自组装ELP多肽的从头设计及其在生物医药领域中的应用进展

重组类弹性蛋白多肽(elastin-like polypeptides, ELPs)是一种通过基因工程方法合成的多肽聚合物,其结构由类弹性蛋白的肽段单元重复串连组成,具有刺激响应性、自组装特性、显著的弹性和良好的生物学特性,如低血小板黏附性和低免疫原性等,因此ELPs材料已被广泛应用于组织工程、药物输送和纳米生物器件制备等生物医药应用领域。本文综合了国内外文献报道,简要介绍了ELPs的性质,重点阐述了ELPs自组装的设计原理、结构及其在药物控释等生物医药领域中的研究与应用进展。     

溶剂特性对三臂星型类弹性蛋白多肽相变温度的影响

本文利用SpyTag/SpyCatcher特性构建了三臂星型结构的类弹性蛋白多肽(elastin like polypeptides, ELPs),考察其在不同溶剂,如分子拥挤试剂、osmolytes及深共熔溶剂(deep eutectic solvents,DESs)中的相变温度及行为,并与含有相同ELPs重复数的线性ELPs120作对比。结果表明：在不同浓度拥挤试剂PEG2000作用下,两种结构的ELPs相变温度均降低,当其各自浓度均为25 μmol/L时,三臂星型ELPs相变温度降低3℃～13℃,而ELPs120相变温度仅降低1.5℃～10.8℃。此外,在添加PEG2000后,三臂星型ELPs相变缓慢;在不同类型和浓度的osmolytes溶液中,25 μmol/L三臂星型ELPs相变温度明显要比线性ELPs高8℃左右;在DESs体系中,三臂星型ELPs有类似与水溶液中的相变行为,且其相变温度受到抑制,另外三臂星型ELPs和ELPs120在DESs/PBS体系中,与在(氯化胆碱+尿素)/PBS体系中的相变行为一致,其中当DESs体积含量为50%,ELPs120相变温度是最低的。由于ELPs在非单一缓冲液体系中的相变行为不同,这丰富了ELPs作为纯化标签的应用,且在非单一缓冲液体系中因降低了相变温度,节约了纯化融合蛋白的经济成本,同时也为研究ELPs拓扑结构与其相变行为之间的关系奠定理论基础。     

拥挤试剂PEG2000对不同拓扑结构类弹性蛋白相变的影响

类弹性蛋白（Elastin-like polypeptides,ELPs）是属于弹性蛋白中的一种且具有温控性的生物大分子,本文研究拥挤试剂对不同拓扑结构ELPs相变温度的影响,利用温控-紫外分光光度计研究其相变特性,结果发现,随着PEG2000浓度的增加,T-E-F的相变温度下降11.9~17.1℃;在固定Tadpole-like-E浓度下,随着PEG2000浓度的增加,Tadpole-like-E的相变温度降低11.5~16℃,其中,25 μmol/L的Tadpole-like-E其相变速度缓慢;ELPs浓度越大,其相变温度降低愈大,且PEG2000影响ELPs相变温度的趋势与ELPs的拓扑结构关系不大。另外,在简单的PBS缓冲溶液中加入PEG2000,可以使E-C在浓度＜0.5 mol/L的Na2CO3中发生相变,且随着PEG2000浓度的增加,E-C相变温度逐渐降低。本研究为今后ELPs在复杂体系的应用提供前期的基础研究。     

类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

旨在克隆、表达与纯化类弹性蛋白多肽,并测定类弹性蛋白的相变温度对不同的盐敏感程度。从头设计了类弹性蛋白多肽的序列并人工合成其编码基因片段,克隆至改造后的表达载体pET-22b中,构建重组表达载体,转化至大肠杆菌BL21(DE3) 中并诱导表达,采用可逆相变循环经高速离心对其进行纯化,并考察了盐类型及浓度对类弹性蛋白相变温度的影响。结果表明：0.4 mmol/L的Na2CO3能使25 μmol/L类弹性蛋白多肽 [KV8F-20] 相变温度降低至19 ℃,此类弹性蛋白多肽序列有望开发成一新型纯化标签,为今后     

纳米丝素蛋白微球的制备及性能研究

以家蚕丝素蛋白为原料,基于丝素自组装理论,通过酶解-干燥-溶解法制备不同尺寸的丝素蛋白微球,制备出的微球具有良好的水不溶性和稳定的分散性。对微球的形貌和结构表征结果表明,用该方法制备的丝素蛋白微球为纳米微球,当酶的添加量为2%且蛋白自组装时间为4 h时,丝素蛋白微球的平均粒径最小,仅为(32±11)nm。红外光谱(FT-IR)和X射线衍射(XRD)结果显示,微球中β-折叠结构的多少决定了微球晶体的大小,β-折叠越多,微球中晶体的体积越大。通过调控丝素蛋白自组装过程,可以制备平均粒径在30～140 nm之间的纳米丝素蛋白微球,且不引入任何有机溶剂和无机溶剂,制备过程绿色环保,制备出的丝素蛋白微球安全无毒。     

