重组人PCNA突变体的克隆与稳定表达

目的:构建重组人PCNA突变体(mutant PCNA,mPCNA)的真核表达质粒,并建立稳定高表达该目的蛋白的宫颈癌细胞系Hela.为进一步研究PCNA在DNA损伤修复中重要的生物学功能奠定基础.方法:将舍有定点突变的PCNA cDNA序列克隆到T载体中,然后再亚克隆到真核表达栽体pCDNA3.1/V5-His A上,构建真核表达载体质粒pCDNA3.1/V5-His A-mPCNA,稳定转染到Hela细胞中;Western Blotting法检测蛋白的表达情况;白细胞记数法测定细胞的生长速率.结果:真核表达质粒pCDNA3.1/V5-His A-mPCNA经酶切、测序分析与实验设计的序列完全一致;稳定建系后,Western Blotting结果显示在相应位置可见清楚的目的条带;稳定高表达mPCNA的细胞系与野生型细胞相比两者的生长速率基本一致.结论:成功构建了真核表达质粒pCDNA3.1/V5-His A-mPCNA;建立了稳定高表达该突变体的Hela细胞系;PCNA突变体的高表达不影响Hela细胞的正常生长.     

Toll样受体5激动剂CBLB502-Fc融合蛋白的表达纯化及初步鉴定

目的:构建Toll样受体5激动剂CBLB502-Fc融合蛋白的真核表达载体,并在CHO细胞中表达,纯化得到具有生物学活性的目的蛋白。方法:首先从合成的pUC57-CBLB502质粒中扩增出CBLB502基因片段,酶切后克隆至pUC57-Fc质粒中,然后双酶切pc DNA3.1和pUC57-CBLB502-Fc,将CBLB502-Fc基因片段克隆至载体pc DNA3.1,挑选阳性克隆并测序,再将重组质粒转染CHO细胞,筛选高表达细胞系,用免疫印迹鉴定表达情况,Protein A亲和柱纯化目的蛋白,SDS-PAGE鉴定目的蛋白,用小鼠辐射试验初步验证目的蛋白的生物学活性。结果:构建了pc DNA3.1-CBLB502-Fc真核表达载体;转染CHO细胞,筛选得到CBLB502-Fc高表达细胞系,并经免疫印迹鉴定培养上清;纯化得到较高纯度的融合蛋白并经SDS-PAGE鉴定;小鼠辐射实验表明CBLB502-Fc具有抗辐射作用。结论:真核表达并纯化了具有抗辐射作用CBLB502-Fc融合蛋白,为后续研究CBLB502-Fc的生物学功能奠定了重要基础。     

人胃癌细胞系中FHIT基因mRNA的表达及pcDNA3．1-FHIT的构建与转染

目的:检测人胃癌细胞系中FHIT基因mRNA的表达状况及构建pcDNA3.1-FI-IIT真核表达载体.方法:RT-PCR法检测三种不同类型人胃癌细胞系中FHIT基因mRNA的表达,构建真核表达质粒pcDNA3.1-FHIT,通过酶切法、PCR扩增法和DNA序列分析鉴定重组质粒后,用脂质体转染至FHIT基因mR.NA阴性表达人胃癌细胞系MKN-45,经G418筛选后RT-PCR鉴定.结果:FHIT基因在人胃癌细胞系BGC-823中呈阳性表达,在MGC-803、MKN-45细胞系中呈阴性表达.FHIT基因cDNA正确克隆到真核细胞表达栽体pcDNA3.1中,并成功转染FHIT基因mRNA阴性表达人胃癌细胞系MKN-45.结论:FHIT基因在不同类型人胃癌细胞系中表达各异.成功构建pcDNA3.1-FHIT,并转染到FHIT基因mRNA阴性表达人胃癌细胞系MKN-45,使其唧T基因阳性表达.     

