重组hIL—12病毒在Sf9细胞传代中遗传稳定性的研究

将含重组白细胞介素12(hIL—12)的杆状病毒(Ac—hIL12)经空斑纯化后,在草地贪夜蛾Sf9细胞中进行连续无稀释传代到P55代,收集被P15、P25、P35、P45、P55代重组病毒感染的细胞,抽提胞内病毒(ICV)DNA。根据重组病毒构建的原理,在P35cDNA和P40 cDNA的3′末段设计一对引物进行PCR,扩增出了包括P35cDNA、Polyhedrin启动子、P10启动子和P40 cDNA序列在内的全长约2．0kb片段,克隆至T Vector进行序列测定后发现,在第PmP25和P35代所扩增出的序列没有发生任何突变。但在P45代,P35 cDNA中就有3个碱基发生了点突变(461T→C,517A→G以及630C→T),Polyhedrin启动子的 1位后插入了一个碱基T,P40 cDNA与P10启动子区(230bp)没有变化;而第P55代除了以上碱基突变以外,P10启动子区—168位的G替换突变为T,—136与—135位之间插入一个碱基T,以及—122位缺失一个碱基T。以上结果表明杆状病毒在体外细胞连续传代过程中可导致外源基因本身的突变。     

重组人白介素-12在银纹夜蛾中的表达纯化及其生物活性

KB细胞经PDBu刺激,采用异硫氰酸胍一步法提取细胞总RNA,RT-PCR法获得重组人白介素-12(rhIL-12) P35和P40 cDNA.将hIL-12 P35 cDNA和P40 cDNA分别克隆到pAcUW51载体的Polyhedrin和P10启动子下游,构建pAcUW51-IL12转移载体.pAcUW51-IL12与坏死缺陷型线性苜蓿银纹夜蛾核型多角体病毒基因DNA共转染Sf9细胞,获得重组杆状病毒Ac-hIL12.该病毒经血腔感染银纹夜蛾幼虫,采用亲和层析法纯化rhIL-12;SDS-PAGE(银染法)、Western blot鉴定表达和纯化的产物;ELISA检测rhIL-12含量;MTT法检测rhIL-12样品生物活性.rhIL-12分子量为75kD.rhIL-12在Sf9细胞培养中表达水平为17.8μg/106细胞;在银纹夜蛾幼虫中表达水平为200-300mg/L血淋巴.纯化的重组rhIL-12样品对经PHA-P激活的PBMC有明显的增殖活性,且具有明显的促NK细胞杀伤活性的生物活性.     

鹅源新城疫病毒ZJI株基因组cDNA克隆的序列修饰

将鹅源新城疫病毒ZJI株全基因组cDNA克隆通过酶切切下包含T7启动子区域和转录载体的片段,将其自身环化后获得约6．5kb的质粒。设计引物,利用基因定点突变技术,在此质粒上T7启动子与NDV Leader序列之间突变插入额外的3个G碱基,将此突变最终引入到原基因组cDNA克隆中。应用RT—PCR技术从尿囊液中扩增NDV基因组F／HN基因区域部分片段,利用限制性内切酶BsmBI将扩增片段连接,最终将原cDNA克隆中相应片段替换下。测序结果表明,原基因组cDNA克隆中特定位置碱基插入突变成功,F／HN基因区域碱基突变均得以纠正。以上cDNA克隆的修饰与替换为该毒株的反向遗传研究打下了基础。     

重组人白介素—12在银纹夜蛾中的表达纯化及其生物活性

KB细胞经PDBu刺激 ,采用异硫氰酸胍一步法提取细胞总RNA ,RT PCR法获得重组人白介素 12 (rhIL 12 )P35和P4 0cDNA。将hIL 12P35cDNA和P4 0cDNA分别克隆到 pAcUW 5 1载体的Polyhedrin和P10启动子下游 ,构建 pAcUW 5 1 IL12转移载体。pAcUW 5 1 IL12与坏死缺陷型线性苜蓿银纹夜蛾核型多角体病毒基因DNA共转染Sf9细胞 ,获得重组杆状病毒Ac hIL12。该病毒经血腔感染银纹夜蛾幼虫 ,采用亲和层析法纯化rhIL 12 ;SDS PAGE(银染法 )、Westernblot鉴定表达和纯化的产物 ;ELISA检测rhIL 12含量 ;MTT法检测rhIL 12样品生物活性。rhIL 12分子量为 75kD。rhIL 12在Sf9细胞培养中表达水平为 17.8μg/10 6细胞 ;在银纹夜蛾幼虫中表达水平为 2 0 0 - 30 0mg/L血淋巴。纯化的重组rhIL 12样品对经PHA P激活的PBMC有明显的增殖活性 ,且具有明显的促NK细胞杀伤活性的生物活性     