以类弹性蛋白多肽为标签的表达质粒构建及其用于木聚糖酶的非色谱纯化

【目的】旨在构建一个能以非色谱纯化目标蛋白的表达质粒,使用自行设计的类弹性蛋白多肽(ELPs)作为非色谱纯化标签,以纯化目标蛋白。该ELPs长度短,对盐非常敏感。【方法】从头设计了木聚糖酶,将其通过一段无规则卷曲同ELPs相连,合成了编码上述序列的基因,并构建重组表达载体pET-22b-SoxB-M2-S-ELP,转化至大肠杆菌BLR(DE3)中诱导表达,采用可逆相变循环经高速离心纯化木聚糖酶,并考察纯酶的酶学性质。【结果】成功构建了表达载体并表达,在pH=7.0时0.5 mol/L碳酸钠可使ELPs的相变温度降至22℃。在上述条件下,对木聚糖酶进行了非色谱纯化,其纯化倍数为3.2,回收率为21.2%,纯度为64.3%。经测定,未连接ELPs的酶、粗酶及纯化酶学性质基本一致,其最适温度为60℃,最适pH为6.0,最适反应时间为30 min,粗酶70℃保温1 h相对酶活仍有50%,为嗜热木聚糖酶,与预期相符。【结论】ELPs作为非色谱纯化标签纯化重组木聚糖酶具有操作简单、易于放大、成本较低的优势,故所构建的重组质粒可望通用于分离多种重组蛋白,具有较广泛的用途。     

一种溶酶体靶向的原位自组装短肽设计及抗肿瘤活性研究

肿瘤已成为威胁人类生命的一大杀手,目前主要采用手术和放、化疗等手段进行治疗,但由于放、化疗的细胞选择性差、毒副作用明显且易引起肿瘤细胞产生耐受(/药)性,不利于肿瘤的持续治疗,因此亟待研发具有定向定位优势、毒副作用低的新型靶向药物.原位自组装多肽能识别肿瘤部位的特异性高表达物质,在肿瘤部位靶向性聚集形成稳定的纳米结构,实现精准和高效治疗,有望成为一种新型的抗肿瘤药物.本研究基于多肽原位自组装的设计理念,利用溶酶体内组织蛋白酶L的催化活性,设计了靶向溶酶体且能够原位自组装的多肽分子Fmoc-FFRIKFERQ-OH,研究了该分子的自组装特性及抗肿瘤活性.结果显示,在体外酸性条件下,组织蛋白酶L能精准切割Fmoc-FFRIKFERQ-OH分子,其酶切产物FmocFFR-OH自组装形成长纳米纤维结构,对肿瘤细胞A375和SH-SY5Y均具有较好的杀伤作用.该分子通过靶向溶酶体杀伤肿瘤细胞且对正常细胞的毒性较低,有望成为一种新型的抗肿瘤药物.     

温敏类弹性蛋白多肽的基因设计及重组克隆表达

温敏类弹性蛋白多肽(elastin-like polypeptides, ELPs)作为新型的药物载体,在肿瘤治疗中具有广阔的应用前景。本研究根据大肠杆菌密码子的偏好性和简并性,将高度连续重复氨基酸的多种遗传密码引入基因序列中,利用依次插入单体基因和递归定向连接技术,成功构建了以缬氨酸为客座氨基酸的ELPs基因的表达质粒库,即p ET28-ELP-V_5～pET28-ELP-V₅₀。将pET28-V₅₀转化至表达宿主Escherichia coli BL21(DE3)中,经重组菌的培养和IPTG诱导,SDS-PAGE结果显示ELP-V₅₀在大肠杆菌中成功获得了可溶性表达,与预期分子量大小一致。本研究为进一步构建不同分子量的ELPs基因库提供了新的思路,并为重组表达获得特定相变特性的多肽分子奠定了基础。     

利用SpyTag/SpyCatcher构建胞内自组装多酶复合体实现高效生物合成

SpyTagr和SpyCatche可通过自发反应形成共价键,产生稳定的分子自组装体。酶分子自组装体因具有高效有序的催化特性在合成生物学和纳米技术领域具有重要的应用价值。为探索SpyTag/SpyCatcher在大肠杆菌胞内多酶复合体系形成有序自组装分子能力,将SpyTagr和SpyCatche分别与P450BM3m单加氧酶和葡萄糖脱氢酶GDH进行融合表达,以期产生具有辅酶再生循环系统、高效生物合成靛蓝分子的SpyTag/SpyCatcher双酶自组装复合体。首先,通过电泳及质谱对重组工程菌表达蛋白进行分析,证实SpyCatcher-P450BM3m与SpyTag-GDH在胞内成功形成了自组装多酶复合体;然后,系统分析不同培养条件下组装体合成靛蓝的能力。结果发现,经0.5mmol/L IPTG诱导后,菌体在16℃继续培养18h后,工程菌对吲哚(2mmol/L)与葡萄糖(4mmol/L)的全细胞催化能力最强,靛蓝产量最高达258mg/L,是未组装多酶系统的1.9倍,比P450BM3m单酶表达系统高约2.4倍;反应70min后达到反应平衡,转化率为52%。成功实现了SpyTag/SpyCatcher介导的多酶体系在大肠杆菌细胞中的自组装和高效转化体系,为胞内多酶复合物组装体的设计提供了新思路。     

基于万古霉素建立荧光酶联免疫吸附法检测金黄色葡萄球菌

以猪IgG作为捕获抗体固定金黄色葡萄球菌,修饰有万古霉素的量子点荧光微球作为"检测抗体",建立荧光酶联免疫吸附法检测金黄色葡萄球菌。文中制备了平均粒径为100 nm的量子点荧光微球并与万古霉素偶联;摸索了反应最佳盐离子浓度为0.01 mol/L,反应最佳pH为6.0。在该实验条件下,金黄色葡萄球菌的检测灵敏度为104 CFU/m L,与其他致病菌无交叉反应。以上结果表明,该方法可用于快速检测金黄色葡萄球菌,为金黄色葡萄球菌的临床监控和食品检测提供参考。     

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.