对双歧杆菌DM9227株的实验研究 Ⅲ.双歧杆菌DM9227株与蜡样芽胞杆菌DM423株共生关系的初步研究

本文对厌氧的双歧杆菌DM9227株与需氧的蜡样芽胞杆菌DM423株的共生关系进行了初步研究。将DM9227株与DM423株分别单独及按一定比例等量混合接种于BS肉汤内进行培养,间隔一定时间检测两菌在培养基内菌数的消长情况。结果表明DM423株与DM9227株之间呈偏生关系,即DM423株对DM9227株有促进作用,且在两菌比例适宜(1:100)情况下促进作用更明显,而DM9227株对DM423株有抑制作用。进一步将接种DM423株的Nb琼脂平板与接种DM9227株的BS琼脂平板在普通环境下一起密闭培养,可见厌氧的DM9227株生长良好,提示DM423株可能是通过消耗氧气,降低培养基的Eh,从而促进DM9227株生长的。     

产脲酶芽胞杆菌的微波诱变育种

巴氏芽胞杆菌是目前微生物诱导碳酸钙沉淀(MICP)方法中应用最为热门的一种细菌。为提高巴氏芽胞杆菌尿素分解以及矿化能力,以巴氏芽胞杆菌YB-B为出发菌株,采用微波诱变育种技术,通过诱变菌株特性筛选及其遗传稳定性检测,成功选育出2株突变菌株YB-3和YB-4。与出发菌株相比,诱变菌株尿素分解能力较原菌株提高1.5倍左右,矿化能力提高114%。诱变菌株具有生长速度快,环境适应性强,矿化能力高等优点,这为MICP更广层次的应用奠定了坚实的基础。     

耐唑类药白念珠菌ERG11基因点突变

ERG11是唑类抗真菌药对白念珠菌作用靶酶羊毛甾醇14α-去甲基化酶（14DM）的编码基因,ERG11基因的有义突变会使靶酶发生氨基酸置换,影响酶与药物分子的结合,是导致菌株耐药的原因之一。14DM具有特定的空间构型,突变所处的位置决定能否导致菌株耐药,已发现的60余种ERG11点突变里面,仅有数种被实验证实可致耐药。     

酸奶生产菌株的诱变选育

通过诱变选育高产酸力菌株;首先采用紫外线和亚硝基胍对乳酸菌进行单因素诱变,确定紫外线照射剂量为150s,并得到一株产酸力为57.85°T的菌株,比原菌株产酸力提高了3.05%;NTG的诱变剂量是0.3mg/mL,处理时间为60min,得到产酸力为58.02°T的菌株,较出发菌株提高了6.52%。接着以紫外线和NTG对高产酸菌株进行了2轮复合诱变,最终得到产酸力达83.66°T的诱变株,较出发菌株产酸力提高了30.64%。     

球孢白僵菌几丁质酶诱发突变的探讨

从球孢白僵菌Beauveria bassiana 017菌株获得了几丁质水解活性较高的突变株。诱变原为紫外线及高能电子束。诱变体为分生孢子及芽生孢子。通过测定在几丁质琼脂平板上透明圈的大小、透明圈直径与菌落直径之比值,选出酶高产菌株,并通过摇瓶培养选育出EU-120高酶活性突变株,其几丁质酶活性比原始菌株提高了近3倍。并发现以上诱变原均能获得较好的诱变效果,在选育过程中加入猩红液可提高透明圈的清晰度。并通过比较几丁质酶对胶体几丁质及“晶体”几丁质的活性,探索酶调节的可能机理。     

产纤溶酶少根根霉菌株的诱变筛选

目的:通过对自南方小酒药中筛选得到的1株产纤溶酶的少根根霉Or株的诱变筛选,提高原有菌株的产酶能力。方法:以Or为出发菌株,进行亚硝基胍、紫外线、Co60诱变,以血纤维蛋白平板法为检测方法,筛选高产酶突变株。结果:经诱变传代后得到高产突变株8B,其产酶活性稳定为291.05U/mL,为原菌株产酶活力的6.34倍。该菌在血琼脂平板上不产生溶圈,诱变后孢子成熟提前8h。结论:物理诱变和化学诱变交替应用,能显著提高少根根霉Or株单位体积发酵液的产酶量,并能缩短孢子的成熟周期。该菌不具有溶血性,与已有报道的产纤溶酶的菌株不同。     

miR-423-3p通过靶向SUFU调节结肠癌细胞对5-Fu的敏感性

目的 研究miR-423-3p在结肠癌5-氟尿嘧啶(5-Fu)耐药中的作用和机制.方法 逆转录实验分析5-Fu耐药结肠癌组织和细胞中miR-423-3p的表达水平,MTT和流式细胞实验分析抑制miR-423-3p对5-Fu耐药结肠癌细胞活性和凋亡的影响,双荧光素实验检测5-Fu耐药结肠癌细胞中miR-423-3p的靶基...     

