Man8GlcNAc2糖基化的酿酒酵母菌株的构建

酿酒酵母糖蛋白的N-糖基化经过高尔基体的修饰后形成聚合度约150-200的甘露寡糖,高尔基体N-糖基化的糖基转移酶Mnn1p和Och1p在甘露寡糖的形成过程中起关键作用。通过同源重组置换敲除了酵母中的MNN1和OCH1基因阻断高尔基体N-糖基化修饰,分离纯化了mnn1 och1突变株中的N-糖蛋白,糖酰胺酶PNGaseF酶解释放的N-糖链经过2-氨基吡啶衍生后,利用HPLC和MALDITOF/MS结合的方法分析了突变株糖蛋白上的N-糖链。结果显示mnn1 och1突变株中的糖蛋白的N-糖链为结构单一的糖链,分子量为1794.66,推测为Man₈GlcNAc₂。     

重构酿酒酵母N-糖基化途径生产人源化糖蛋白

【目的】为了在酿酒酵母(Saccharomyces cerevisiae)中生产人源化的糖蛋白,必须对N-糖基化途径进行基因工程改造。作者通过敲除一些酵母N-糖基化途径中的特异性糖基转移酶,得到一株可以用于继续表达人类糖基转移酶的重组菌,并通过生长适应性进化技术回复其细胞生长能力。【方法】首先运用酵母遗传学和分子生物学技术敲除酿酒酵母的α-1,3-甘露糖基转移酶基因(ALG3)、α-1,6-甘露糖基转移酶基因(OCH1)和α-1,3-甘露糖基转移酶基因(MNN1)。采用蔗糖酶(invertase)活性染色实验初步检测N-糖链的变化,然后通过高效液相色谱和甘露糖苷酶酶切实验对其糖链结构进行鉴定。重组菌通过在高温条件下进行生长适应性进化,筛选出生长能力回复突变的菌株。【结果与结论】构建了Δalg3Δoch1Δmnn1菌株得到人类糖基化中间体Man5GlcNAc2,并对上述三缺陷型菌株进行适应性进化提高其细胞生长能力和环境适应能力。此外,作者还发现,该重组菌存在少量Man6GlcNAc2结构的糖链。经体外α-1,2-甘露糖苷酶切处理后,糖链Man5GlcNAc2和Man6GlcNAc2均转化为Man3GlcNAc2,表明形成Man3GlcNAc2之后的甘露糖之间均通过α-1,2-糖苷键连接。Δalg3Δoch1Δmnn1菌株的构建获得了生产人源化糖蛋白的酿酒酵母表达系统,为进一步糖基化改造和工业应用提供了良好的基础。     

毕赤酵母X-33OCH1基因敲除菌株的构建及其表达低糖基化蛋白GM-CSF

【目的】酵母表达外源糖蛋白时会对蛋白进行过度N-糖基化修饰,产生高甘露糖型糖链,影响蛋白的活性,其中α-1,6-甘露糖转移酶(och1p)在这一过程中起着关键作用。通过敲除毕赤酵母X-33的α-1,6甘露糖转移酶(och1p)基因,获得一个对糖蛋白进行低糖基化修饰的毕赤酵母表达系统。【方法】采用双交换同源重组敲除目的基因的方法,首先敲除毕赤酵母X-33的URA3基因,获得一个尿嘧啶营养缺陷型的X-33(ura3-)菌株;然后用URA3基因作为选择标记,敲除X-33(ura3-)的α-1,6甘露糖转移酶(och1p)基因,获得OCH1基因敲除的X-33(och1-)菌株。用X-33(och1-)菌表达糖蛋白GM-CSF,分析GM-CSF蛋白糖链的变化。【结果】首次成功敲除了X-33的URA3和OCH1基因,与野生型相比,X-33(och1-)菌表达的GM-CSF蛋白过度糖基化修饰程度明显降低。【结论】X-33(och1-)菌株的构建提供了一个对蛋白低N-糖基化修饰的毕赤酵母表达系统,也为进一步的糖基化改造提供了良好的基础。     

