新型冠状病毒S1蛋白的表达及活性鉴定

旨在获得具有免疫活性的SARS-CoV-2S1蛋白,为COVID-19的免疫预防及临床诊断提供较好的候选抗原.该研究利用PCR技术扩增出刺突蛋白(Spike,S)的S1亚基序列,通过同源重组法将目的片段克隆至冷休克表达载体pCold I,转化至大肠杆菌BL21感受态细胞内诱导表达S1蛋白,经Ni2+柱亲和层析纯化后,通...     

克隆、表达及纯化核糖体蛋白S6用于体外激酶活性检测

目的:构建40S核糖体蛋白S6的原核表达载体,表达并纯化S6蛋白,将其作为底物用于S6激酶(S6K)的体外活性测定。方法:采用RT-PCR方法从人胚肾细胞HEK293中获取S6 cDNA,将扩增产物克隆至大肠杆菌表达载体中,进行酶切及测序鉴定;IPTG诱导GST-S6融合蛋白在大肠杆菌中表达,用谷胱甘肽亲和层析纯化GST-S6,免疫沉淀法检测该蛋白是否可作为底物用于S6K的体外激酶活性测定。结果:酶切及测序鉴定表明构建了S6原核表达载体,并表达及纯化出GST-S6融合蛋白,相对分子质量为55×103。该蛋白可用于S6K的体外激酶活性测定,特异性强。结论:S6蛋白的克隆、表达与纯化成功,可用于S6K的体外激酶活性测定,为研究S6K的功能奠定了基础。     

重组人CK2β亚基的原核表达、纯化与鉴定

将构建成功的人蛋白激酶CK2β亚基cDNA的重组质粒, 转化大肠杆菌BL21 (DE3), 在IPTG诱导下表达. 表达蛋白大多数以不溶形式存在. 6L(约10.2 g)表达菌抽提得到约20 mg的可溶性表达产物, 通过P11磷酸纤维素一步层析分离, 得到6.8 mg纯化蛋白. SDS-聚丙烯酰胺凝胶电泳结果显示纯化的蛋白为一分子质量26 ku的单一蛋白带.蛋白质印迹结果证明：纯化的表达产物与抗人CK2β抗体可发生特异性免疫反应. CK2β亚基对CK2α有激活作用, 纯化的CK2α和β亚基在等摩尔混合时即可组成有最大生物活性的全酶. 实验结果有力地证明了克隆表达与纯化的重组蛋白是人蛋白激酶CK2β亚基.     

酿酒酵母ATP合酶δ亚基融合蛋白的构建、表达及生物学功能分析

利用分子克隆手段构建了酿酒酵母ATP合酶δ亚基和流感病毒血凝素(haemagglutinin, HA)标签融合蛋白的表达质粒.该融合蛋白在酿酒酵母细胞中能够正常表达,而且能够互补编码δ亚基的ATP16基因缺失株在利用非发酵性碳源方面的表型缺陷,表明该融合蛋白具有野生型δ亚基的功能.同时,构建了在大肠杆菌细胞中表达该δ亚基的ScAtp16p-His6融合蛋白,并用纯化的融合蛋白在家兔中制备了其多抗血清.结果表明此多抗可以很好地与ScAtp16p-His6和HA-ScAtp16p两种融合蛋白特异性结合.这些研究材料的获得为深入研究ATP合酶的解偶联机制和磷酸化调控机理奠定了基础.     

一次大强度运动后大鼠骨骼肌收缩蛋白降解和26S蛋白酶体活性的变化

目的：探讨一次大强度运动对骨骼肌收铺蛋白降解和26S蛋白酶体活性的影响。方法：36只雄性Sprague—Dawley大鼠随机分为6组：安静对照组、运动0．5小时组、运动1小时组、运动后1小时组、运动后2小时组、运动后6小时组,每组6只。运动方式采用一次大强度跑台运动,速度为25m／min,坡度为5％,运动时间分别为0．5小时和1小时。每组按规定时间取材大鼠腓肠肌,观察其3．甲基组氨基酸（3-MH）含量、26S蛋白酶体活性和26S蛋白酶体C2亚基mRNA表达的变化。结果：（1）运动后即刻和运动后6小时大鼠腓肠肌中3-MH含量比安静时高（P〈0．01）,其中运动0．5小时即刻含量最高（P〈0．01）;（2）运动0．5小时后即刻、运动后6小时大鼠腓肠肌26S蛋白酶体活性高于安静水平（P〈0．05）。（3）运动后6小时大鼠腓肠肌26S蛋白酶体C2亚基mRNA表达高于安静水平（P〈0．01）,其它时间点低于安静水平。结论：运动后26S蛋白酶体活性增高可能促使骨骼肌收缩蛋白降解增强,其活性受亚基基因调控。     