抗性突变他克莫司高产菌株的选育

研究利用核糖体工程育种方法结合紫外诱变处理产他克莫司链霉菌SIIA-9818,以期筛选得到发酵水平有较大提高的新菌株。首轮采用链霉素抗性筛选,第二轮组合链霉素和利福平抗性结合紫外诱变育种,进一步巩固育种成效。采用链霉素抗性诱变得到突变株T-122,其气生菌丝丰满程度明显好于原对照株,发酵效价提高22.8%;采用组合链霉素和利福平抗性结合紫外诱变T-122,得到多株高产菌株,其中H-493发酵水平较原出发菌株提高82.6%;在50 L发酵罐通过增大搅拌转速,使菌种H-493发酵单位进一步提高31.0%。本方法简单经济,得到的突变株发酵单位显著提高,组分无明显变化,传代稳定。     

紫外线和硫酸二乙酯复合诱变选育PHB高产菌株

【目的】获得高产聚β-羟基丁酸酯(Poly-β-hydroxybutyricacid,PHB)菌株。【方法】以假单胞菌属Pseudomonas koreensis PK3菌株为出发菌株,采用硫酸二乙酯和紫外线相结合的诱变方法进行多轮诱变。【结果】经过初筛和复筛得到一株高产PHB突变菌株,命名为Pseudomonas koreensis UVCN-18。连续传代9次后,发酵28 h条件下,PHB产量达到15.94 g/L,占细胞干重69.54%,较原出发菌株Pseudomonas koreensis PK3(4.42 g/L)提高了2.61倍,并且该菌株具有良好的遗传稳定性。【结论】采用硫酸二乙酯和紫外线相结合的诱变方法,成功获得了一株高产PHB突变菌株。     

乳酸菌DM9054、DM9057的安全性评价

目的实验评价的2株乳酸菌DM9054和DM9057均分离于泡菜,分别具有降胆固醇和降血压功能,由于其功能性强,性状优良,故本实验对其安全性进行评价,为应用于食品药品生产提供参考依据。方法通过急性毒性试验、耐药性、机体指标、有害代谢产物的评价试验以及其他安全性评价试验检测两实验菌株的安全性。结果在一次性分别使用DM9054和DM9057大剂量灌胃后,动物体征指标无异常;两菌株对部分抗生素具有抗性,但不携带可转移的耐药质粒;使用实验菌株后,机体指标MDA、GSH和SAA的变化基本正常;菌株代谢不产生胆盐羟化酶、硝基还原酶和氨基脱羧酶,但产生乳酸;无溶血作用,长期使用,不发生易位现象。结论乳酸菌DM9054和DM9057总体上是安全的。     

固氮红细菌(Rhodobacter azotoformans) 423 nm特征吸收峰成因与定位

【背景】在不产氧光合细菌中,因420-425nm特征峰位于类胡萝卜素(Carotenoid,Car)吸收区域,通常被认为是由Car积累引起,但固氮红细菌R7菌株呈现的423 nm特征峰不具备Car三指峰特征。【目的】阐明R7菌株423 nm特征吸收峰形成的物质基础及胞内定位。【方法】采用吸收光谱、薄层层析、高效液相色谱、质谱、超速离心和离子交换层析等方法阐明423 nm吸收峰形成原因。【结果】谷氨酸钠明显促进R7菌株活细胞呈现423 nm特征峰,色素提取液中该峰蓝移至415 nm,但其生长、细菌叶绿素(Bacteriochlorophyll,BChl)和Car含量大幅度降低,而添加酵母提取物则反之。色素组成分析表明,在检测到的色素成分中,只有镁卟啉单甲基酯Ⅸ (Magnesium Protoporphyrin Ⅸ Monomethylester,MPE)呈现415 nm特征吸收峰。MPE可定位于光合膜上并呈现出423 nm特征峰。对色素蛋白复合体(Pigment Protein Complex,PPC)的研究显示,添加谷氨酸和酵母提取物的菌体细胞虽然都检测到3种PPC组分[2个外周捕光复合体(Peripheral Light Harvesting Complex 2,LH2)和1个光反应中心(Reaction Center,RC)],但源自谷氨酸菌体细胞的RC和1个LH2则呈现423 nm特征吸收峰,表明R7菌株可产生2种不同类型的LH2,且MPE可定位于一种LH2和RC。【结论】R7菌株所呈现的423 nm特征峰不是由Car积累所致,而是由MPE积累所形成,且能与LH2和RC结合定位于光合膜上。MPE是BChl合成的中间产物,其合成受严格调控,不容易获得。MPE代谢调控的深入研究可为光合作用光氧化损伤与保护机理增添新内容。     

