皖南尖吻蝮蛇毒Ⅰ型金属蛋白酶AcutolysinA的表达、重折叠及其生物学活性

将皖南尖吻蝮蛇毒Ⅰ型金属蛋白酶acutolysinA基因克隆进原核表达载体pBASD/gⅢA后得到重组表达质粒pDS,在0.02%的L-（ ）-阿拉伯糖的诱导下,质粒pDS在E.coliTOP10中表达了重组acutolysinA。与其天然形式相比,重组蛋白质在N端和C端各增加了22个和8个氨基酸残基（均源于载体序列）。并以包含体的形式存在于胞质中,表达量约占菌体总蛋白质的5%-10%。在经8mol/L尿素或6mol/L盐酸胍变性及体外复性后,重组蛋白质获得了良好的免疫学及生物学活性,Western印迹及ELISA实验表明,天然金属蛋白酶acutolysinA与重组蛋白质具有良好的免疫反应相关性。动物实验表明,复性的重组蛋白质能明显引起皮下出血,其最低出血剂量约为20μg左右,1mmol/LEDTA,1mmol/LEGTA及3mmol/L咪唑均能不同程度的抑制天然及重组金属蛋白酶的出血活性,PMSF没有明显的抑制效果。并对出血毒金属蛋白酶的出血机制进行了探讨。     

MMP-9-PEX原核表达、纯化及其对结肠癌细胞侵袭能力的影响

用RT-PCR法扩增出基质金属蛋白酶-9(MMP-9)C末端血红素结合蛋白样结构域(PEX),克隆至表达载体pET32a中并转化至E.coilBL21(DE3),经IPTG诱导后在约为42kDa处发现有外源基因的表达,密度扫描显示表达蛋白含量占菌体总蛋白的50%左右。重组蛋白质pET32a/PEX主要以包涵体形式表达,其在上清液中的含量极微。含8mol/L尿素和10mmol/LDTT的裂解缓冲液溶解的包涵体采用金属整合层析有效分离出目标蛋白,纯化后pET32a/PEX蛋白质的纯度大于90%。在Transwell实验中发现复性纯化后的重组蛋白质pET32a/PEX能抑制结肠癌细胞的侵袭,且呈剂量依赖性。     

人肾小球系膜细胞纤溶酶原激活物抑制物cDNA克隆和高效表达

利用逆转录 聚合酶链式反应 (RT- PCR)方法 ,从中国正常人肾小球系膜细胞总RNA中扩增出人纤溶酶原激活物抑制物 (PAI 1 )基因cDNA编码区序列 ,并定向亚克隆至pUC1 9质粒 ,克隆的PAI -1cDNA去除了信号肽核苷酸序列并加入新的起始密码ATG ,编码区序列与文献报道的人内皮细胞PAI -1cDNA序列完全相同 .将PAI -1cDNA定向亚克隆至原核表达质粒 pBV2 2 0 ,构建了重组PAI -1基因表达质粒pBV2 2 0 PAI -1 ,在大肠杆菌中得到了高效表达 ,重组PAI -1蛋白表达占菌体总蛋白 45 % .Westernblotting检测 ,在分子量约为 43.0ku处出现一特异性蛋白质条带 .对形成包涵体的表达产物进行变复性处理及FPLC纯化 ,获得纯度 97%以上的潜伏态重组PAI -1 .经 4mol/L盐酸胍激活后 ,重组PAI- 1具有与天然PAI- 1同样的生物学活性 ,对尿激酶型纤溶酶原激活物 (u- PA)具有显著抑制活性 .     

