人源抗狂犬病毒中和性全抗体在昆虫细胞中的表达

将来源于噬菌体抗体库的人源狂犬病毒糖蛋白特异性单抗G10Fab的基因 ,克隆入杆状病毒人源IgG抗体表达载体 ,通过转染将重组质粒导入昆虫细胞 ,以全抗体的形式表达了一株人源抗狂犬病毒基因工程抗体G10。用亲和层析的方法纯化了表达产物 ,经与一株鼠源糖蛋白特异性单抗竞争证实 ,该单克隆抗体特异性识别狂犬病毒糖蛋白 ,亲和力约为 10 -9M。体外中和实验证明 ,该单抗对狂犬病毒aG株具有体外中和活性     

人源抗狂犬病毒中和性全抗体在昆明细胞中的表达

将来源于噬菌体抗体库的人源狂犬病毒糖蛋白特性异特单抗G10Fab基因的,克隆入杆状病毒人源IgG抗体表达载体,通过转染将重组质粒导入昆虫细胞,以全抗体的形式表达了一株人源抗狂犬病毒基因工程抗体R10。用亲和层析的方法纯化了表达产物,经过一株鼠源糖蛋白特异性单抗竞争证实,该单克隆抗体特异性识别狂犬病毒糖蛋白,亲和力约为10^-9M.体外中和实验证明,该单抗对狂犬病毒aG株具有体外中和活性。     

共表达甲型流感病毒M1和HA抗原的重组杆状病毒的构建

为构建同时表达流感病毒M1和HA抗原的重组杆状病毒,采用PCR扩增流感病毒A/PR/8/34株的M1基因和去除信号肽的HA基因,将两基因克隆到杆状病毒转座载体pFastBac Dual的两个启动子下游的多克隆位点,筛选出阳性重组转座载体pFastBac Dual-M1-HA。将其转化含有杆状病毒穿梭载体(Bacmid)的DH10Bac感受态细胞,通过抗生素、蓝白斑筛选和PCR鉴定获得重组杆状病毒穿梭载体rBacmid-M1-HA,在脂质体介导下转染Sf9昆虫细胞,获得重组杆状病毒rBac-M1-HA。提取重组病毒基因组,通过PCR鉴定外源基因插入成功。间接免疫荧光和Western-blot检测表明,该重组杆状病毒在Sf9昆虫细胞中成功地表达了M1和HA。应用杆状病毒/昆虫细胞系统成功共表达流感病毒M1和HA抗原,为研究流感病毒VLP的形成机制和开发新型流感疫苗奠定了基础。     

HBV pre-c-Fc融合蛋白在杆状病毒表达系统中的表达及其生物学活性研究

目的:在杆状病毒表达系统中表达融合蛋白乙型肝炎病毒前C蛋白-小鼠IgG Fc蛋白(HBV precore protein-mouse IgG Fc,HBV pre-c-Fc),并鉴定其免疫原性。方法:目的基因HBV prec-Fc连接到pFastBac1载体,获得的pFastBac1-HBV pre-c-Fc质粒转化DH10Bac感受态,通过Tn7转座子将目的基因转座到Bacmid中,得到Bacmid-HBV pre-c-Fc穿梭载体,脂质体包被后转染Sf9昆虫细胞获得P1代病毒,重复转染Sf9获得高滴度病毒。收集细胞上清超滤后通过Protein G亲和层析柱纯化得到目的蛋白HBV pre-c-Fc。纯化的蛋白大腿内侧肌肉注射免疫BALB/c小鼠并检测血清中乙型肝炎病毒核心蛋白抗体产生量。结果:HBV pre-c-Fc在昆虫细胞中成功表达,纯化后蛋白纯度达90%以上,蛋白产量约为3.03mg/L,纯化蛋白能有效刺激BALB/c小鼠产生特异抗体。结论:成功地在杆状病毒表达系统中表达了具有免疫原性的HBV pre-c-Fc蛋白,为生产乙肝治疗性疫苗奠定了基础。     

16型人乳头瘤病毒衣壳蛋白在真核细胞中的表达

为了构建HPV16型晚期蛋白重组杆状病毒,并使其在昆虫细胞中获得高效表达.首先构建2株重组杆状病毒转移质粒,分别携带人乳头瘤病毒晚期基因L1及L1和L2,再用线性化的杆状病毒DNA与该重组杆状病毒转移质粒共转染sf9昆虫细胞进行同源重组,获得2株重组杆状病毒.经鉴定该重组病毒中有目的基因存在且可表达所编码的L1或L2晚期蛋白.结果表明HPV16型晚期蛋白在昆虫细胞中获得成功表达,为HPV16型预防性基因工程亚单位疫苗的研制和诊断试剂的研究开发奠定了基础.     