Knockdown of miR-423-5p simultaneously upgrades the eNOS and VEGFa pathways in ADSCs and improves erectile function in diabetic rats

This study aimed to explore the possibility of miR-423-5p modified adipose-derived stem cell (ADSCs) therapy on streptozotocin (STZ)-induced diabetes mellitus erectile dysfunction (DMED) rats. MiR-423-5p was knocked down in ADSCs. ADSCs, NC-miR-ADSCs and miR-ADSCs were co-cultured with human umbilical vein endothelial cells (HUVECs). Normal and high glucose media were supplemented. The supernatant and HUVECs were collected for assessment of eNOS and VEGFa expression, cell proliferation, and apoptosis. HUVECs co-cultured with ADSCs or miR-ADSCs exhibited higher eNOS and VEGFa protein expression levels compared to DM groups. MiR-ADSCs enhanced HUVEC proliferation compared to the ADSCs and NC-miR-ADSCs. Lower apoptotic rates were observed when HUVECs were co-cultured with miR-ADSCs, compared to ADSCs and NC-miR-ADSCs. Fifteen male Sprague-Dawley (SD) rats aged 12 weeks were induced to develop diabetes mellitus by intraperitoneal injection with STZ, and five healthy SD rats were used as normal controls. Eight weeks after developing diabetes, the rats received ADSCs and miR-ADSCs via injection into the corpora cavernosa, whereas normal controls and DM controls were injected with saline. Erectile function and histological assessment of penile tissues were performed 8 weeks after injection. The ICP/MAP indicated that erectile function was impaired in the DM rats compared with the normal group. Injection of ADSCs and miR-ADSCs improved erectile function significantly and was associated with the overexpression of eNOS and VEGFa. MiR-423-5p knockdown in ADSCs ameliorated high glucose-mediated damage to HUVECs and improved erectile function in DM rats by inducing eNOS and VEGFa overexpression, indicating that miR-423-5p may be a potential target in the treatment of DMED.     

一株溴氰菊酯降解菌的分离鉴定及其降解条件优化

【背景】拟除虫菊酯类农药的降解已成为食品安全和环境卫生领域的研究热点,而生物降解被认为是一种绿色高效的解决方法。【目的】从长期受拟除虫菊酯类农药污染的草莓根系土壤分离一株溴氰菊酯(deltamethrin,DM)降解菌,并优化其培养基及降解条件,从而提高DM降解菌的降解效率。【方法】采用富集驯化、分离纯化法筛选DM降解菌,通过形态学和生理生化特征,以及16S rRNA基因序列分析进行鉴定。通过Plackett-Burman因素筛选试验、最陡爬坡试验和Box-Behnken试验优化菌株降解条件。【结果】筛选获得一株DM降解菌LH-1-1,96h对DM(100mg/L)的降解率为53.43%,经鉴定为琼氏不动杆菌(Acinetobacter junii);通过优化后,在DM浓度75mg/L、胰蛋白胨3 g/L、pH值6.8、硫酸铵1.5 g/L、氯化铁0.01 g/L、接种量为5%(体积比)、菌龄12 h、培养温度30℃条件下,菌株LH-1-1对DM降解率达82.36%,较未优化前提高了28.93%。【结论】A. junii LH-1-1具有较高的DM降解能力,该菌可为生物修复受DM或拟除...     

