd1基因成功插入到乳酸乳球菌中。在乳链菌肽诱导下进行scd1的表达,转化株提取脂肪酸,进行脂肪酸含量的气相色谱分析。结果显示,SCD1转化菌株中的C16∶1n-7和C18∶1n-7脂肪酸组分比转化pNZ8149的对照组乳酸菌分别提高了92%~169%和53%~127%。文中以scd1基因为例,尝试并证明了脂肪酸脱氢酶类基因能够在食品级乳酸菌中有效表达,为后续研究奠定了基础。     

转基因小鼠乳腺表达人胰岛素基因的研究

转基因动物乳腺生物反应器表达和生产医用蛋白是国际上的研究热点,目前已有很多成功的转基因动物乳腺表达出外源蛋白质［１］。在转基因动物乳腺表达的蛋白质包括凝血因子Ⅸ、组织纤溶酶原激活物（ｔＰＡ）、α１抗胰蛋白酶原、白介素２、蛋白质Ｃ、超氧化物歧化酶...     

Lactobacillus casei磷脂酶A_2基因在大肠杆菌中的重组表达

以Lactobacillus casei染色体基因组为模板,PCR扩增获得磷脂酶A2基因pla2,以pET-28a(+)为载体构建重组表达质粒pET-28a(+)-pla2。通过IPTG诱导实现磷脂酶A2在E.coli DE3中的重组表达。对诱导条件初步优化后,重组菌酶活最大可达2.8 U/mL。通过Ni-螯合柱对目的蛋白进行纯化,SDS-PAGE分析重组磷脂酶A2相对分子质量为1.7×104。通过酶学性质分析,最适温度为37℃,最适pH 8,比酶活为110 U/mg。     

变形链球菌致龋抗原基因在乳酸乳球菌的表达

目的 克隆变形链球菌葡聚糖结合蛋白B(GbpB)功能区的基因片段,并在乳酸乳球菌中表达.方法 在实验中利用了分子克隆技术构建携带GbpB基因的重组原核表达质粒pNI1,将重组质粒转化乳酸乳球菌YF02株,筛选鉴定阳性菌落,诱导表达的GbpB蛋白用SDS-PAGE进行鉴定.结果 成功克隆了GbpB功能区的基因片段,并在乳酸乳球菌中得到其融合蛋白的表达.结论 利用分子生物学技术能够成功克隆GbpB功能区基因并获得乳酸乳球菌融合蛋白的表达,为后续研究奠定了基础.     

乳酸菌素Gassericin T基因的合成及其在大肠杆菌中的融合表达

目的:在大肠杆菌系统中表达有抗菌活性的乳酸菌素Gassericin T。方法:根据乳酸菌素Gassericin T的基因序列,把Gassericin T的结构基因gatA编码的氨基酸的密码子转换成大肠杆菌偏爱的形式;用人工合成的寡核苷酸片段,通过重叠PCR法扩增得到gatA片段(gat基因);将合成的gat基因插入pGEX-4T-1,构建pGEX-4T-1-gat融合表达载体,转化大肠杆菌DH5α株,IPTG诱导表达,经超声裂解后获得包涵体蛋白,经溶解、变性、复性处理后获得GST-Gassericin T融合蛋白;用琼脂扩散法测定其对金黄色葡萄球菌、大肠杆菌、李斯特菌、枯草杆菌等的抗菌活性。结果与结论:采用pGEX-4T-1融合表达系统在大肠杆菌中表达了有活性的Gassericin T,融合蛋白以包涵体形式存在。复性的融合蛋白对金黄色葡萄球菌和大肠杆菌有明显的抑制作用,对李斯特菌的抑制作用不明显。     