毕赤酵母X-33OCH1基因敲除菌株的构建及其表达低糖基化蛋白GM-CSF

摘要: 【目的】酵母表达外源糖蛋白时会对蛋白进行过度N-糖基化修饰,产生高甘露糖型糖链,影响蛋白的活性,其中α-1,6-甘露糖转移酶(och1p)在这一过程中起着关键作用。通过敲除毕赤酵母X-33的α-1,6甘露糖转移酶(och1p)基因,获得一个对糖蛋白进行低糖基化修饰的毕赤酵母表达系统。【方法】采用双交换同源重组敲除目的基因的方法,首先敲除赤酵母X-33的URA3基因,获得一个尿嘧啶营养缺陷型的X-33(ura3-)菌株;然后用URA3基因作为选择标记,敲除X-33(ura3-)的α-1,6甘露糖转移酶(och1p)基因,获得OCH1基因敲除的X-33(och1-)菌株。用X-33 (och1-)菌表达糖蛋白GM-CSF,分析GM-CSF蛋白糖链的变化。【结果】首次成功敲除了X-33的URA3和OCH1基因,与野生型相比,X-33(och1-)菌表达的GM-CSF蛋白过度糖基化修饰程度明显降低。【结论】X-33(och1-)菌株的构建提供了一个对蛋白低N-糖基化修饰的毕赤酵母表达系统,也为进一步的糖基化改造提供了良好的基础。     

利用伴刀豆球蛋白检测O-甘露糖基化

目的:基于伴刀豆球蛋白A(ConA)特异性识别并结合甘露糖的特性,建立一种检测O-甘露糖基化的方法,为酵母等宿主表达蛋白的O-糖基化提供一种高效筛选和分析的方法。方法:利用糖苷酶F(PNGF)切除检测蛋白的N-糖链,排除N-糖基化的干扰;通过Q阴离子交换柱和ConA Sepharose 4B柱纯化Western印迹膜封闭蛋白牛血清白蛋白(BSA),除去BSA中甘露糖修饰的蛋白的干扰,优化膜封闭条件;利用辣根过氧化物酶标记的ConA检测具有低甘露糖型N-糖基化修饰能力的毕赤酵母GJK01-HL(Δoch1)表达的抗Her-2抗体是否存在O-甘露糖基化现象。结果:通过PNGF酶切处理,可以完全去除糖蛋白的N-糖链的干扰;BSA经过Q阴离子交换柱和ConASepharose 4B柱纯化后,除去了大部分甘露糖蛋白,可作为封闭蛋白;用建立的方法检测,发现毕赤酵母工程菌GJK01-HL(Δoch1)表达的抗Her-2抗体存在O-甘露糖基化现象。结论:本方法是研究糖蛋白是否发生O-甘露糖基化的有效检测手段,可用于酵母等表达蛋白的O-糖基化的高效筛选和分析。     

酵母N-糖基化工程研究进展

酵母表达系统可用来生产具生物活性的重组糖蛋白,但其在N-糖基化过程中会生成高甘露糖型糖链。通过引入相关的甘露糖苷酶和糖基转移酶基因、切断酵母自身的高甘露糖链形成通道能够改变酵母宿主N-糖基化的类型。本对酵母N-糖基化工程的研究状况、最新进展及存在问题作简要阐述。     

α-1,6-甘露糖转移酶基因敲除的毕赤酵母菌株构建及其用于融合蛋白HSA/GM-CSF 表达的研究

酵母对蛋白的糖基化修饰过程不同于哺乳动物,其特点为产生高甘露糖型糖基且易发生过度糖基化。本研究通过两步基因重组敲除目标基因的方法成功敲除了毕赤酵母中的α-1,6-甘露糖转移酶(och1p)基因,获得了och1敲除的菌株。以此为基础,构建了高效表达人血清白蛋白与粒细胞-巨噬细胞集落刺激因子融合蛋白(HSA/GM-CSF)的工程酵母,与野生型毕赤酵母表达的过度糖基化HSA/GM-CSF不同,och1敲除菌表达的该融合蛋白糖基化程度明显降低,这为该融合蛋白的开发提供了重要基础。och1敲除菌株的构建不仅提供了一个对糖蛋白进行低糖基化修饰的毕赤酵母表达系统,而且为进一步的酵母糖基工程改造提供了基础。     