Adhesion of Lactobacillus plantarum 423 and Lactobacillus salivarius 241 to the intestinal tract of piglets, as recorded with fluorescent in situ hybridization (FISH), and production of plantaricin 423 by cells colonized to the ileum

AIMS: To determine which intestinal section of pre and postweaned piglets are colonized by Lactobacillus plantarum 423 and Lactobacillus salivarius 241, and follow production of plantaricin 423 in a gastro-intestinal model. METHODS AND RESULTS: Lactobacillus plantarum 423 and Lact. salivarius 241, single or in combination, were administered to 1-, 14- and 28-day-old (postweaned) piglets. According to results obtained by fluorescent in situ hybridization (FISH), Lact. plantarum 423 adhered strongly to the ileum and posterior colon and Lact. salivarius 241 to the duodenum in preweaned piglets. High numbers of strain 241 were recorded in the duodenum and posterior colon of postweaned piglets, whereas strain 423 remained localized to the ileum. Lowering in Enterococcus faecalis cell numbers were recorded when preweaned piglets were challenged with strain 241. Plantaricin 423 was produced for 96 h in the ileum section of a gastro-intestinal model. CONCLUSIONS: Lactobacillus plantarum 423 and Lact. salivarius 241 adhere to different sections of the intestinal tract, depending on the piglet's age. Ent. faecalis were inhibited in vivo, probably by plantaricin 423. SIGNIFICANCE AND IMPACT OF THE STUDY: Fluorescent in situ hybridization proved valuable in the detection of probiotic bacteria adhered to the intestine. This is the first report of bacteriocin production in a model simulating the porcine gastro-intestinal tract.     

人胰岛素基因在乳酸菌中的表达及其对非肥胖糖尿病(NOD)小鼠的作用

为实现人胰岛素基因在乳酸菌中的表达及探索其用作口服疫苗治疗Ⅰ型糖尿病(T1DM)的可行性,首先将人胰岛素基因密码子替换为乳酸菌偏爱密码子,同时在A,B链序列间加入连接短肽序列,经引物退火拼接合成人胰岛素基因。克隆至乳酸菌表达载体中后,利用电击转化法实现了带信号肽SPUsp45的人胰岛素基因在乳酸乳球菌(Lactococcus lactis)MG1363和干酪乳杆菌(Lactobacillus casei)ATCC27092中的表达。Western blot检测显示重组胰岛素位于细胞壁上,当菌体生长到OD600为0.4时达到最大表达量。用含有表达重组人胰岛素的Lactobacillus caseiATCC27092/pSW501菌体饲喂非肥胖糖尿病(NOD)小鼠,发现可刺激小鼠产生特异性抗体,同时使与免疫耐受相关的细胞因子IL-4水平明显升高(38.583±2.083pg/mL,P<0.05),提示其对NOD小鼠产生免疫耐受有一定的作用,为研制乳酸菌口服疫苗防治T1DM的可行性进行了有益的探索。     

对回春生生产菌种DM8504菌株的研究总结报告

本文对回春生生产菌种双歧杆菌ＤＭ８５０４株的研究作了总结报道。ＤＭ８５０４林是青春型双歧杆菌的一个变种,该菌株具有明显的生态效应,对调整肠道菌群失调有明显的效果。该菌淋经过试管内、动物体内及人体内试验证明是一个理想的微生态调节剂生产菌株。通过实验和临床观察证明,该菌株对降低血内毒素,治疗肝炎等多种疾病均有明显的辅助治疗作用。该菌株是从健康人的大便标本经分离培养并加以驯化等复杂过程而获得的生产菌株。     

对双歧杆菌DM9227菌株的实验研究IV对双歧杆菌DM9227菌株生物学特性的研究

本文对双歧杆菌ＤＭ９２２７菌株进行了形态学、生化反应特性、代谢酸分析及抗生素敏感性的研究。综合有关试验结果,初步认为该菌属婴儿双歧杆菌（Ｂｉｆｉｄｏｂａｃｔｅｒｉｕｍｉｎｆａｎｔｉｓ）。     

Molecular cloning of the Pseudomonas putida glyoxalase I gene in Escherichia coli

The glyoxalase I gene of Pseudomonas putida was cloned onto a vector plasmid pBR 322 as a 7.5 kilobase Sau 3AI fragment of chromosomal DNA and the hybrid plasmid was designated pGI 318. The gene responsible for the glyoxalase I activity in pGI 318 was recloned in pBR 322 as a 2.2 kilobase Hin dIII fragment and was designated pGI 423. The P. putida glyoxalase I gene on pGI 318 and pGI 423 was highly expressed in E. coli cells and the glyoxalase I activity level was increased more than 150 fold in the pGI 423 bearing strain compared with that of E. coli cells without pGI 423. The E. coli transformants harboring pGI 318 or pGI 423 could grow normally in the presence of methylglyoxal, although the E. coli cells without plasmid were inhibited to grow and showed the extremely elongated cell shape.