PA28γ及其胞浆定位突变体的构建及在肝癌细胞HepG2中的表达

目的：野生型PA28γ和核定位序列突变的PA28γ真核表达质粒并检测其在人肝癌细胞株HepG2中的表达,为进一步研究PA28γ的功能提供实验基础。方法：采用RT-PCR法从293T细胞中克隆得到PA28γ cDNA开放阅读框全长序列,并将该片段亚克隆到pMD-18T载体中,在该载体中利用定点突变获得核定位序列缺失的克隆。分别将野生型（WT）和突变型（MT）PA28γ克隆克隆到真核表达载体pDsRed1-C3中,脂质体法转染293T细胞确定两种克隆的细胞定位。再将野生型和突变型PA28γ分别克隆到真核表达载体pcDNA3.1(-)Flag中,构建好的真核表达质粒采用脂质体法转染人肝癌细胞株HepG2,经G418筛选、Western Blot鉴定后,获得分别高表达野生型PA28γWT和PA28γMT的HepG2细胞系。结果：双酶切及DNA测序结果表明,成功克隆和构建了含PA28γWT和PA28γMT的pDsRed1-C3和pcDNA3.1(-)Flag重组质粒;荧光显微镜观察证实在293T细胞中pDsRed1-C3/PA28γWT定位在胞核中而pDsRed1-C3/PA28γMT定位在胞浆中;Western Blot检测HepG2/ pcDNA3.1(-)Flag/PA28γWT和HepG2/ pcDNA3.1(-)Flag/PA28γMT,证实所筛选的HepG2细胞高表达相应目标蛋白。结论：成功构建了高表达野生型PA28γ和胞浆定位PA28γ的HepG2细胞系,为进一步研究人类PA28γ基因的功能奠定了基础。     

用于活体影像技术稳定表达荧光素酶的肺癌细胞系的建立

目的:建立稳定表达萤火虫荧光素酶的人肺癌细胞系并进行体外验证.方法:亚克隆萤火虫荧光素酶基因到真核表达载体pRc/CMV2上,构建重组质粒pRc/CMV2.1uc+,转染到肺癌细胞株spc-a-1,经过G418筛选获得单细胞抗性克隆.抗性克隆连续传代,并通过荧光素酶活性检测筛选出高水平表达荧光素酶的稳定细胞克隆,在体外检测细胞的生物发光能力与细胞数量的相关性.结果:构建的pRc/CMV2-1uc+真核表达质粒转染spe-a-1细胞后,经G418筛选出多个抗性克隆;传代至40代时确定有3株细胞的荧光素酶活性最高;活体影像系统体外检测证实阳性细胞株发光强度与数量呈正相关.结论:结果表明成功构建了稳定表达萤火虫荧光素酶的spc-a-1细胞系.     

pcDNA3．1表达载体转染对细胞生长的影响

为了探讨真核表达载体转染对细胞生长的影响,通过脂质体介导将pcDNA3.1( )表达载体DNA转染鼻咽癌细胞系HNE1,G418筛选后,Southern杂交鉴定稳定表达细胞株,以HNE1细胞为对照,观察pcDNA3.1( ）／HNE1克隆细胞的生物学特性;结果显示,在pcDNA3.1( )/HNE1阳性克隆中,一株细胞克隆培养过程中发生自溶性死亡,一株细胞生长明显受到抑制,另一株细胞生长无明显影响,揭示在宿主细胞中pcDNA3.1( )DNA与宿主基因组DNA发生了随机整合,从而表现不同的细胞生物学改变。     

用表达T7RNA聚合酶细胞系拯救麻疹病毒微复制子

构建稳定表达T7RNA聚合酶的细胞系,用于提高拯救麻疹病毒微复制子效率.PCR扩增T7RNA聚合酶基因,克隆到真核表达载体,转染Vero细胞,用G418筛选到稳定表达的细胞株Vero/pcDNA3-T7.用Westernblotting证明了T7RNA聚合酶在细胞中的表达.将T7启动子控制绿色荧光蛋白表达的质粒转染该细胞后,绿色荧光蛋白在细胞株中得到表达.反向插入报告基因的微复制子转染感染麻疹病毒的Vero/pcDNA3-T7细胞后,细胞中能够检测到报告基因的表达.用细胞系取代痘苗病毒系统,可以提高拯救效率.     

Smad4基因siRNA表达载体的构建与鉴定

目的:以Smad4基因为靶标构建小干扰RNA(siRNA)真核表达载体.方法:根据GenBank公布的人Smad4核酸序列及SiRNA设计原则,用AmbionRNAi在线软件,筛选得到2个19 bp片段为靶序列,合成两端带有Bam H Ⅰ、HindⅢ酶切位点的发夹结构寡核苷酸序列,经过退火,将此序列克隆到真核表达质粒pSilencerTM3.1-H1 neo vector中,构建成重组质粒,酶切及测序验证.脂质体介导转染人原代增生性瘢痕成纤维细胞,经G418筛选细胞克隆,并用RT-PCR检测Smad4基因的表达.结果:成功构建了pSilencerTM3.1.H1 neo-Smad4 shRNA表达载体克隆,插入片段测序结果与合成的siRNA序列一致.RT-PCR结果显示转染shRNA1、shRNA2重组质粒的成纤维细胞内Smad4 mRNA水平均降低,其中以pSilencerTM3.1-H1-Smad4 shRNA1更为明显(P＜0.01).结论:构建P-Smad4 shRNA袁达载体成功,为进一步研究Smad4基因的RNA干扰奠定了基础.     