鹅源新城疫病毒ZJI株基因组cDNA克隆的序列修饰*

将鹅源新城疫病毒ZJI株全基因组cDNA克隆通过酶切切下包含T7启动子区域和转录载体的片段,将其自身环化后获得约6.5kb的质粒。设计引物,利用基因定点突变技术,在此质粒上T7启动子与NDV Leader序列之间突变插入额外的3个G碱基,将此突变最终引入到原基因组cDNA克隆中。应用RT-PCR技术从尿囊液中扩增NDV基因组F/HN基因区域部分片段,利用限制性内切酶BsmB I将扩增片段连接,最终将原cDNA克隆中相应片段替换下。测序结果表明,原基因组cDNA克隆中特定位置碱基     

nut R 中的突变对λ噬菌体早期转录抗终止作用的影响

将λDNA的nutR序列置于Lac启动子的下游,在nut R和β-半乳糖苷酶基因(gal K)之间插入λ噬菌体依赖rho的终止子tR1,使ga1 K的表达取决于N蛋白介导的转录抗终止作用。为研究nutR对抗终止的影响,系统地进行了该序列中每个碱基的点突变(A→C,G→T,C→A及T→G)。结果表明boxA中有两个碱基(位置2和5)的突变对抗终止作用是至关重要的,使抗终止效率降低了10倍;而其他位置的改变影响甚微。boxA的缺失在nus~ 宿主中使抗终止效率降低了40％,但在nusB宿主中却恢复到野生型水平。boxB茎环结构中,茎部顶端的碱基对及环中邻近基部的两个碱基的突变对抗终止作用影响很大,被认为是N蛋白的识别序列。boxA和boxB之间的间隔序列中有两个碱基的突变几乎使抗终止作用丧失。     

HLA-B*2736等位基因的序列分析

使用FLOW-SSO、PCR-SSP以及测序等分型技术, 发现一个与HLA-B*270401基因相关的未知基因。设计基因特异性引物单独扩增B*27基因的外显子2-5, 包括内含子2-4, 并进行双向测序, 分析与B*270401基因序列的差异。该基因的扩增产物为1 815 bp。与B*270401相比在外显子3和4共有10个碱基的改变, 从而使相应氨基酸发生错义或同义突变。碱基634 A→C (密码子130丝氨酸→精氨酸); 670 A→T (密码子142苏氨酸→丝氨酸); 683 G→T (密码子146色氨酸→亮氨酸); 698 A→T (密码子151谷氨酸→缬氨酸); 774 G→C (密码子176谷氨酸→天冬氨酸); 776 C→A (密码子177苏氨酸→赖氨酸); 781 C→G (密码子179谷氨酰胺→谷氨酸); 789 G→T (密码子181丙氨酸同义突变); 1 438 C→T (密码子206甘氨酸同义突变); 1 449 G→C (密码子210甘氨酸→丙氨酸)。在IMGT/HLA数据库中B*27组只有3个基因（B*270502 / 2706 / 2732）提交了内含子序列。该未知基因的内含子2序列与B*2706相同, 显示了与B*27组基因的同源性, 但其同源性在内含子3、4均未得到支持, 与B*27组基因相比, 内含子3的第106个碱基C→G, 碱基168缺失, 碱基179 G→A, 碱基536 G→A; 内含子4中碱基82 T→C。但其内含子3、4序列却与B*070201完全相同。该基因序列已提交GenBank, 编号为被DQ915176, 被WHO确认为HLA-B*2736等位基因。     

麻疹病毒全长cDNA构建及其感染性的研究

为发展新型疫苗和改造目前使用的麻疹病毒疫苗,以麻疹病毒疫苗株为模板,构建了具有感染性的麻疹病毒cDNA克隆.用RT-PCR分6段扩增出麻疹病毒全长基因,通过酶切、拼接构建麻疹病毒疫苗株CC-47的全长正链cDNA序列,并精确地置于T7启动子控制下与丁型肝炎病毒核酶序列之前.克隆麻疹病毒CC-47株蛋白N、P、L编码区质粒并置于T7启动子控制下,用4个质粒共转染哺乳动物细胞,在表达T7 RNA聚合酶的重组痘苗病毒VTF7-3的作用下进行病毒拯救.经免疫荧光、PCR等方法检测证实,获得了具有感染性的麻疹病毒.所拯救的病毒在哺乳动物细胞连续传3代后,仍能检出病毒抗原和核酸.     

脂蛋白脂酶基因的克隆、序列测定及定点突变

 以人的脂肪组织总RNA为模板 ,参考已报道的脂蛋白脂酶 (lipoproteinlipase ,LPL)cDNA设计引物 ,利用RT PCR方法扩增得到了LPLcDNA ,并经序列测定证实其序列是正确的 .在冠心病患者LPL基因第 5外显子的 830位碱基处发现了G→A的转换 ,该变异导致LPL基因第 192位的密码子CGA被CAA取代 ,使LPL第 192位精氨酸改变为谷氨酰胺 .在变异碱基附近设计合成两条引物 ,其中一条包含所要改变的碱基 ,利用基于PCR的定点突变技术和体外重组的方法获得了G830A变异的LPLcDNA     

表达H5N1亚型禽流感病毒HA基因重组腺病毒的遗传稳定性分析及滴度测定

研究表达H5N1亚型禽流感病毒HA基因重组腺病毒pAd-H5的遗传稳定性及重组病毒的滴度测定。将重组腺病毒pAd-H5在293细胞上连续传代20次,取第5、10、15和20代的重组病毒采用PCR 方法扩增禽流感病毒HA基因,并进行基因序列测定分析;用标记为GFP的快速测定法计算出20代次时重组病毒的滴度。从各代重组病毒DNA 中均扩增出了约1 700bp的目的条带,与HA基因片段长度一致,基因序列分析表明：第5 、10 、15 代重组病毒中的HA基因序列与原始转移载体序列完全一致,第20代重组病毒插入基因有1处发生了点突变(即HA基因417位A→G),但其编码的氨基酸未发生变化(即同义突变),表明表达的目的蛋白抗原表位未发生变化;计算出的重组病毒滴度为108.875pfu/0.1ml。重组腺病毒pAd-H5在293细胞上连续传代20次,具有良好的遗传稳定性,重组腺病毒的病毒滴度相对较高。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

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.