人类细小病毒B19壳蛋白VP2在昆虫杆状病毒表达系统中的表达

利用BAC-TO-BAC系统获得了人类细小病毒B19壳蛋白VP2的重组昆虫杆状病毒,并在sf9细胞中表达出VP2。用蚀斑法纯化病毒,终末稀释法测定病毒滴度为3.6×108。Western印迹检测证实了表达蛋白的特异性,间接免疫荧光法可观察到细胞胞浆中的表达蛋白颗粒。     

截短型人乳头瘤病毒58型L1蛋白的表达及其体外生物活性研究

利用PCR技术克隆截短型HPV58 L1基因并重组入杆状病毒表达系统穿梭质粒pFastBac-Htb,通过转座反应,将目的基因片段重组入杆状病毒基因组,分离重组的Bacmid DNA, 并转染Sf-9昆虫细胞,收集被转染的Sf-9细胞,提取细胞蛋白,SDS-PAGE检测可见在大约58Kda处出现一新生蛋白条带,Western blot 证实为HPV58L1蛋白。用ProBondTM纯化系统纯化所表达的蛋白。小鼠红细胞凝集试验证实纯化的蛋白可介导小鼠红细胞凝集,透射电镜观察证实纯化蛋白可自组装成VLP。结果表明昆虫杆状病毒表达系统可高效表达截短型HPV58L1蛋白,纯化后的截短型HPV58L1蛋白在体外可自组装VLP,并具有介导小鼠红细胞凝集的生物学活性。     

鸡白细胞介素18成熟蛋白在昆虫细胞/杆状病毒系统中的表达

将编码鸡的白细胞介素 1 8(chickeninterleukine 1 8,ChIL 1 8)成熟蛋白的基因亚克隆到杆状病毒转移载体pMelBacB上 ,构建真核转移载体pMelBacBChIL 1 8,经限制性内切酶消化、ChIL 1 8特异引物PCR鉴定和确证性序列测定 ,证明目的基因正确克隆到载体的预期位点。将纯化的pMelBacBChIL 1 8质粒与杆状病毒DNA(Bac N BlueTM DNA)共转染sf9昆虫细胞 ,经四轮蓝斑筛选纯化 ,获得了重组杆状病毒 ,命名为rBaculovirusChIL 1 8。提取病毒染色体DNA ,经ChIL 1 8特异引物和重组杆状病毒特异引物PCR鉴定 ,证明获得了纯化的重组杆状病毒。用该重组病毒接种sf9昆虫细胞 ,收获接种后不同时间的细胞进行SDS PAGE电泳。结果表明ChIL 1 8基因在昆虫细胞中获得了表达 ,表达的重组蛋白分子量约为 2 3kDa。应用在大肠杆菌原核表达系统中表达的重组蛋白制备的兔抗ChIL 1 8多克隆抗体进行Westernblot分析 ,表明本研究真核系统表达的ChIL 1 8成熟蛋白和前期原核系统表达的ChIL 1 8成熟蛋白均具有生物学活性。     

重组嵌合抗人CD22四价基因工程抗体在昆虫杆状病毒中的表达

重组嵌合抗人CD22四价基因工程抗体是由一条短肽链将两个鼠源抗CD22mAb的scFv连接起来,再与人IgG1的CH3片段连接所获得重组基因工程抗体(cRFB4-CH3),是目前开发治疗B细胞系淋巴瘤的人源化基因工程抗体。为探讨该重组基因工程抗体高效表达技术,本研究利用DNA重组技术将含有人IgG1的CH3段的抗人CD22四价基因工程抗体基因克隆到含信号肽的重组杆状病毒载体pAcSG2中,构建重组质粒pAcSG2-cRFB4-CH3并转染到Sf9细胞中,构建携带有重组嵌合抗人CD22四价基因工程抗体基因的重组杆状病毒AcNPV-cRFB4-CH3。通过对该重组毒进行PCR和IFA鉴定,证实获得了可以稳定表达抗人CD22四价基因工程抗体的重组杆状病毒。以蚀斑试验进一步纯化病毒,经过3次病毒蚀斑克隆,获得毒价达到4.5×107pfu/mL重组病毒,为治疗白血病药物的开发和应用打下了基础。     

甲型H1N1流感病毒HA基因在昆虫细胞中的截短表达

目的:利用Bac-to-Bac Baculovirus Expression System表达重组HA蛋白,Western blot及IFA方法鉴定其表达。方法:采用PCR方法扩增A/California/04/2009(H1N1)HA基因,将其克隆到pFastBacHT A载体上,重组质粒pFastBacHT-HA经双酶切及测序鉴定正确后,转化阳性重组载体进入E.coli DH10Bac感受态细胞中,通过Bluo-gal蓝白斑筛选、PCR鉴定获得重组转座子rBacmid-HA。从重组转座子中提取rBacmid-HA质粒DNA转染sf 9昆虫细胞,制备重组杆状病毒。重组杆状病毒感染sf 9细胞表达重组蛋白,Western blot及IFA鉴定重组蛋白表达情况。结论:成功构建了甲型H1N1流感病毒HA基因的昆虫杆状病毒表达载体,该表达载体转染昆虫细胞后制备的重组杆状病毒病毒滴度较高,重组杆状病毒表达的重组蛋白经Western blot 及IFA 鉴定后具有良好的免疫反应原性。     

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.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

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.