人谷胱甘肽硫转移酶A1在乳酸乳球菌中的表达及活性研究

用RTPCR技术从人肝总RNA中分离扩增了人谷胱甘肽硫转移酶A1基因的cDNA序列,克隆至大肠杆菌表达质粒pET23b,采用蛋白表达筛查法及DNA测序证明该cDNA序列完全正确。重组质粒pET23bhgst转化大肠杆菌BL21(DE3),经IPTG诱导获得高效表达的可溶性hGSTA1产物,其表达量约为大肠杆菌可溶性总蛋白的40%。将hGSTA1cDNA亚克隆至乳酸乳球菌表达载体pMG36e,电穿孔法转化乳酸乳球菌MG1363获得hGSTA1乳酸乳球菌表达株。SDSPAGE及Western杂交分析表明该菌株表达预期大小的hGSTA1融合蛋白,经谷胱甘肽琼脂糖亲和层析纯化获得的hGSTA1蛋白具有较高的谷胱甘肽硫转移酶活性。具hGSTA1酶活性的乳酸乳球菌工程菌可望应用于研制防癌保健乳制品。     

糖尿病小鼠胚胎早期发育的差异表达基因

目的利用小鼠糖尿病模型,探讨母体糖尿病环境对早期胚胎基因表达的影响。方法ICR雌性小鼠腹腔注射150mg／kg剂量STZ诱发糖尿病,与正常雄鼠交配受孕,取14d胎龄的胚胎,提取胚胎的总RNA。将Cy3和Cy52种荧光分别标记到实验组和对照组的RNA上,制成RNA探针,并与包含24859个基因的表达谱芯片进行杂交及扫描,重复3次实验,采用Agilent扫描仪进行扫描软件读取数据。结果筛选出差异表达基因397个,其中有328个基因在实验组表达量比对照组大2倍,69个基因在实验组表达量比对照组小2倍。结论母体糖尿病环境能影响早期胎儿的基因表达,通过上调代谢相关基因和下调发育相关基因影响小鼠胚胎的早期发育。为深入探讨糖尿病胚胎病理和代谢疾病的分子机理提供了基本数据和研究的方向。     

不同质粒电转三种乳酸菌的比较研究

为比较研究不同质粒针对不同乳酸茵的电转效率的差异,分别以乳酸乳球菌NZ9000、干酪乳杆菌LC2W和植物乳杆菌WCFS1三种乳酸菌为受体,以七种不同质粒为载体,进行电转实验.结果表明在乳酸乳球菌NZ9000中,转化效率最高的是pIB 184质粒,达到了1.53×107 cfg/μgDNA,在干酪乳杆菌LC2W中,pSIP403质粒的电转化效率最高,达到了6.42×105 cfg/μgDNA,在植物乳杆菌WCFS1的电转化中,是pNZ44质粒达到8.68×105 cfg/μgDNA的最高转化效率.其中质粒pIB184和pNZ44在三种菌株中均有较高的电转化效率,超过了103 cfg/μgDNA,另一方面T-pAMS100、pSIP403、pSIP409三个质粒在干酪乳杆菌与植物乳杆菌中的电转化效率明显高于乳酸乳球菌.不同质粒针对不同乳酸茵的转化效率为乳酸茵的高效电转和表达栽体的选择与构建提供了可行依据.     

Food-grade gene expression in lactic acid bacteria

In the 1990s, significant efforts were invested in the research and development of food-grade expression systems in lactic acid bacteria (LAB). At this time, Lactococcus lactis in particular was demonstrated to be an ideal cell factory for the food-grade production of recombinant proteins. Steady progress has since been made in research on LAB, including Lactococcus, Lactobacillus and Streptococcus, in the areas of recombinant enzyme production, industrial food fermentation, and gene and metabolic pathway regulation. Over the past decade, this work has also led to new approaches on chromosomal integration vectors and host/vector systems. These newly constructed food-grade gene expression systems were designed with specific attention to self-cloning strategies, food-grade selection markers, plasmid replication and chromosomal gene replacements. In this review, we discuss some well-characterized chromosomal integration and food-grade host/vector systems used in LAB, with a special focus on sustainability, stability and overall safety, and give some attractive examples of protein expression that are based on these systems.     

