具有抗HIV活性的突变型天花粉蛋白TCSYFF-KR的构建及原核表达

目的：构建具有抗HIV活性的突变型天花粉蛋白（TCS）,并将其在原核系统内进行表达与纯化。方法：借助计算机预测TCS分子上可能的抗原决定簇（YFF81-83和KR173-174）,并依此设计适当的突变引物;以栝楼基因组DNA为模板,利用重组PCR技术扩增双突变型TCS全长基因,经BamHI和EcoRI双酶切后与原核表达载体pRSET-A连接,转化感受态大肠杆菌DH5α,提取质粒进行酶切鉴定及测序;将所获阳性重组质粒转化感受态大肠杆菌BL21（DE3）,经IPTG诱导表达后,对表达产物进行Western印迹鉴定;用Ni-NTA亲和层析柱对所获突变型TCS蛋白进行纯化。结果：构建了突变型TCSYFY-KR,并获得了该蛋白在大肠杆菌内的可溶性高效表达;经Ni-NTA亲和层析柱纯化,产生大量均一的突变型TCS蛋白。结论：TCS的定点突变及其在原核系统内的表达,为基因工程方法改造TCS提供了一条新途径。     

带有穿膜结构域的转录因子蛋白Oct4和Sox2的融合表达

目的:克隆、表达及纯化带有穿膜结构域的转录因子蛋白Oct4和Sox2。方法:根据GenBank中的Oct4和Sox2基因序列,在其3’端引入穿膜结构域11R,并在其两端引入NdeⅠ和XhoⅠ酶切位点,进行全基因合成;将目的基因克隆至pET41a载体,进行酶切鉴定及测序;将所获阳性重组质粒转化感受态大肠杆菌BL21(DE3),经IPTG诱导表达后,对表达产物进行Western印迹鉴定;最后用Ni-NTA亲和层析柱对所获目的蛋白进行纯化。结果:质粒酶切鉴定结果表明带有目的基因的重组质粒构建成功;SDS-PAGE结果显示有相对分子质量约42×103和38×103的特异性蛋白表达条带,经Western印迹证实为目的蛋白;用Ni-NTA亲和层析柱纯化后,得到均一的Oct4和Sox2目的蛋白。结论:得到带有穿膜结构域的转录因子融合蛋白Oct4和Sox2,为今后安全开展诱导性多能干细胞研究奠定了基础。     

小鼠精囊自身抗原的原核表达和纯化

目的：在原核细胞中表达小鼠精囊自身抗原（SVA）,并对表达产物进行鉴定和纯化。方法：提取小鼠附睾组织总RNA,RT-PCR获得SVA的cDNA,设计并合成特异引物序列,进一步扩增出不含信号肽的SVA编码序列,连入原核表达载体pET28a中,经酶切和测序鉴定正确的重组质粒转化大肠杆菌Rosetta（DE3）感受态细胞,IPTG诱导表达,Western印迹分析表达产物His-SVA,采用Ni-NTA纯化融合蛋白His-SVA。结果：原核表达获得融合6个组氨酸的SVA,用抗His单克隆抗体进行Western印迹鉴定,检测到相对分子质量约18×10^3的目的蛋白,与理论值一致;经Ni-NTA纯化获得较高纯度的His-SVA融合蛋白。结论：获得了在大肠杆菌中表达的小鼠附睾蛋白SVA,为后续研究其对小鼠生殖的影响奠定了基础。     

柔嫩艾美耳球虫第二代裂殖子表面抗原基因（λMZ5—7）在大肠杆菌中的表达

将重组克隆质粒（PGEM－λMZ）用EooRⅠ酶切后,电泳回收目的的片段,克隆到经EooRⅠ酶切、CIAP处理的表达载体pET－28a中,转化大肠杆菌JM109感受态细胞,得到的转子化经PCR鉴定和酶切分析,筛选出符合正确阅读框的重组子,构建成重组表达质粒（PET－λMZ）,并在大肠杆菌BL21（DE3）表达菌中成功地表达了含目的蛋白的融合蛋白,融合蛋白的分子量的34KDa,加入IPIG诱导6h后,蛋白表达接近最高 水平。表达产物经Ni-NTA亲和层析柱纯化,SDS－PAGE检测为要带。经Western-blotting杂交实验,纯化出的目的蛋白能与兔抗E．tenella第二代裂殖子抗血清发生反应,说明MZP蛋白是E．tenella第二代裂殖子抗原蛋白,具有一定的免疫原性,为进一步研究重组疫苗创造了一定的条件。     

