Characterization of an i-type lysozyme gene from the sea cucumber Stichopus japonicus, and enzymatic and nonenzymatic antimicrobial activities of its recombinant protein

Because sea cucumbers lack a well-developed immune system and can ingest pathogenic bacteria together with food, some form of active antibacterial substances must be present in the body for defense. In this study, the cDNA of an i-type lysozyme from the sea cucumber Stichopus japonicus (designated SjLys) was cloned by RT-PCR and RACE PCR techniques. The full length cDNA of SjLys was 713 bp with an open reading frame of 438 bp coding for 145 amino acids. Two catalytic residues (Glu34 and Asp47), conserved in i-type lysozymes, and a highly conserved region near the active site, MDVGSLSCG(P Y)(Y F)QIK, were detected in SjLys. In addition, the domain structure analysis of SjLys showed that it is highly similar to the medicinal leech destabilase, which belongs to a new phylogenetic family of invertebrate lysozymes possessing both glycosidase and isopeptidase activities. To gain insight into the in vitro antimicrobial activities of SjLys, the mature peptide coding region was heterologously expressed in Escherichia coli. The recombinant SjLys protein displayed an inhibitive effect on the growth of the tested Gram-positive and Gram-negative bacteria. A remarkable finding is that the recombinant SjLys exhibited more potent activities against all tested bacterial strains after heat-treating at 100 °C for 50 min. These results indicated that the S. japonicus lysozyme is an enzyme with combined enzymatic (glycosidase) and nonenzymatic antibacterial action.     

植物乳杆菌天然质粒分类

为建立植物乳杆菌天然质粒分类方法,以质粒复制起始蛋白(replication initiation protein,Rep)作为分类标记,通过系统进化树分析方法,将植物乳杆菌53个编码Rep天然质粒划分为6个质粒类型,包括5个质粒家族和1个新的复制类型质粒,之后进一步提出了每个质粒家族特有的骨干序列,最终建立了一种简单、有效的植物乳杆菌天然质粒的分类方法和标准。与以往质粒功能和不相容性分类方法相比较,本方法具有较好的实用性和通用性。     

The regulatable MAL32 promoter in Saccharomyces cerevisiae: characteristics and tools to facilitate its use

Here we describe a set of tools to facilitate the use of maltose and the MAL32 promoter for regulated gene expression in yeast, alone or in combination with the GAL1 promoter. Using fluorescent protein reporters we find that under non‐inducing conditions the MAL32 promoter exhibits a low basal level of expression, similar to the GAL1 promoter, and that both promoters can be induced independently of each other using the respective sugars, maltose and galactose. While their repression upon glucose addition is immediate and complete, we found that the MAL32 and GAL1 promoters each exhibit distinct induction kinetics. A set of plasmids is available to facilitate the application of the MAL32 promoter for chromosomal modifications using PCR targetting and for plasmid based gene expression. Copyright © 2016 John Wiley & Sons, Ltd.     

重组质粒DNA D-GPEi工程菌发酵工艺的优化

目的优化重组质粒D-GPEi工程菌发酵工艺条件。方法控制相关发酵参数,观察溶解氧浓度、比生长速率和培养时间对工程菌生长和质粒浓度的影响。结果发酵培养对数生长期溶解氧浓度控制在30%~50%。补料前比生长速率为0.5~0.7/h,6 h开始补料,比生长速率下降,12 h后比生长速率为0.1~0.2/h。连续发酵3批工程菌,21 h后收集菌体,得到菌体平均产量为89.4 g/L(A=52.6),质粒平均产量为359.8 mg/L。3批工程菌浓度和质粒拷贝数差异无显著意义。结论此发酵工艺得到工程菌浓度和质粒拷贝数较高,重复性好,适用于大规模工业化生产。     

重组人白细胞介素-31表达条件的优化

目的优化重组人白细胞介素-31(rhIL-31)的表达条件。方法以不同浓度IPTG、不同诱导时间、不同诱导温度对含有pET32a/rhIL-31的工程菌E.coliBL21(DE3)进行诱导表达,用SDS-PAGE对表达产物进行分析。结果诱导时间为5h,IPTG浓度为1.5mmol/L,诱导温度为37℃时,rhIL-31的表达量最高。结论已初步优化了重组蛋白rhIL-31的表达条件。     

重组戊型肝炎疫苗抗原工程菌发酵工艺的优化

目的优化重组戊型肝炎疫苗抗原工程菌的发酵工艺,为其规模化生产奠定基础。方法以目的蛋白的表达量及菌体浓度作为综合评价指标,对重组戊型肝炎疫苗抗原工程菌的摇瓶发酵温度、初始pH值、溶解氧、IPTG浓度及诱导时间进行优化;以摇瓶发酵结果为基础,进一步对发酵罐培养工艺中诱导时机、诱导时间及补料培养基配方进行优化;以确定的罐发酵工艺条件连续发酵5批,验证该发酵工艺。结果最适摇瓶发酵工艺条件为:发酵温度为37℃,初始pH值为7.0,高溶氧,IPTG诱导浓度为0.05 mmol/L,诱导时间为4 h;最适罐发酵工艺条件为:菌体A600值约为40时开始诱导,诱导时间为4 h,补料培养基配方为葡萄糖25%,MgSO4.7H2O 0.5%。按照确定的工艺连续发酵5批,最终菌体A600均达50.0以上,目的蛋白的表达量均高于15%。结论优化的重组戊型肝炎疫苗抗原工程菌的发酵工艺稳定性良好,已达到中试生产规模的要求。     

