黑曲霉果胶裂解酶基因毕赤酵母在Pichia pastoris GS115中的表达

利用RT-PCR技术从黑曲霉(EIM-6)中扩增得到去除信号肽的果胶裂解酶基因A,将其插入到毕赤酵母表达载体pPIC9k上,构建重组表达质粒pPIC9K-pelA,电击转化毕赤酵母GS115,得到了表达成功的工程菌株。用终浓度为1.5%的甲醇对其进行诱导,将发酵上清液浓缩后,用盐酸法测定其酶活可以达到2.3U/mL。通过对重组毕赤酵母诱导表达产物进行SDS-PAGE鉴定,发现重组毕赤酵母分泌了1个约38kD的蛋白,与该酶基因产物的理论值相符,并通过水解圈法测定验证,均说明果胶裂解酶得到正确的分泌表达.     

共表达分子伴侣PDI和Ero1对灰盖鬼伞过氧化物酶在毕赤酵母中表达的影响

来源于灰盖鬼伞长度为1 092 bp的CiP目的基因与AOX1启动子一起整合进酵母染色体基因组中。重组蛋白CiP在酿酒酵母信号肽的引导下成功分泌到胞外,质谱鉴定为目的蛋白,成功在毕赤酵母中表达灰盖鬼伞过氧化物酶(CiP)。将伴侣蛋白内质网氧化还原酶1(Ero1)、二硫键异构酶(PDI)分别单独及同时转入CiP酵母受体菌中,研究它们对CiP在毕赤酵母中表达的影响。结果表明:在摇瓶中,相对于无分子伴侣的菌株,单独整合PDI及同时整合Ero1、PDI菌株的CiP酶活分别提高了2.43和2.62倍,活力达到316 U/m L和340 U/m L。挑选同时整合Ero1、PDI伴侣蛋白的CiP菌株,5 L发酵罐进行高密度发酵,酶活最高达到3 379 U/m L,比摇瓶提高约10倍。本实验结果较目前已报道的1 200 U/m L已是最高水平。     

不同信号肽对毕赤酵母表达漆酶的影响

【目的】从毕赤酵母X-33的内源分泌蛋白中寻找高分泌能力的信号肽,以提高漆酶POXA1c的表达水平。【方法】通过二维电泳的数据分析,以及毕赤酵母的基因组测序结果筛选出7种毕赤酵母中高分泌水平的蛋白,利用其引导漆酶的分泌,通过测定漆酶的活力,来确定信号肽的分泌能力。【结果】七种信号肽都能引导漆酶的分泌,其中PHO5和FLO10信号肽,其引导下漆酶POXA1c的活力分别是自身信号肽引导下的2.5倍和2倍。组合信号肽FLO10-αpro和PHO5-αpro引导下漆酶的活力分别为自身信号肽引导下的3倍和3.5倍,分别比在α-MF引导下提高了20%和40%。【结论】毕赤酵母内源分泌蛋白的信号肽可以有效的引导外源蛋白的分泌,漆酶POXA1c在PHO5-αpro信号肽引导下,通过高密度发酵培养后,其漆酶活力达到57.98 U/m L。     

嗜热毛壳菌CT2纤维二糖水解酶Ⅰ在毕赤酵母中的高效表达

嗜热毛壳菌ChaetomiumthermophilumCT2可产生具有重要工业生产价值的纤维素酶类。RT-PCR扩增cbh1成熟蛋白的编码基因,利用基因重组的方法构建可在毕赤酵母分泌表达系统中表达纤维二糖水解酶的重组表达载体,并转化毕赤酵母得到重组子。在毕赤酵母醇氧化酶AOX1基因启动子的作用下,重组蛋白得到高效表达,小规模发酵量达1.42mg/mL。表达蛋白分泌到培养基中,分子量约80kD;以脱脂棉为底物测得酶活为21U/mL。表达蛋白在60℃稳定,70℃保温60分钟仍保持90%的酶活力,具有较高的热稳定性。     

基因拷贝数对重组毕赤酵母产谷氨酰胺转胺酶的影响

基于毕赤酵母核糖体DNA序列 (rDNA),构建多拷贝谷氨酰胺转胺酶基因表达载体pPICZα-rDNA- mtg,并转化到表达前导肽 (Pro peptide或pro) 的宿主菌pGAP9-pro/GS115,得到共表达菌株pro/rDNA-mtg (GS115)。实时荧光定量PCR (qPCR) 分析了4株阳性表达菌株中mtg基因拷贝数,进一步研究了不同基因拷贝数对重组毕赤酵母产酶的影响及高产菌株在3 L发酵罐高密度发酵。结果表明,被检测的4株阳性表达菌株中mtg拷贝数分别为2.21、3.36、5.72和7.62 (mtg-2c、mtg-3c、mtg-6c和mtg-8c),其发酵产酶能力和蛋白质表达水平为mtg-3c>mtg-2c>mtg-6c>mtg-8c;高密度发酵较低和较高拷贝数的两株菌mtg-3c和mtg-6c,发酵上清的最高酶活和单位菌体酶活分别为3.12 U/mL、52.1 U/g湿重和2.07 U/mL、36.5 U/g湿重,其中单位菌体酶活mtg-3c是mtg-6c的1.4倍;mtg-3c纯化酶的最高酶活达到7.21 U/mL,蛋白浓度为437.2 μg /mL。通过分析拷贝数对重组毕赤酵母产酶的影响,发现mtg-3c适合pro/rDNA-mtg中pro和mtg共表达,MTG高酶活与菌株较高分泌蛋白有关。     