Anchoring of proteins to lactic acid bacteria

The anchoring of proteins to the cell surface of lactic acid bacteria (LAB) using genetic techniques is an exciting and emerging research area that holds great promise for a wide variety of biotechnological applications. This paper reviews five different types of anchoring domains that have been explored for their efficiency in attaching hybrid proteins to the cell membrane or cell wall of LAB. The most exploited anchoring regions are those with the LPXTG box that bind the proteins in a covalent way to the cell wall. In recent years, two new modes of cell wall protein anchoring have been studied and these may provide new approaches in surface display. The important progress that is being made with cell surface display of chimaeric proteins in the areas of vaccine development and enzyme- or whole-cell immobilisation is highlighted.     

Characterization of bacteriocin-like inhibitory substances (BLIS) from sourdough lactic acid bacteria and evaluation of their in vitro and in situ activity

AIMS: To identify and characterize bacteriocion-producing lactic acid bacteria (LAB) in sourdoughs and to compare in vitro and in situ bacteriocin activity of sourdough- and nonsourdough LAB. METHODS AND RESULTS: Production of antimicrobial compounds by 437 Lactobacillus strains isolated from 70 sourdoughs was investigated. Five strains (Lactobacillus pentosus 2MF8 and 8CF, Lb. plantarum 4DE and 3DM and Lactobacillus spp. CS1) were found to produce distinct bacteriocin-like inhibitory substances (BLIS). BLIS-producing Lactococcus lactis isolated from raw barley showed a wider inhibitory spectrum than sourdough LAB, but they did not inhibit all strains of the key sourdough bacterium Lb. sanfranciscensis. Antimicrobial production by Lb. pentosus 2MF8 and Lc. lactis M30 was also demonstrated in situ. CONCLUSIONS: BLIS production by sourdough LAB appears to occur at a low frequency, showing limited inhibitory spectrum when compared with BLIS-producing Lc. lactis. Nevertheless, they are active BLIS producers under sourdough and bread-making conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of BLIS has been demonstrated in situ. It may influence the complex sourdough microflora and support the implantation and stability of selected insensitive bacteria, such as Lb. sanfranciscensis, useful to confer good characteristics to the dough.     

In situ activity of a bacteriocin-producing Lactococcus lactis strain. Influence on the interactions between lactic acid bacteria during sourdough fermentation

AIMS: To biochemically characterize the bacteriocin produced by Lactococcus lactis ssp. lactis M30 and demonstrate its effect on lactic acid bacteria (LAB) during sourdough propagation. METHODS AND RESULTS: A two-peptide bacteriocin produced by L. lactis ssp. lactis M30 was purified by ion exchange, hydrophobic interaction and reversed phase chromatography. Mass spectrometry of the two peptides and sequence analysis of the ltnA2 gene showed that the bacteriocin was almost identical to lacticin 3147. During a 20-day period of sourdough propagation the stability of L. lactis M30 was demonstrated, with concomitant inhibition of the indicator strain Lactobacillus plantarum 20, as well as the non-interference with the growth of the starter strain Lact. sanfranciscensis CB1. CONCLUSIONS: In situ active bacteriocins influence the microbial consortium of sourdough LAB and can "support" the dominance of insensitive strains during sourdough fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The in situ bacteriocinogenic activity of selected lactococci enables the persistence of insensitive Lact. sanfranciscensis strains, useful to confer good characteristics to the dough, at a higher cell concentration with respect to other LAB of the same ecosystem.     

Beneficial effect of oral administration of Lactobacillus casei strain Shirota on insulin resistance in diet-induced obesity mice