NAG7基因转染对鼻咽癌细胞生长的影响

为了探讨鼻咽癌表达下调基因NAG7对鼻咽癌细胞系HNE1生长的影响, 构建了NAG7基因的真核表达载体pcDNA3.1(+)/NAG7, 并采用脂质体转染技术将真核重组体pcDNA3.1(+)/NAG7质粒和真核空载体pcDNA3.1(+)质粒分别导入HNE1细胞, 经G418筛选后获得稳定转染细胞克隆, RT-PCR和RNA印迹检测NAG7基因的表达, 并通过细胞生长曲线、裸鼠接种和流式细胞等方法对转染细胞的生物学行为进行检测.结果显示：转染NAG7基因后,基因表达增加,细胞生长倍增时间较空载体转染和HNE1明显延长,流式细胞技术检测表明,NAG7可延缓细胞由G0～G1期进入S期;裸鼠接种实验显示转染NAG7基因后的HNE1细胞致瘤性受到抑制.上述结果表明：NAG7基因转染后鼻咽癌细胞生长受到抑制,提示NAG7基因是一鼻咽癌相关的抑瘤基因候选者.     

人尿激酶型纤溶酶原激活因子截短型突变体与绿色荧光蛋白在真核细胞HEK293F中的融合表达

目的:构建人尿激酶型纤溶酶原激活因子(uPA)截短型突变体与绿色荧光蛋白(EGFP)分泌型融合表达载体并在真核细胞中表达。方法:采用PCR法,分别以质粒pIRES2-EGFP和重组质粒pcDNA3.1(+)/uPA为模板,扩增出带BamHⅠ和XbaⅠ酶切位点的EGFP及带NheⅠ和HindⅢ酶切位点的uPA截短体基因片段,先后将EGFP和截短型uPA基因片段克隆到真核表达载体pcDNA3.1(+)上,转入HEK293F细胞,用G418对转染细胞进行加压筛选,通过共聚焦显微镜观察和ELISA方法鉴定表达产物。结果:DNA测序结果显示,uPA不同截短型突变体基因片段与EGFP基因融合的真核表达载体构建成功,共聚焦显微镜观察发现HEK293F细胞中有绿色荧光且定位于细胞质中,ELISA检测到HEK293F细胞培养上清中分泌型融合蛋白的表达。结论:构建了uPA截短型突变体与EGFP分泌型融合表达载体并在真核细胞中表达,为后期研究uPA的相互作用蛋白及其生理功能奠定了基础。     

Investigation of the mechanism of temperature sensitivity of the electroreceptors of ampullae of lorenzini

In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980.     

Determination of stoichiometry of radioiodination of proteins

A sensitive method for the detection of small quantities of hydrophobic antioxidant free radical scavengers such as butylatedhydroxytoluene (BHT) and butylatedhydroxyanisole (BHA) in aqueous samples is described. The procedure involves extraction of the hydrophobic free radical scavenger into an organic solvent phase, followed by the subsequent reaction of an aliquot of this extract with the stable cation radical tris(p-bromophenyl)amminium hexachloroantimonate (TBACA). In experiments with BHT and BHA, the loss of TBACA absorbance at 730 nm was found to be linearly proportional to the amount of antioxidant added, with quantities of BHT as small as 200 pmol being easily detectable. In aqueous suspensions of dimyristoylphosphatidylcholine vesicles, assays of the aqueous BHT concentration showed that BHT partitioned strongly into the membrane phase, achieving very high BHT/phospholipid ratios. For a given concentration of BHT, partitioning into the membrane phase was greater in large, multilamellar liposomes than in either small, single-walled vesicles or in purified rat brain synaptic vesicle membranes. Direct assay of BHT and BHA in phospholipid membranes, however, was complicated by a nonspecific interaction between TBACA and the phospholipid.