Gsα调控小鼠大脑皮层发育

异源三聚体G蛋白激活alpha亚基(Gsα),是一种普遍存在的鸟苷酸结合蛋白,调节受体介导的胞内cAMP信号通路进而参与调控细胞的生命活动。目前Gsα信号通路的研究主要在生物化学和药物方面,但在小鼠大脑皮层发育中的作用还没有详尽的描述。本研究首先利用cre-loxP系统在小鼠大脑皮层神经前体细胞中成功敲除Gsα基因;其次,通过收集出生后不同天数的正常小鼠和敲除小鼠,统计分析后发现敲除鼠的脑重和体重减轻;最后,对小鼠大脑做切片后染色,发现在小鼠大脑皮层条件性敲除Gsα基因的成年鼠中表达thy1-EGFP(增强绿色荧光蛋白)的神经元的数量减少,皮层形成异常。由此推测,Gsα在小鼠大脑皮层发育中发挥重要作用。     

罗马洋甘菊精油的嗅闻缓解WKY大鼠的抑郁样行为

如今,精油香薰治疗被认为是治疗抑郁症的一种可替代疗法.检测了连续两周的罗马洋甘菊(Anthemis nobilis)精油香薰治疗对抑郁症模型大鼠(Rattus norvegicus)-WKY(Wistar-Kyoto)大鼠的抑郁样行为的影响.发现无论是罗马洋甘菊或是其主要成分α-哌烯的香薰治疗都能缓解WKY大鼠在强迫游泳实验中的抑郁样行为.通过蛋白组学同位素标记相对和绝对定量(iTRAQ)技术发现,α-哌烯治疗的WKY大鼠脑内与氧化磷酸化相关的蛋白表达上升,包括海马中的细胞色素C氧化酶6C-2亚基及7A2亚基、ATP酶抑制剂,以及前额叶皮层中的细胞色素c氧化酶6C-2亚基、ATP合成酶e亚基、酰基载体蛋白,以及细胞色素b-c1复合体6号亚基.进一步应用实时定量PCR技术还发现,α-哌烯的香薰治疗使得WKY大鼠海马内小清蛋白的mRNA表达上升,而iTRAQ分析显示,该蛋白在海马的表达上调2.8倍.以上结果提示,线粒体及小清蛋白相关信号通路在α-哌烯香薰治疗WKY大鼠的抑郁样行为中发挥作用.     

宽频噪音对谷氨酸中毒损伤AD模型大鼠不同脑区NMDAR1(ζ1)、NMDAR2A(ε1)亚基表达的影响

目的:观察AD大鼠模型颞叶和额叶在98 dB宽频噪音暴露5 min后不同脑区NMDAR1(ζ1)、NMDAR2A(ε1) 表达的影响.方法: 采用Western Blot及 RT-PCR技术,结合ABR测定方法.结果:①AD模型组大鼠、空白对照组大鼠在98 dB宽频噪音暴露5 min后额叶、颞区、海马及小脑NMDAR1(ζ1)亚基表达无明显差异,但AD模型组表达明显弱于空白对照组;②生理盐水组加噪音后NMDAR1(ζ1)亚基小脑表达最强,颞叶最弱; NMDAR2A(ε1)表达最强为颞叶,海马最弱.③在海马三组大鼠NMDAR1(ζ1) 、NMDAR2A(ε 1)亚基表达有较明显的下调趋势;④空白对照组大鼠NMDAR1(ζ1)、NMDAR2A(ε1)亚基mRNA表达各区无差异.⑤AD模型组大鼠颞叶、海马NMDAR2A(ε1) 表达明显减弱 ,最弱为小脑,额叶次之.结论:噪音刺激抑制AD大鼠模型海马NMDAR1(ζ1)亚基表达,且不在mRNA水平.噪音刺激抑制AD模型大鼠颞叶、海马NMDAR2A(ε1) 亚基表达,且有部位差异,在mRNA水平已调节.     