人自噬相关蛋白ATG7的原核表达及纯化鉴定

目的:原核表达并纯化自噬相关蛋白ATG7,初步鉴定其生物学活性。方法:利用PCR技术从人乳腺文库中扩增出人ATG7基因的编码序列,插入载体p ET-28a(+)得到重组质粒,经Bam HⅠ和NotⅠ双酶切鉴定后转化大肠杆菌Rossate菌株进行小量诱导,纯化融合蛋白His-ATG7,通过Western印迹和SDS-PAGE检测融合蛋白的纯化效果。结果:用PCR技术从人乳腺文库中扩增得到约2031 bp的目的片段,插入载体p ET-28a(+)后构建出His-ATG7重组质粒,并经酶切鉴定及测序证实无误;转化大肠杆菌Rossate并进行小量诱导,纯化后SDS-PAGE检测显示获得相对分子质量约为78×103的融合蛋白。结论:纯化得到原核系统表达的His-ATG7融合蛋白,为后续研究ATG7在自噬中的作用机制奠定了实验基础。     

羊口疮病毒蛋白ORFV035的表达、纯化和多克隆抗体的制备

克隆表达羊口疮病毒蛋白ORFV035,并制备其多克隆抗体,为后续对病毒复制、装配、形态发生和成熟过程的研究奠定基础。PCR扩增羊口疮病毒ORFV035基因,将其与质粒pET-30a(+)经Bam HⅠ和HindⅢ双酶切后连接,构建重组质粒pET30a-035。重组质粒经双酶切和测序鉴定,转化感受态大肠杆菌BL21,IPTG诱导表达,SDS-PAGE鉴定蛋白表达情况。表达产物进行超声破碎和Ni柱纯化,纯化后目的蛋白免疫小鼠,制备多克隆抗体并对其进行鉴定。成功构建了重组质粒pET30a-035,在大肠杆菌BL21中以包涵体形式高效表达。包涵体洗涤、溶解后进行Ni柱纯化,得到纯度较高的ORFV035-his融合蛋白。以纯化蛋白免疫小鼠获得多克隆抗体。Western blot检测显示该多抗可以识别天然ORFV035蛋白。成功诱导表达、纯化ORFV035蛋白并制备ORFV035多克隆抗体。     

人β肌动蛋白基因原核表达载体的构建及其表达和纯化

目的:构建带GST标签的人β肌动蛋白(β-actin)基因的原核表达产物,纯化出GST-β-actin融合蛋白,为探究β-actin的各项生理功能做准备。方法:以人乳腺文库为模板,利用PCR技术扩增β-actin基因,将其连接到带有GST标签的载体上,经鉴定正确的重组质粒转化大肠杆菌Rossate感受态细胞,小量诱导表达后,利用GST-Sepha-rose 4B亲和珠纯化GST-β-actin融合蛋白,经SDS-PAGE和Western印迹检测。结果:目的基因经PCR技术得以扩增,将其与带GST标签的载体连接后再经双酶切鉴定及测序后确认构建成功;转化大肠杆菌Rossate感受态后获得小量诱导表达,纯化出GST-β-actin融合蛋白,并证实其有生物活性。结论:构建了人β-actin的原核表达载体,并获得了GST-β-actin融合蛋白。     

