昆虫细胞内N-糖基化途径及人源化糖蛋白表达

虽然昆虫杆状病毒表达系统在蛋白表达领域得到了广泛的应用, 但由于不能表达复杂的末端唾液酸化的N-糖链, 使得该系统在生物制药行业的应用受到了很大的限制。通过比较哺乳动物细胞和昆虫细胞内糖基化途径可知, 其起始步骤一致, 之后再发生分化, 主要表现为3方面, 即昆虫细胞内缺乏哺乳动物细胞所具备的N-乙酰葡萄糖氨转移酶II、 半乳糖基转移酶/N-乙酰氨基半乳糖转移酶、α-2,3-唾液酸转移酶和α-2,6-唾液酸转移酶等延长N-糖链的糖基转移酶; 另外, 昆虫细胞内具有能够特异性地将蛋白质末端的N-乙酰氨基葡萄糖残基从GlcNAcMan3GlcNAc(±α3/6-Fuc)GlcNAc上切除的N-乙酰氨基葡萄糖苷酶及核心α-1,3-岩藻糖基转移酶。本文从上述异同出发, 综述了克服昆虫细胞内不能表达人源化糖蛋白这一缺陷所进行的N-糖基化途径的改造研究--主要集中在昆虫细胞内GlcNAcase的抑制和昆虫细胞内GnT2, GalT/ GalNAcT, ST3及ST6等基因的导入等方面, 结果表明经改造的昆虫细胞可表达人源化糖蛋白, 这将极大地拓宽昆虫杆状病毒表达系统的应用领域。本文还探讨了选择特殊细胞系及特殊培养条件以在昆虫细胞内表达唾液酸化蛋白的可行性。     

N-乙酰氨基葡萄糖转移酶干扰慢病毒载体系统的建立及应用

目的构建针对N-乙酰氨基葡萄糖转移酶（GnT）Ⅲ、Ⅳa和Ⅴ的RNA干扰（RNAi）慢病毒系统,并检测干扰慢病毒在体外小鼠肝癌细胞中对不同GnT表达的抑制作用。方法针对三种基因序列设计合成特异的shRNA序列,并构建干扰慢病毒表达载体,利用病毒包装细胞293T包装生产病毒,感染靶细胞Hca-F后,应用RT-PCR和免疫印迹检测干扰慢病毒对三种N-乙酰氨基葡萄糖转移酶表达的抑制。结果经测序证实三种干扰慢病毒表达载体构建成功,并获得高滴度的感染慢病毒。干扰慢病毒感染靶细胞后能够有效下调三种N-乙酰氨基葡萄糖转移酶的表达。结论干扰慢病毒可有效地抑制三种N-乙酰氨基葡萄糖转移酶GnT-Ⅲ、GnT-Ⅳa和GnT-Ⅴ的表达。     

细菌肽聚糖、磷壁酸的教学探讨

由于构成肽聚糖四肽链的氨基酸组成不同,交联方式又变化多端,从而不同种类细菌的肽聚糖结构多种多样。在描绘肽聚糖结构时要注意:1.四肽链第二位的D-谷氨酸与下一个氨基酸的氨基连接成肽键的是r-羧基,而不是α-羧基。2.交联主要是两条邻近的不同聚糖链骨架上与N-乙酰胞壁酸相连的两条相邻四肽链间的交联。3.G-细菌是两条四肽链直接交联,没有桥肽的参与。溶菌酶仅水解N-乙酰胞壁酸c-1和N-乙酰葡糖胺c-4之间的β-1,4糖苷键;而青霉素是抑制肽聚糖合成的最后阶段——转肽反应。由于两者的作用机理完全不     

高效表达N-乙酰高丝氨酸内酯酶-木聚糖酶融合蛋白及其酶学性质

摘要: 【目的】构建N-乙酰高丝氨酸内酯酶-木聚糖酶双酶活性表达毕氏酵母重组菌株,并对经纯化的重组蛋白SL2B 进行N-乙酰高丝氨酸内酯酶及木聚糖酶酶学性质的研究。【方法】利用PCR 拼接技术得到N-乙酰高丝氨酸内酯酶基因aiiA-B546 和木聚糖酶基因xynAS27cd 融合而成的基因Sl2b。构建重组表达载体pPIC9 / Sl2b 转化毕氏酵母,筛选得到同时具有木聚糖酶和N-乙酰高丝氨酸内酯酶活性的重组子,随后对经硫酸铵沉淀、分子筛纯化后得到的重组蛋白SL2B 进行N-乙酰高丝氨酸内酯酶及木聚糖酶     

双歧杆菌肽聚糖结构及分子量的分析

从双歧杆菌细胞壁中分离纯化肽聚糖,研究其化学成分、结构和分子量分析。经氨基酸、多糖和蛋白质含量分析和肽聚糖的溶解实验鉴定,所提取物质的主要成分为肽聚糖;经核磁共振和红外光谱分析,所提取肽聚糖的结构与已知的肽聚糖的结构相吻合,其糖链结构为由D型吡喃糖N-乙酰葡糖胺和N-乙酰胞壁酸以β-1,4-糖苷键连接而组成;经SDS-PAGE和多种染色方法结合,分析经溶菌酶水解后的肽聚糖,其分子量的分布呈弥散状,范围在97.6kD到14.4kD之间。     

UDP-GalNAc:多肽N-乙酰氨基半乳糖转移酶-14

UDP-GalNAc:多肽N-乙酰氨基半乳糖转移酶家族(简称GalNAc-T)是黏蛋白O-糖基化的起始酶,N-乙酰氨基半乳糖转移酶-14(GalNAc-T14)是该家族中最新发现的成员。近年来有人指出,O-糖基化可能与肿瘤的发生发展具有密切关系,因此对N-乙酰氨基半乳糖转移酶家族的研究也受到越来越多重视。本文主要综述了GalNAc-T14的命名、结构、分布、功能以及潜在的应用价值。     