单纯疱疹病毒1型囊膜糖蛋白gD在大肠杆菌中的表达、纯化及其免疫活性的鉴定

目的:在大肠杆菌中表达1型单纯疱疹病毒(HSV-1)囊膜糖蛋白gD,纯化重组蛋白并对其免疫活性进行鉴定。方法:将HSV-1 gD 基因克隆入原核表达载体pET-28b,利用异丙基-B-D-硫代吡喃半乳糖苷(IPTG)诱导重组质粒转化的大肠杆菌,探讨IPTG浓度、诱导时间、诱导温度对重组蛋白表达的影响;盐酸胍裂解变性包涵体,镍柱亲和层析法纯化gD蛋白,并对纯化后的蛋白进行透析复性;Western blot和ELISA检测gD蛋白的免疫活性。结果:酶切和测序结果表明gD基因克隆入pET-28b载体。该重组质粒转化的大肠杆菌经IPTG诱导后重组蛋白主要以包涵体形式存在,大小约40kDa。gD蛋白诱导表达的最佳条件为0.5mmol/L IPTG于37℃诱导8h。镍柱亲和层析法纯化获得的gD蛋白总量为3.1mg/L,透析复性后获得的gD蛋白总量为1.3mg/L,复性率为41.37%。Western blot及ELISA检测表明表达的gD蛋白具有免疫活性。结论:在大肠杆菌中表达并纯化获得具有免疫活性的HSV-1 gD蛋白,为进一步制备HSV-1诊断试剂和预防疫苗奠定了基础。     

甲胎蛋白的原核表达及复性优化

构建甲胎蛋白的原核表达载体p ET32a-AFP,对包涵体形式表达的甲胎蛋白进行复性优化。将构建的重组质粒p ET32a-AFP转化入E.coli,IPTG诱导表达后,经亲和层析纯化获得AFP包涵体,通过对复性过程、p H、添加剂等的研究摸索,获得最佳复性条件。当采用添加0.5 mol/L L-精氨酸的一步法透析复性方法,且透析液p H值为8.5,重组人AFP包涵体蛋白起始浓度为1.0 mg/m L时,复性效率最高。该复性方法获得蛋白质具有较高的回收率且操作简便。     

人乙醛脱氢酶2的原核表达及其活性的鉴定

商品化的乙醛脱氢酶主要从动物细胞、肝细胞线粒体中提取,其来源受到很大的限制,而且价格昂贵。为了解决乙醛脱氢酶原料有限的问题,利用微生物发酵法获取乙醛脱氢酶。将人乙醛脱氢酶2基因(aldh2)克隆至原核表达载体p ET32a中,构建重组载体p ET32a-ALDH2。将构建的重组载体转化大肠杆菌E.coli BL21(DE3),用异丙基硫代-β-D-半乳糖苷(IPTG)进行诱导,SDS-PAGE电泳结果显示目的蛋白得到大量表达;且对诱导表达条件进行优化。结果显示,在37℃,1.0 mmol/L IPTG诱导表达6 h为最优表达条件。由于目的蛋白几乎都是以包涵体形式表达,为了得到有活性的乙醛脱氢酶,对包涵体变性、复性和纯化进行了研究。试验数据显示,酶的最终得率为14.49 U/L。并通过用Bradford法,测定复性蛋白的浓度约为77μg/m L,进行活性检测表明,该复性蛋白具有乙醛脱氢酶活性,酶活性约为1.449 U/m L。通过构建重组载体p ET32a-ALDH2和优化表达条件,aldh2在原核表达宿主E.coli BL21(DE3)得到大量表达;此外,利用透析复性法得到的复性蛋白酶活性有所提高。     

ATF-PAI2CD融合蛋白的生物学功能

为了解尿激酶型纤溶酶激活物 (urokinase typeplaminogenactivator,uPA)的氨基末端片段 (amino terminalfrag ment,ATF)和纤溶酶激活物抑制剂 2 (plasminogenactivatorinhibitortype 2 ,PAI 2 )突变体PAI 2CD融合蛋白在大肠杆菌的表达情况及进一步研究其生物学活性、将ATF PAI2CD融合基因与大肠杆菌表达载体pLY 4重组得到表达质粒pZWE ATF PAI2CD ,以其转化大肠杆菌JF112 5 ,经温度诱导 ,ATF PAI2CD获得较高水平表达 ,融合蛋白质以包涵体形式存在 ,占菌体总蛋白质的 15 %。包涵体经洗涤、8mol L尿素溶解、稀释复性及离子交换色谱一步分离 ,纯度达 90 % ,分子量与理论值相符。每升发酵液得重组融合蛋白质 5 0mg。经牛奶板法检测具有纤溶抑制活性 ,比活性达 12 0 0 0IU mg ;应用放射竞争法证实融合蛋白质能与肿瘤细胞表面的uPA受体特异性结合。双功能融合蛋白质的纤溶抑制活性与野生型PAI 2 (或PAI 2突变体 ,PAI 2CD)基本一致 ,与肿瘤细胞表面的uPA受体的结合能力同pro uPA。     

