重组人淋巴毒素随机点突变组合文库的构建

构建重组人淋巴毒素（rhLT）随机点突变组合文库以进行体外分子进化及结构和功能的研究。应用含随机核苷酸序列的引物,通过Overlap PCR的方法分别对rhLT 的46、106和130位氨基酸进行定点随机突变,获得各单点随机突变体库。通过基因操作将这三个单点随机突变体库拼接并克隆于Pmd_18T载体建立三点组合突变体文库,DNA测序鉴定突变位点的随机性和多样性,原核表达该变异体库,体外测定生物学活性。成功获得rhLT三点随机点突变组合文库,其转化克隆数达到1.5×10⁵,是多样性理论值的4.5倍。50个样品的序列分析显示各个位点的核苷酸和氨基酸序列的突变都呈随机性分布。对原核表达的30个样品进行生物学活性测定,结果70%(21个)的样品无活性、23.3%(7个)的样品活性低于rhLT、6.7%(2个)的样品活性高于rhLT。成功构建了rhLT随机点突变组合文库,该库不仅在一级结构上具有良好的随机性和多样性,而且具有生物学活性的多样性,为应用噬菌体展示等高通量筛选策略对淋巴毒素进行体外分子进化和结构与功能的深入研究打下了基础。     

低毒且高效杀伤人肿瘤细胞的新型人肿瘤坏死因子突变体的构建

采用定点突变的方法构建了两株新型人肿瘤坏死因子突变体MT1 (32Trp157Phe)和MT2(2Lys30Ser32Trp157Phe), 并在大肠杆菌中得以高效表达, 表达量占菌体总蛋白的50%且为可溶性蛋白. 表达产物经蛋白印渍法检测, 均能与抗野生型hTNF-a单抗结合且经分离纯化后样品的纯度在95%以上. 各突变体虽然对鼠成纤维瘤细胞L929的生物学活性比野生型下降了4～5个数量级, 但却能高效杀伤人肿瘤细胞, 杀伤程度与野生型相差不明显. 测定突变体MT1的LD50值降至野生型的0.34%, 而MT2对小鼠30%致死剂量则低于野生型LD50的1/700.     

低毒且高效杀伤人肿瘤细胞的新型人肿瘤

采用定点突变的方法构建了两株新型人肿瘤坏死因子突变体MT1 (32Trp157Phe)和MT2(2Lys30Ser32Trp157Phe), 并在大肠杆菌中得以高效表达, 表达量占菌体总蛋白的50%且为可溶性蛋白. 表达产物经蛋白印渍法检测, 均能与抗野生型hTNF-a单抗结合且经分离纯化后样品的纯度在95%以上. 各突变体虽然对鼠成纤维瘤细胞L929的生物学活性比野生型下降了4~5个数量级, 但却能高效杀伤人肿瘤细胞, 杀伤程度与野生型相差不明显. 测定突变体MT1的LD50值降至野生型的0.34%, 而MT2对小鼠30%致死剂量则低于野生型LD50的1/700.     

噬菌体显示技术改造人干扰素α1c/86D的研究

利用噬菌体表面显示技术表达具生物活性的干扰素α1c,并通过构建和筛选突变文库 ,以得到高受体结合能力和高比活性的新α1c干扰素 .利用噬菌粒载体pCANTAB5E表达了人IFNα1c/ 86D基因 ,利用随机 6肽库和干扰素的中和抗体推导干扰素α1c的受体结合区及关键性结合残基 ,运用盒式突变构建了基于AB环的突变库 ,其中氨基酸 2 9,3 1,3 2 ,3 5位完全随机化 ,并直接利用WISH细胞选择出高抗病毒活性的干扰素突变体 .结果获得了 3个重组噬菌体干扰素突变体 ,其抗病毒活性是干扰素母体的 4～ 16倍 .这一结果为国内外首次报道利用噬菌体表面显示技术的筛选表达优势改造干扰素以提高抗病毒活性 ,实验结果说明了其可行性 ,并提示干扰素突变体的比活性有可能较母体高 .     

从噬菌体展示肽库中筛选脂质A的结合肽

以脂质A为靶标,筛选噬菌体展示十二肽库,三轮后随机挑取14个噬菌体克隆进行结合活性鉴定,并对5个克隆进行序列分析。结果表明,14个克隆全部是阳性克隆,测序结果显示4个阳性克隆的序列完全一样。说明筛选到了一个脂质A的结合多肽。     

保守的第52位色氨酸突变引起的胰高血糖素样肽1受体N端片段活性丧失

通过易错PCR方法建立了一个鼠肺不同长度的nGLP-1R(从第21个氨基酸开始到第145个氨基酸)的噬菌体随机突变展示肽库,通过噬菌体表面展示技术检测胰高血糖素样肽1受体N端片段(nGLP-1R)在缺失一段或两段基因后是否还具有结合Exendin-4的活性.经ELISA分析发现了一株无结合活性的突变株,命名为EP16.经测序比对,发现EP16缺失了前20个和后10个氨基酸,且第52位色氨酸突变为精氨酸.为确定EP16与Exendin-4无结合活性的原因,重新构建了无前20个和后10个氨基酸的EP16野生型及第52位色氨酸变为精氨酸的全长nGLP-1Rw52R与EP16进行对比分析.结果表明,EP16的活性丧失是由保守的第52位色氨酸突变为精氨酸引起的,缺失的前20个和后10个氨基酸没有影响其生物学活性.关键位点单个氨基酸残基的突变可以改变胰高血糖素样肽1受体N端片段整个蛋白质的生物学活性.     