钝齿棒杆菌中异源表达N-乙酰鸟氨酸脱乙酰基酶合成L-鸟氨酸的研究

目的:对一株产鸟氨酸的钝齿棒杆菌Corynebacterium crenatum SYPA5-5/△proB/△argF(SYPO-1)进行代谢工程改造,筛选不同细菌来源的N-乙酰鸟氨酸脱乙酰基酶在大肠杆菌中克隆与表达,纯化后对其进行酶学性质的比较;将黏质沙雷氏菌Serratia marcescens Y213来源的Smarg E基因编码的N-乙酰鸟氨酸脱乙酰基酶在L-鸟氨酸生产菌株C.crenatum SYPO-1中过量表达,进一步提高L-鸟氨酸的产量。方法:通过利用pDXW10穿梭质粒对不同来源的N-乙酰鸟氨酸脱乙酰化酶进行克隆表达和酶学性质比较,选择性质最优来源的N-乙酰鸟氨酸脱乙酰基酶编码基因Smarg E在产L-鸟氨酸重组钝齿棒杆菌中表达,考察重组菌株发酵过程中参数的变化。结果:来源于S.marcescens Y213的N-乙酰鸟氨酸脱乙酰基酶比酶活最高为798.98U/mg,最适pH为7,最适温度为37℃,0.1mmol/L的Mg~(2+)、Li~+、Mn~(2+)促进酶的比酶活提高了50%;在钝齿棒杆菌中表达N-乙酰鸟氨酸脱乙酰基酶酶活达到128.4U/ml,显著提高了钝齿棒杆菌中胞内乙酰基循环水平;5L发酵罐发酵重组菌株96h,L-鸟氨酸的产量达到38.5g/L,比出发菌株,L-鸟氨酸的产量提高了33.2%,产率达0.401g/(L·h)。结论:筛选出最佳来源的N-乙酰鸟氨酸脱乙酰基酶,在鸟氨酸生产菌株C.crenatum(SYPO-1)中过量表达,可以促进鸟氨酸的前体物质N-乙酰鸟氨酸的快速消耗,实现鸟氨酸的积累。     

N-乙酰半胱氨酸在改善糖尿病心肌病大鼠心功能中的机制研究

本研究以60只清洁级雄性SD大鼠为实验对象,通过分析健康大鼠(对照组)、Ⅱ型糖尿病大鼠(Ⅱ型糖尿病模型组)、不同剂量N-乙酰半胱氨酸治疗的Ⅱ型糖尿病大鼠(N-乙酰半胱氨酸低剂量组、中剂量组和高剂量组)的心功能、CHOP、NLRP3蛋白表达差异,来探讨N-乙酰半胱氨酸在改善糖尿病心肌病大鼠心功能中的作用机制。研究表明,糖尿病大鼠心功能参数及血流动力学参数存在明显的异常,心肌组织中PERK、CHOP、IL2、Bax、Caspase6、NLRP3蛋白表达明显升高而Bcl2的表达减弱非常明显,并且,与对照组以及Ⅱ型糖尿病的模型组相比,差异明显,差异有统计学意义(p0.05);而经N-乙酰半胱氨酸作用后上述异常指标明显恢复(p0.05),该结论表明抗氧剂N-乙酰半胱氨酸能通过抑制CHOP及NLRP3蛋白高表达来促进糖尿病大鼠心功能异常的改善。本研究揭示了N-乙酰半胱氨酸改善糖料病大鼠心功能的作用机理,为寻求有效治疗糖尿病的药物提供了理论支撑和研究方向。     

从大肠杆菌中表达、纯化宁乡猪N-乙酰谷氨酸合成酶并制备其多克隆抗体

N-乙酰谷氨酸合成酶催化生成的N-乙酰谷氨酸(NAGS)对于哺乳动物尿素循环第一个酶—氨基甲酰磷酸合成酶I变象异构激活是必需的。N-乙酰谷氨酸合成酶定位于肝脏和小肠线粒体基质中,通过提供N-乙酰谷氨酸调节氨基甲酰磷酸合成酶I的活性来调节尿素合成。我们用RT-PCR方法从宁乡猪肝脏中扩增了N-乙酰谷氨酸合成酶的开发阅读框,并将此基因连接到原核表达载体上,构建了pET-NAGS质粒。将重组质粒转化到Ec.oliBL21(DE3),在IPTG诱导下表达His-NAGS融合蛋白。通过SDS-PAGE,得出NAGS分子量约为40kDa。一步亲和层析纯化后,我们将纯化后的NAGS蛋白注射到新西兰大白兔中制备多克隆抗体。通过免疫组化和免疫印迹测试抗体,结果表明此抗体有较好的抗原性和特异性。据我们所知,这是第一次在大肠杆菌中表达来源于宁乡猪的NAGS。     

重组人溶菌酶研究进展

与其他来源的溶菌酶相比,人溶菌酶具有独特的优越性和多种多样的药理作用效果,在临床上具有多种重要应用价值。但天然人溶菌酶来源极其困难,利用重组DNA技术进行生产是解决这一难题的有效途径。迄今人们已利用化学合成或从人类细胞组织中制备cDNA等途径获取人溶菌酶基因,并在大肠杆菌、酵母菌和真菌表达系统中进行了表达,最高水平为40mgL,低于人乳中溶菌酶含量(50～250mgL),虽然距离工业化生产仍有一定距离。但重组人溶菌酶发展前景看好。     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失.自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的.     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.