毕赤酵母蛋白质糖基化途径的改造

【背景】以酵母为宿主生产的蛋白往往发生过糖基化，形成高甘露糖型的N-糖基化。高甘露糖型的结构易在人体中引起免疫反应，这是酵母不能用于绝大部分糖蛋白药物生产的主要限制因素。因此，构建表达人源糖基化糖蛋白的酵母底盘细胞将为糖蛋白药物的生产提供强有力的工具。库德里阿兹威氏毕赤酵母(Pichia kudriavzevii)具有极强的抗逆性且生长迅速，是一种近年来备受关注的非典型性酵母，对其进行糖基化途径的改造将具有巨大的应用前景。【目的】对酵母N-糖基化途径的改造，首先要使其N-糖基化转变为Man₅GlcNAc₂核心结构，本研究对P. kudriavzevii的och1基因进行敲除并引入源自曲霉的msd S基因，以改变其分泌糖蛋白N-糖链的糖型结构。【方法】通过基因编辑对P. kudriavzevii的N-糖基化途径进行改造，获得P. kudriavzeviiΔura3Δoch1::msd S菌株，分析P. kudriavzeviiΔura3Δoch1::msd S菌株分泌糖蛋白上N-糖组的变化。【结果】与野生型P. kudriavzevii相...     

Man5GlcNAc2哺乳动物甘露糖型糖蛋白的毕赤酵母表达系统构建

蛋白的糖基化对蛋白的活性、高级结构及功能都有重要的影响。酵母表达的糖蛋白不同于哺乳动物表达的杂合型或复杂型糖蛋白,而是高甘露糖型或过度甘露糖化糖蛋白。在前期成功敲除毕赤酵母α-1,6-甘露糖转移酶(Och1p)基因、阻断毕赤酵母过度糖基化,获得毕赤酵母过度糖基化缺陷菌株GJK01 (ura3、och1) 的基础上,通过表达不同物种来源的α-1,2-甘露糖苷酶I (MDSI) 的活性区与酵母自身定位信号的融合蛋白,并通过DSA-FACE (基于DNA测序仪的荧光辅助糖电泳) 分析筛选报告蛋白HSA/GM-CSF (人血清白蛋白与粒细胞-巨噬细胞集落刺激因子融合蛋白) 的糖基结构,发现当编码酿酒酵母α-1,2-甘露糖苷酶 (MnsI) 基因的内质网定位信号与带有完整C-端催化区的拟南芥MDSI基因融合表达时,毕赤酵母工程菌株能够合成Man5GlcNAc2哺乳动物甘露糖型糖蛋白。这为在酵母体内合成类似于哺乳动物杂合型或复杂型糖基化修饰的糖蛋白奠定了基础。     

基于超滤膜辅助的糖蛋白全N-连接糖链的富集和质谱解析

糖基化作为一种常见的蛋白质翻译后修饰,对蛋白质的空间结构、生物功能等具有重要的影响.解析糖蛋白糖链结构有助于更清楚地认识糖蛋白及其功能.本研究建立了一种基于超滤膜富集血清中糖蛋白全N-连接糖链,并利用质谱技术对糖链结构进行分析的方法.根据糖蛋白及其糖链结构之间的分子质量差异,利用Millipore公司的10 ku超滤膜富集血清糖蛋白上酶解(PNGase F)释放的全N-连接糖链,并使用MALDI-TOF/TOF-MS解析糖链结构.通过该技术可以从血清中富集并鉴定到23种独特的N-连接的糖链结构,并且利用二级质谱进行了结构确认.该方法可以被用于从大量生物样本中富集糖蛋白全N-连接糖链,可以达到快速、高通量地解析糖蛋白N-连接糖链的目的.     

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     

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.     

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.     

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.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

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).     

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.