共表达HAC1和分子伴侣基因对甘露聚糖酶在毕赤酵母中表达的影响

为了提高甘露聚糖酶ManA在毕赤酵母中分泌表达的酶活,选择毕赤酵母内质网未折叠蛋白反应(Unfolded protein response,UPR)激活调控因子HAC1与5种毕赤酵母蛋白折叠相关的分子伴侣ERO1、PDI、PDI1、CPR5、BiP,通过构建pPICZA-HAC1等6种胞内表达重组质粒,分别电转化至分泌表达ManA的毕赤酵母重组菌中胞内共表达,并分析其重组菌摇瓶发酵时ManA表达的影响。结果发现在摇瓶发酵水平,胞内共表达HAC1、ERO1、PDI的重组菌发酵上清液中的ManA酶活力分别提高了26%、15%、20%,其重组菌发酵上清液的酶活力分别达到1 014 U/mL、925 U/mL、965 U/mL。通过对各重组菌上清液酶活力、胞内滞留酶活力、上清液蛋白浓度数据进行分析,进一步选择将HAC1、ERO1、PDI进行两基因或三基因组合,并分别在分泌表达ManA的重组菌胞内共表达,但各共表达重组菌发酵上清液的酶活力都没有进一步的提升。单独共表达HAC1或者分子伴侣ERO1、PDI可以辅助ManA的正确折叠,提高其蛋白表达。     

黄孢原毛平革菌木质素过氧化物酶基因在甲醇毕赤酵母中的表达

将编码黄孢原毛平革菌木质素过氧化物酶(lip)的cDNA克隆到酵母整合型质粒pMETA上,电转化Ade缺陷型甲醇毕赤酵母(Pichiamethanolica)PMAD16,通过MD平板及PCR方法筛选和鉴定重组子。重组子发酵液经SDSPAGE分析和木质素过氧化物酶活力测定等方法鉴定,表明带自身信号肽的黄孢原毛平革菌木质素过氧化物酶基因(lip)在甲醇毕赤酵母中得到表达。优化其发酵培养条件,以藜芦醇为底物进行酶活测定,其酶活可达932U/L。相应发酵指数为12.94U/h·L。比出发菌株提高了24.18%。     

信号肽序列对毕赤酵母表达外源蛋白质的影响

乙醇氧化酶启动子被分离、克隆 ,并建立了转化方法后 ,毕赤酵母已被发展成为一种高效的外源蛋白表达宿主。为了进一步提高外源蛋白质的分泌表达 ,对信号肽序列进行了研究。首先按毕赤酵母的偏爱密码合成了酿酒酵母的α因子信号肽序列MF4I,随后在MF4I信号肽序列的N端分别引入 1～ 10个毕赤酵母Aox1蛋白质的N端氨基酸 ,构成 10种不同的分泌信号肽序列 ,10种不同的分泌信号肽序列被用于植酸酶基因的毕赤酵母分泌表达。以上新的信号肽序列都可使植酸酶的分泌表达量增加 ,而以N端增加A、I、P三个氨基酸的信号肽序列引起的提高最大 ;和野生型的酿酒酵母α因子信号肽序列相比 ,使植酸酶分泌表达量增加 5倍 ,摇瓶中植酸酶的分泌表达量为 90mg/L。此外在MF4I信号肽的引导序列和内切蛋白酶间增加了EEAEAEAEP和K共 10个氨基酸 ,进一步提高信号肽的分泌效率 ,使表达又提高约 35 % ,使得摇瓶中酸性植酸酶的表达量达到 12 0mg/L ,是pPCI9K表达量的 8倍。     

来源于Escherichia coli的高比活植酸酶基因的高效表达

高效表达高比活植酸酶是进一步提高植酸酶发酵效价、降低植酸酶生产成本的一个有效途径。对源于Escherichiacoli的高比活植酸酶基因appA ,按照毕赤酵母 (Pichiapastoris)密码子的偏爱进行了密码子优化改造。该改造后的基因appA m按正确的阅读框架融合到毕赤酵母表达载体pPIC9上的α 因子信号肽编码序列 3′端 ,通过电击转化得到重组转化子。对重组毕赤酵母的Southernblotting分析证实植酸酶基因已整合到酵母基因组中 ,并确定了整合基因的拷贝数。Northernblotting分析证实植酸酶基因得到了正常转录。SDS PAGE分析和表达产物的研究表明 ,植酸酶得到了高效分泌表达 ,在 5L发酵罐中植酸酶蛋白表达量达到 2 5mg mL发酵液 ,酶活性 (发酵效价 )达到 7 5× 10 6 IU mL发酵液以上 ,大大高于目前报道的各种植酸酶基因工程菌株的发酵效价。     

采用混合策略提高葡萄糖氧化酶在毕赤酵母中的表达水平

为了提高葡萄糖氧化酶 (GOD) 在毕赤酵母中的表达水平,提出了甲醇/山梨醇混合碳源诱导和共表达分子伴侣二硫键异构酶 (PDI) 和透明颤菌血红蛋白 (VHb) 两种策略。利用对照菌株X33/pPIC9k–GOD 在5 L发酵罐放大培养时,采用甲醇/山梨醇混合碳源诱导,GOD最终酶活为456 U/mL,比只采用甲醇作为单一碳源诱导时GOD最终酶活提高了20%。利用整合伴侣蛋白菌株X33/pPIC9k-GOD/pPICZ-PDI-VHb在5 L发酵罐进行高密度发酵,采用甲醇/山梨醇混合碳源诱导,GOD最终酶活达到716 U/mL,蛋白浓度为7.4 g/L。研究结果对提高外源蛋白在毕赤酵母中的表达有重要参考价值。     

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.