猪细小病毒NS1基因的原核表达及重组蛋白的复性

利用PCR技术扩增出PPVNS1基因抗原区。将目的基因与原核表达载体pGEX-4T-1进行连接并转化,重组质粒经鉴定并测序。测序结果表明,目的基因插入的位置、大小和读码框均正确,通过试验摸索并确定了表达NS1基因的最佳诱导条件:IPTG终浓度为1.0mmol/L,诱导时间为10h,温度为37℃,其表达量占全菌蛋白的29.8%。表达产物经SDS-PAGE分析,得到分子量约为52kDa的重组蛋白且以包涵体形式存在。重组蛋白经Westernblot检测,结果证明重组蛋白可被PPV阳性血清识别。用8mol/L尿素变性溶解包涵体,再用稀释方法和还原型、氧化型谷胱甘肽系统相结合的方法对重组蛋白进行复性。ELISA检测表明,复性后的重组蛋白有良好的生物活性。     

融合牛肠激酶轻链EKL包涵体蛋白的复性纯化

大肠杆菌高密度发酵表达肠激酶轻链融合蛋白DsbA-rEKL,主要以包涵体形式存在。包涵体经4mol/L尿素和0.5%TritonX-100洗涤,以6mol/L盐酸胍、100mmol/LDTT溶解,在胱氨酸存在下,以脉冲加样方式复性。融合蛋白复性在6mmol/L胱氨酸存在下、脉冲加量0.03mg/mL和复性终蛋白浓度0.3mg/mL为最佳复性方案。复性的融合蛋白加2mmol/LCaCL2后快速自切。经IDA-Sepharose及Q-Sepharose纯化,rEKL纯度可达95%以上,可高效酶切重组瑞特普酶融合蛋白Trx-rPA。实现了大规模生产rEKL,每升发酵液经复性及纯化后,可得rEKL60mg/L以上,使以融合蛋白表达rPA等药用蛋白成为现实。     

SUMO蛋白酶Ulp1的高效表达纯化并通过His-SUMO标签制备scFv

SUMO蛋白酶(Ulp1)是切割小分子泛素修饰(SUMO)融合蛋白获得天然N端靶蛋白的一种工具酶,具有酶切效率高、特异性好等优点。但现有市售SUMO蛋白酶Ulp1价格昂贵、操作复杂,限制了SUMO融合体系的运用。利用基因工程技术,合成基因ulp1(Leu403-Lys621),并在N端和C端加入多聚组氨酸标签(His_6),构建重组表达载体psv T7-ulp1,将重组质粒转入大肠杆菌BL21(DE3)和BL21 trx B(DE3)中。经过高通量筛选技术快速确定最优的表达条件为采用BL21(DE3)作为表达宿主,转接后7h加入IPTG,IPTG的终浓度为0.1mmol/L,诱导时间为16h,最终蛋白质表达量占菌体总蛋白质量的34.5%,重组蛋白Ulp1的表达量为190mg/L,通过Ni-NTA一步纯化即可得到纯度95%以上的Ulp1。通过酶切反应,测定酶活为5.19U/μl,比酶活为5.23×10~4U/mg,是先前报道比酶活的1.87倍,通过酶活动力学分析,Ulp1的表观米氏常数K_m=0.359g/L,V_m=5.10μg/(ml·min)。将SUMO融合表达体系用于单链抗体(single-chain antibody fragment,scFv)的表达,得到可溶的SUMO-scFv融合蛋白,使用表达的Ulp1进行酶切并纯化,获得纯度高于90%且N端不含多余氨基酸的scFv,操作步骤简单,显著改善了scFv在大肠杆菌中难以高效可溶性表达纯化的现状。     

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.