人三叶因子1大肠杆菌及毕赤酵母表达载体的构建与鉴定

目的:构建人三叶因子1(hTFF1)的大肠杆菌及毕赤酵母表达载体。方法:从人胃窦部提取总RNA,经RT-PCR得到hTFF1 cDNA,用PCR方法扩增hTFF1基因,并将其分别克隆到大肠杆菌的表达载体pET32α及毕赤酵母的表达载体pCAPZαA中,构建原核及真核重组表达质粒pET32α-hTFF1和pGAPZαA-hTFF1。结果:通过双酶切和基因序列分析确定插入pET32α和pGAPZαA中的片段为hTFF1基因片段。结论:重组质粒pET32α-hTFF1和pGAPZαA-hTFF1成功构建,为在比较原核和真核细胞中hTFF1高效表达的情况及下一步的功能研究奠定了基础。     

人肝再生增强因子在毕赤酵母中的表达、纯化和生物学活性的研究

肝再生增强因子（ALR）是一类胞源性肝细胞生长因子。为在毕赤酵母中分泌表达人肝再生增强因子（rhALR）,以色谱法分离纯化后进行体外活性研究,构建表达载体pPICZαA- ALR,经电穿孔转入毕赤酵母中,用0.5％甲醇诱导表达;重组酵母培养上清经SDS-PAGE电泳和western blot鉴定后表明, rhALR以分子量为30kD的二聚体为主;定量分析结果表明,重组酵母培养上清中rhALR约占总蛋白的66％,表达量约为40mg/L;经DEAE柱和G75柱纯化后,获得的rhALR纯度大于95％,得率为52％;体外生物学活性实验表明,rhALR能明显促进HepG2、SMMC-7721和NIH-3T3细胞的增殖。     

人GDNF在甲醇酵母及家蚕幼虫中的表达

将人胶质细胞源性神经营养因子(GDNF)基因克隆入酵母分泌型表达载体pPIC9K中,酶切线性化后电穿孔导入酵母细胞进行整合,经G418筛选得到多拷贝转化子,甲醇诱导表达。将人GDNF基因克隆入昆虫病毒转移载体pBacPAK8中,与线性化Bm-BacPAK6修饰病毒基因组DNA共转染家蚕细胞,经体内重组,筛选到重组病毒。用重组病毒感染家蚕幼虫,5d后收集血淋巴。SDS-PAGE和蛋白质印迹杂交结果证实了酵母培养上清液及家蚕幼虫血淋巴中含有GDNF蛋白。活性研究表明,甲醇酵母及家蚕幼虫表达的GDNF蛋白能促进多巴胺能神经元的存活和突起生长。     

人三叶因子3在毕赤酵母中表达条件的研究

为提高人三叶因子 3 (HumanTrefoilfactor 3 ,hTFF3 )在毕赤酵母中的表达量 ,研究了转化子生长的培养条件 ,包括不同碳源对转化子生长的影响和接种量、甲醇浓度、pH值、摇瓶转速及不同诱导时间对人三叶因子 3表达的影响。结果表明转化子在生长阶段加入葡萄糖生长旺盛 ,培养 14h后OD₆₀₀ 就可达到 50。在 100mL生长培养基上的菌液以 1∶1接入诱导培养基时蛋白表达量最高 ;转化子在 1%的甲醇、pH60、摇瓶转速240r/min的条件下诱导4 8h ,菌体密度OD₆₀₀为 15 ,目的蛋白表达量达到 20mg L。用 5L发酵罐进行了高密度发酵 ,经2%甲醇32h诱导 ,最终菌体密度OD₆₀₀ 达到 120 ,每升发酵液中含目的蛋白100mg。     

人可溶性gp190在酵母Pichia pastoris中的表达

人可溶性gPl90(sgp190)是白血病抑制因子(LIF)的可溶性受体,可能在LIF行使众多生物学功能中扮演着重要的角色。为获得这一蛋白以研究它在人体内的生物学活性,在酵母Pichia pastoris中实现了重组sgp190分泌表达。利用基因重组技术,将编码人可溶性gP190(LlF受体α亚基gP190胞外区)cDNA克隆到毕赤酵母Pichia pastoris分泌表达载体pPIC9K,构建了重组表达质粒pPIC9K-sgp190。原生质体法转染Pichia pastoris GS115菌株,经过G418筛选,得到了高效分泌表达sgp190蛋白的毕赤酵母菌株GS115／pPIC9K-sgp190,sgp190蛋白占摇瓶培养表达上清中总蛋白质的26％。经初步纯化,对表达产物的性质鉴定表明,其分子量约125kD,具有免疫活性。对sgp190体外活性分析表明它能够抑制LIF诱导滋养层细胞分泌hCG。     