Aims: This study aimed at determining whether oral administration of a probiotic strain, Lactobacillus casei strain Shirota (LcS), can improve insulin resistance, which is the underlying cause of obesity‐associated metabolic abnormalities, in diet‐induced obesity (DIO) mice. Methods and Results: DIO mice were fed a high‐fat diet without or with 0·05% LcS for 4 weeks and then subjected to an insulin tolerance test (ITT) or oral glucose tolerance test (OGTT). Oral administration of LcS not only accelerated the reduction in plasma glucose levels during the ITT, but also reduced the elevation of plasma glucose levels during the OGTT. In addition, plasma levels of lipopolysaccharide‐binding protein (LBP), which is a marker of endotoxaemia, were augmented in the murine models of obese DIO, ob/ob, db/db and KK‐A^y and compared to those of lean mice. LcS treatment suppressed the elevation of plasma LBP levels in DIO mice, but did not affect intra‐abdominal fat weight. Conclusions: LcS improves insulin resistance and glucose intolerance in DIO mice. The reduction in endotoxaemia, but not intra‐abdominal fat, may contribute to the beneficial effects of LcS. Significance and Impact of the Study: This study suggests that LcS has the potential to prevent obesity‐associated metabolic abnormalities by improving insulin resistance.     

Analysis of promoter sequences from Lactobacillus helveticus CNRZ32 and their activity in other lactic acid bacteria

AIMS: To clone and analyse seven putative promoter fragments (pepC, pepN, pepX, pepO, pepE, pepO2, hsp17) from Lactobacillus helveticus CNRZ32 for their expression in Lact. helveticus CNRZ32, Lact. casei ATCC334 and Lactococcus lactis MG1363. METHODS AND RESULTS: Promoter fragments were fused to the promoter-less beta-glucuronidase (gusA) gene on pNZ272(RBS-) (ATG-). The resulting constructs were evaluated for their ability to drive the expression of active GusA with 0.5 mmol l(-1) 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. All promoters except P(pepN)::gusA were active in the examined strains. Northern hybridization was performed to examine the promoter strength. Sequence analysis of these promoters identified well conserved putative ribosomal binding and putative -10 hexamers sites. CONCLUSIONS: Seven promoter fragments from Lact. helveticus CNRZ32 were recognized in the lactic acid bacteria, Lact. casei ATCC334 and L. lactis MG1363, as well as in Escherichia coli. P(pepN)::gusA could not be maintained in the strains examined because of toxicity associated with heterologous protein over-expression driven by P(pepN). SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed that desirable levels of heterologous food-grade protein production in GRAS organisms can be obtained with the application of natural promoter fragments from closely related organisms.     

The role of lactic acid bacteria in accelerated cheese ripening

Abstract: The ripening of cheese is a slow and consequently an expensive process. The economic advantage of rapid development of more intense cheese flavour in shorter periods of time would be substantial. Lactic acid bacteria play a key role during ripening and can therefore be used as accelerating agents. This review describes the different strategies where lactic acid bacteria or their enzymes were used to reduce the ripening time of cheese. The advantages, limitations and technical feasibility as well as the commercial potential of the different approaches are also considered.     

Preventive effect of Ninjin-to (Ren-Shen-Tang), a Kampo (Japanese traditional) formulation, on spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice

We previously found that ingestion of an extract of Ninjin-to (NJT; Ren-Shen-Tang) suppressed the development of autoimmune diabetes in C57BL/KsJ mice induced by multiple low doses of streptozotocin. To verify this effects on spontaneous autoimmune diabetes, the effects of NJT on NOD mice were investigated in the present study. NJT, provided in drinking water (0.25%, 450 mg/kg/day) from 6 weeks of age, significantly prevented the incidence of spontaneous diabetes in female NOD mice at 30 weeks of age (2/10) compared with that of the controls (7/10), with no effects on body growth or food intake. Even in non-diabetic mice, the blood glucose levels of the NOD controls gradually increased with age, while such increase in NJT-treated mice was significantly suppressed by preventing any deficiency of glucose tolerance. NJT also significantly suppressed the progression of insulitis, which causes insulin deficiency and diabetes. It is well known that NOD mice develop insulitis and diabetes because of their Th1-dominant autoimmune response. IFN-gamma production from splenic T lymphocytes stimulated with anti-CD3 monoclonal antibodies was increased, whereas IL-4 production was decreased in NOD controls compared to age- and sex-matched normal ICR mice. NJT-treatment reduced these deviations of cytokine production in NOD mice. These data all suggest that NJT can prevent spontaneous insulitis and diabetes by the modification of deviated cytokine production in NOD mice.