利用噬菌体展示肽库筛选鸡传染性支气管炎病毒模拟抗原表位

目的：利用噬菌体展示肽库技术筛选鸡传染性支气管炎病毒（IBV）的模拟抗原表位。方法：用IBV阳性血清纯化IgG作为靶标。对噬菌体展示随机12肽库进行筛选，通过ELISA和竞争抑制ELISA鉴定筛选克隆的结合特性，并对阳性克隆提取ssDNA进行测序分析。结果：3轮生物淘洗后，目标噬菌体得到125倍富集。随机挑选50个克隆进行ELISA和竞争抑制ELISA。其中有12个噬菌体克隆可以与IBV阳性血清高特异性结合。测序分析发现，这12个克隆带有2种氨基酸序列。即KSPKHSSSALHF和SFFQLNLHRPTS。且未发现这2种序列与GenBank中已发表的IBV氨基酸序列有同源性。结论：结果提示。这2个肽可能是IBV抗原的模拟表位。     

具有GCSF活性的噬菌体展示肽

从NFS 6 0细胞中克隆了小鼠粒细胞集落刺激因子 (granulocytecolony stimulatingfactor,G CSF)受体的细胞因子受体同源区 (cytokinereceptorhomologous ,CRH)结构域 ,采用GST融合表达策略 ,实现了CRH结构域的表达 .以纯化的GST CRH融合蛋白为靶 ,从噬菌体随机环七肽库中筛选CRH结构域的结合肽 ,找到两组具有核心序列的噬菌体展示肽 .其中C2和C13噬菌体展示肽能刺激NFS 6 0细胞增殖 ,说明C2和C13噬菌体展示肽具有G CSF活性     

噬菌体展示HIV-1 Tat38-61碱性区51和55位随机突变体文库的构建

为了构建噬菌体展示Tat38-61(51N/55N) 碱性区突变体文库,进一步研究HIV-1 Tat38-61表位的分子进化筛选,采用含随机核苷酸序列的引物,通过Overlap PCR的方法获得51和55位氨基酸随机突变的全长Tat编码序列,再以此为模板PCR扩增出两端含有Xba I识别序列的Tat38-61突变体片段HIV-1 Tat38-61(51N/55N),克隆至噬菌体展示载体pCANTAB5S上,转化大肠杆菌TG1,经M13K07辅助噬菌体拯救,构建噬菌体展示Tat38-61(51N/55N) 碱性区突变体文库。结果显示文库的库容量为5.0×106,滴度为2.65×1012 TU/mL,阳性克隆率为56.50％;序列分析显示文库中51、55位核苷酸与氨基酸均呈随机性分布,达到了对文库进行分子进化筛选的要求,为获得可用作疫苗候选物的新型Tat突变体奠定基础。     

从抗TNF抗体的CDR肽库中获得拮抗TNF的短肽

为设计来自抗体的短肽 ,以抗肿瘤坏死因子 (TNF)嵌合抗体 (cA2 )CDRs为模板 ,在其两侧各加 3个随机氨基酸残基 ( X3 CDR X3 ) ,构建了 6个以CDR为基础的肽库 .经过 3轮亲和选择 ,挑取单克隆 ,进一步经ELISA检测TNF阳性噬菌体克隆 ,分离得到 7个ELISA阳性较好的噬菌体肽克隆 ,分别命名为CDR2L1、CDR2L2、CDR2L3、CDR1L1、CDR2H1、CDR3H1、CDR3H 2 .应用MTT方法 ,检测 7个克隆对TNF生物学活性的拮抗作用 .结果显示 :来自CDR2L ,CDR3H肽库中的CDR2L2、CDR2L3,CDR3H2噬菌体肽具有明显的拮抗TNF诱导L92 9细胞的细胞毒作用 ,其中以CDR2L2噬菌体肽的拮抗活性最强 .而来源于CDR1L ,CDR2H肽库的CDR1L1和CDR2H1噬菌体肽和来自CDR2L ,CDR3H肽库中的CDR2L1和CDR3H1噬菌体肽没有明显的拮抗TNF作用 .研究结果初步表明 :从cA2抗体CDR肽库中筛选得到的噬菌体CDR模拟肽具有亲本抗体相似的结合活性和生物学效应 ,从而为开发已知抗体 (特别是治疗用抗体 )CDR为基础的肽药物创建一个技术平台奠定基础     

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.