人αA干扰素在克鲁氏乳酸酵母中的表达和分泌

pE1是由酵母天然质粒pKD1衍生出来的重组穿梭质粒,在克鲁氏乳酸酵母中具有高拷贝,高稳定性等特点。把人αA干扰素分泌表达单元克隆到pE1载体中,得到分泌型表达质粒pE-IFN1。pE-IFN1在克鲁氏乳酸酵母中相当稳定,在非选择性培养基中生长50世代后,大多数酵母细胞仍带有质粒。结果表明,分泌表达单元中的酿酒酵母α因子的分泌信号肽能被克鲁氏乳酸酵母的蛋白质分泌系统所识别,人αA干扰素被分泌到细胞外。在摇瓶培养条件下每升发酵液中含有1—2毫克αA干扰素。     

玉米叶绿体偶联因子ε亚基基因在E.coli中的表达

玉米叶绿体偶联因子ε亚基基因（ａｔｐＥ）被克隆到载体ｐＪＬＡ５０５和ｐＷＡ的多聚接头处,形成重组质粒ｐＪＬＡ５０５－ａｔｐＥ和ｐＷＡ－ａｔｐＥ,转化Ｅ．ｃｏｌｉ,并进行温敏诱导表达研究。在大肠杆菌中温敏诱导的基因表达产物经ＳＤＳ－ＰＡＧＥ测定,表达量达全菌蛋白质的３０％以上．Ｗｅｓｔｅｒｎ－ｂｌｏｔ分析结果表明温敏诱导的表达产物可特异性地与ε亚基抗体反应,在免疫双扩散中也观察到沉淀线。大肠杆菌表达的玉米叶绿体ａｔｐＥ基因产物以包含体形式存在于细菌中。经对包含体处理后;获得的粗产物可达８０％以上纯度,并具有天然ε亚基蛋白质的生物功能．     

利用两种不相容质粒在大肠杆菌中共表达DFF45和DFF40

DNA断裂因子（DNA fragmentation factor,DFF）是细胞凋亡过程中起重要作用的蛋白质之一,它由分子量为45kD和40kD的两个亚基构成,分别称为DFF45和DFF40。利用RT－PCR技术从人宫颈癌细胞系HeLa的总RNA中扩增了DFF45和DFF40的cDNA,分别克隆到到卡那霉素抗性表达载体pET-28a( )中,构建了pET28a-DFF45和pET28a-DFF40。用它们单独转化大肠杆菌BL21（DE3）后,经IPTG诱导都可获得高效表达。表达的重组蛋白质各自约占菌体总蛋白质的56％和22％。再将DFF45的cDNA克隆到氨苄青霉素抗性表达载体pET-21a( )中,得到了pET21a-DFF45。利用二者的不同抗性,将pET28a-DFF40和pET21a-DFF45共同转化大肠杆菌BL21（DE3）,工程菌经IPTG诱导后实现了DFF45和DFF40的共表达,表达产物各约占菌体总蛋白质的30％和17％。为了研究这两种不相容质粒在细菌中共存的稳定性,我们将共转化子在同时含有卡那霉素和氨苄青霉素的液体培养基中连续培养14h,发现此时仍有75％以上的细菌可同时耐受两种抗生素,即同时含有pET28a-DFF45和pET28a-DFF40,说明利用两个具有不同抗性的不相容载体进行蛋白质共表达的方法是可行的。     

人胰岛素原基因在酵母中的分泌表达

 我们研究了外源基因——合成的人胰岛素原基因在酵母α因子系统中的表达和分泌。用表达载体YTI-15转化酵母63号株可得到每升约2毫克的人胰岛素原分泌产物。     

人Tumstatin在毕赤酵母中的表达和活性分析

利用PCR技术从重组质粒pET-3c-tum中扩增人tumstatin的cDNA片段,连入pPICZαA酵母表达载体,获得的重组质粒pPICZα-tum电激法转化毕赤酵母GS115。经表型鉴定、诱导表达筛选,得到可分泌表达人tumstatin的重组酵母转化子,表达蛋白质的相对分子量约30kD,表达量约25mg/L。表达上清经超滤浓缩和离子交换法初步纯化,所得产物具有免疫活性,能够抑制内皮细胞增殖,诱导其发生细胞凋亡,并能抑制鸡胚尿囊膜血管生成。     

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.