重组氨基酰化酶1与N-乙酰鸟氨酸脱乙酰酶酶学性质比较

利用基因工程手段重组表达了牛肾来源的氨基酰化酶1(ACY1),并与实验室之前构建的N-乙酰鸟氨酸脱乙酰酶(NAO)进行比较,以N-乙酰-DL-蛋氨酸为底物,研究了其酶学性质,考察了pH、温度、反应时间、表面活性剂、金属离子等因素对拆分DL-蛋氨酸的影响。结果表明,重组NAO和ACY1拆分300 mmol/L的N-乙酰-DL-蛋氨酸所需时间分别是2 h和6 h;重组NAO的酶活及N-乙酰-L-蛋氨酸的转化率分别为1 139.41 U/g和98%,高于重组ACY1的671.24 U/g和58.78%,约是重组ACY1的1.7倍。     

Expression of recombinant Ara h 6 in Pichia pastoris but not in Escherichia coli preserves allergic effector function and allows assessment of specific mutations

Scope: Ara h 6 has recently been recognized as an important peanut allergen. Recombinant allergens have been used for analysis of IgE binding, but have not been used to analyze the allergic effector activity that is more relevant to allergic reactions. Methods and results: Ara h 6 was expressed as a recombinant protein in both Escherichia coli and Pichia pastoris (rAra h 6‐E. coli and rAra h 6‐Pichia, respectively). Effector activity was assayed by measuring degranulation of RBL SX‐38 cells sensitized with IgE from patients with severe peanut allergy. Compared to native Ara h 6 (nAra h 6), rAra h 6‐Pichia had intact effector function whereas rAra h 6‐E. coli had significantly reduced function. The lower effector activity in rAra h 6‐E. coli compared to nAra h 6 and rAra h 6‐Pichia did not appear to be due to differences in posttranslational modificatons (analyzed by mass spectrometry and staining for carbohydrates) and may be due to subtle alteration(s) of folding seen on CD analysis and on nonreduced gels. Finally, we introduced point mutations in four important IgE‐binding linear epitopes of Ara h 6 and found dramatically reduced allergic effector activity. Conclusion: Our studies demonstrate the utility of fully functional rAra h 6‐Pichia as a starting point for analysis of specific mutations that adversely affect allergic effector function.     

嘌呤核苷磷酸化酶不同表达方式对发酵利巴韦林的影响

嘌呤核苷磷酸化酶(PNPase,由pup G基因编码)是合成利巴韦林的关键酶,本研究考察该酶的质粒过表达和基因组整合表达对鸟苷生产菌解淀粉芽孢杆菌(Bacillus amyloliquefaciens)TA208发酵生产利巴韦林的影响。以B.amyloliquefaciens TA208为出发菌株,分别利用基因过表达和无标记整合技术,成功构建了B.amyloliquefaciens TA2081和TA2082。通过实时定量PCR测定这3株菌pup G基因的转录水平,结果表明与B.amyloliquefaciens TA208相比,TA2081和TA2082的转录水平均有提高,但TA2081提高的更为显著;通过测定PNPase活性发现,B.amyloliquefaciens TA2081和TA2082的PNPase活力分别为出发菌株TA208的2倍和1.30倍。7.5 L分批补料发酵实验表明,与出发菌B.amyloliquefaciens TA208相比,工程菌TA2081鸟苷到利巴韦林的摩尔转化率从20.41%提高到98.63%,TA2082的转化率从20.41%提高到40.95%。综上所述,增加pup G的表达量能提高利巴韦林的产量,本研究为利巴韦林生产菌的代谢工程改造提供了理论依据和技术指导。     

一株食源性多重耐药单核细胞增生李斯特菌的耐药性研究

本研究对食品样本中分离的一株多重耐药单核细胞增生李斯特菌进行耐药机制的探讨,以期对食源性单核细胞增生李斯特菌多重耐药现象的控制提供理论依据。本文通过聚合酶链式反应筛选耐药决定因子,质粒消除及自然转化实验对耐药决定因子进行定位及传播能力的探讨,最后通过传代实验验证该菌株多重耐药性传播的稳定性。结果表明,对检测到的多重耐药菌株LM78(耐受氯霉素、红霉素、链霉素、四环素、复方新诺明)进行相关耐药基因检测,检测到cat、erm B、tet S 3个耐药基因。质粒消除后MIC值下降到敏感范围,且该质粒可通过自然转化在不同菌属间传递,说明这些耐药基因存在于质粒上。该质粒在无抗生素选择压力下连续传代,仍具有较高稳定性。食源性致病菌多重耐药性有可能通过不同细菌种属间转移,进而由食物链向人类传播,对人类健康造成潜在的威胁。