肠出血性大肠杆菌转移紧密黏附素受体的表达及其活性鉴定

目的:在原核表达基因工程茵中实现肠出血性大肠杆菌(EHEC)O157:H7转移紧密黏附素受体(Translocated Intimin Receptor,Tit)蛋白的高效表达,并对其活性进行初步鉴定.方法:采用PCR方法从EHEC O157:H7基因组中调取tir基因,插入pEASY-T1 克隆栽体.克隆质粒测序鉴定后,采用Nde Ⅰ、Xho Ⅰ限制性核酸内切酶双酶切pEASY-Tl-tit质粒获得tir基因,连接同样经过双酶切的pET-22b(+).表达质粒转化E.coli BL21(DE3),IPTG诱导表达,SDS-PAGE检测相对分子质量,Western blotting验证抗原活性.荧光显微镜观察蛋白是否具有嵌入细胞膜的活性.结果:PCR扩增得到1686bp的目的片段.构建的原核表达质粒pET-22b(+)-tir经酶切鉴定及测序与预期序列一致.目的蛋白以裂解上清形式表达,表达量约3mg/ml.经镍柱纯化后纯度达90%以上.重组表达的Tir具有嵌入细胞膜的生物学功能.结论:成功表达了具有生物活性的重组Tir蛋白,为Tir的功能研究奠定基础.     

人铜锌超氧化物歧化酶在大肠杆菌中的融合表达与纯化

目的:在大肠杆菌中表达并纯化人铜锌超氧化物歧化酶(HuSOD1)。方法:合成HuSOD1编码基因,PCR扩增后连入pMAL-p5x质粒构建融合表达载体,转化大肠杆菌BL21(DE3)感受态,IPTG诱导表达,NBT法测定HuSOD1酶活,利用麦芽糖结合蛋白亲和层析柱纯化MBP-HuSOD1融合蛋白,经因子Ⅹa酶切及分子筛柱层析纯化HuSOD1蛋白。结果:构建了pMAL-p5x-HuSOD1表达载体,在大肠杆菌中实现了高表达,目的蛋白占全菌蛋白的30%,其中可溶性表达占63%,具有超氧化物歧化酶活性;通过亲和层析纯化得到纯度大于95%的融合蛋白MBP-HuSOD1,经因子Ⅹa酶切后纯化得到纯度约90%的HuSOD1蛋白。结论:在大肠杆菌中表达并纯化获得有活性的MBP-HuSOD1,经进一步酶切、纯化后得到HuSOD1。     

重组蓖麻、相思子毒素A链嵌合突变体的制备与分析

目的:构建蓖麻毒素(RIC)、相思子毒素(ABR)A链突变体的嵌合体蛋白,实现嵌合体蛋白的可溶性表达、纯化及抗原性分析。方法:采用柔性linker连接RIC A链突变体(mRICAD75AV76MY80A)和ABR A链突变体(mABRAE164AR167L),构建嵌合体基因mRICA/mABRA,将该嵌合体基因亚克隆至原核载体pQE80L构建表达质粒pQE80L-mRICA/mABRA,再转化至大肠杆菌M15获得表达工程菌株M15/pQE80L-mRICA/mABRA,工程菌在18℃经0.1 mmol/L的IPTG诱导14 h,表达的嵌合体蛋白经Ni-NTA亲和层析柱纯化,通过ELISA和Western印迹检测嵌合体蛋白的抗原性。结果:所获得的mRICA/mABRA嵌合体基因经一致性比对分析,与预计嵌合基因的序列一致性为100%,其开放读框全长1572 bp,编码524个氨基酸残基;重组表达质粒pQE80L-mRICA/mABRA经PCR及双酶切鉴定证明构建正确,嵌合体蛋白相对分子质量约为62×103,与预测相符,可溶性的嵌合体蛋白经Ni-NTA亲和层析柱纯化,纯度可达99%;间接ELISA和Western印迹结果表明,嵌合体蛋白能同时与抗RIC多克隆抗体和抗ABR多克隆抗体发生特异的抗原抗体反应。结论:得到的mRICA/mABRA嵌合体蛋白具有良好的抗原性,为研制新型RIC和ABR双价疫苗奠定了重要基础。     

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.