高活性F1-ATP酶单分子旋转初步观察

从基因突变的F1 ATP酶 (基因突变质粒 ,α C193S ,γ S10 7C ,β亚基带有 10个组氨酸标记 (His Tag) ,转入到菌株大肠杆菌JM10 3)的菌株中筛选出一高表达菌株 .该菌株表达的F1 ATP酶经纯化后其水解活性明显高于文献值 .从单分子水平上进行观察 ,发现在水解ATP过程中 ,γ亚基上连接的荧光标记蛋白微丝 ,其旋转速度要比文献中同样条件下快约一倍     

中东呼吸综合征冠状病毒S1蛋白在昆虫细胞中的重组表达及免疫效果评价

目的:利用昆虫杆状病毒表达系统重组表达中东呼吸综合征冠状病毒(MERS-Co V)S1蛋白,并对其免疫效果进行评价。方法:构建含有MERS-Co V S1基因的重组杆状病毒质粒,转染Sf9细胞包装杆状病毒;重组病毒传代3次获得种子病毒,感染Sf9细胞,收获感染上清,通过镍离子亲和层析纯化获得S1重组蛋白;用纯化的S1蛋白免疫BALB/c小鼠,采用ELISA检测免疫小鼠血清抗原特异性的抗体水平;采用假病毒中和试验检测血清中抗体的中和活性。结果:获得了表达MERS-Co V S1蛋白的重组病毒株,在昆虫细胞中表达并纯化了S1重组蛋白;利用重组表达的S1蛋白免疫小鼠3次,血清S1特异性Ig G抗体滴度可达1∶102 400,免疫小鼠血清稀释至1/5120后中和百分比仍达50%以上。结论:利用昆虫细胞重组表达的MERS-Co V S1蛋白具有良好的免疫原性,并能有效诱导产生高滴度中和抗体,为发展MERS-Co V重组蛋白疫苗奠定了基础。     

非洲猪瘟病毒CP204L基因的原核表达与单抗制备

由非洲猪瘟病毒(ASFV)引起的非洲猪瘟(ASF)给我国养猪业带来了不可估量的经济损失,严重阻碍了我国养猪业的发展,研发ASFV快速诊断试剂是目前最重要的内容之一。CP204L基因编码ASFV结构蛋白p30。本研究以克隆ASFV的CP204L基因为基础,通过基因重组技术,加入His标签,将构建的重组质粒命名为pET-28a-CP204L。将重组质粒转化至大肠杆菌BL21(DE3)感受态细胞,37℃经1mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达6h,表达蛋白进行SDS-PAGE鉴定和Western Blot检测。重组蛋白纯化后免疫小鼠制备筛选单克隆抗体,Western Blot和IFA验证单抗的结合特异性。结果表明,重组的pET-28a-CP204L诱导后表达蛋白为30kD,以不可溶性包涵体形式存在;表达蛋白利用His标签进行纯化,获得纯化蛋白2mg,单克隆抗体筛选获得5株IgG亚型的ASFV p30蛋白的单抗,且均具有良好的结合活性。本研究为发展ASFV检测方法提供了基础。     

Purification of recombinant activin A using the second follistatin domain of follistatin-related gene (FLRG)

Activins are multifunctional growth factors belonging to the transforming growth factor-beta superfamily. Isolation of activins from natural sources requires many steps and only produces limited quantities. Even though recombinant preparations have been used in recent studies, purification of recombinant activins still requires multiple steps. To purify recombinant activin A, we have developed a simple method using the second follistatin domain of an activin-binding protein follistatin-related gene (FLRG). An affinity column was prepared with a partial FLRG fusion protein. The partial FLRG protein contained the second follistatin domain and the C-terminus acidic domain, and was tagged with six histidine residues at its N-terminus. The fusion protein was expressed in Escherichia coli and purified with nickel affinity column. Thereafter, the purified fusion protein was coupled to NHS-activated column. Recombinant activin A was produced in Chinese hamster ovary (CHO) cells, which were stably transfected with rat inhibin/activin betaA-subunit cDNA. After 48-h suspension culture of the cells in a serum free medium, the culture media was recovered and passed through the FLRG-coupled column. After washing with phosphate-buffered saline, bound protein was eluted out with an acidic buffer. Any significant contaminations were not detected when the purified protein was analyzed by SDS-PAGE. Apparent sizes of the protein were 14 and 28 kDa under the reduced and non-reduced conditions, respectively. Western blot analysis confirmed that the purified protein was activin A. The purified recombinant activin stimulated p3TP-lux reporter activity in CHO cells and follicle-stimulating hormone secretion from rat pituitary cells.     

huGM-CSF(9-127)-IL-6(29-184)融合蛋白的复性及纯化研究

利用Q Sepharose H.P.离子交换柱层析在8mol/L尿素变性条件下对huGM-CSF(9-127)-IL-6(29-184）融合蛋白进行初步纯化,然后再利用Sephacryl S-200分子筛柱层析复性及纯化后获得目的蛋白,其纯度达到95％以上。该纯化方案成功地解决了稀释复性或透析复性产物在进行Q Sepharose H.P.离子交换柱层析时目的蛋白不稳定而沉积于柱上的问题,获得了较好的复性效果,复性率达到80％以上。使用该纯化方案,1天内便可基本完成重组蛋白的复性及纯化过程,而且也便于扩大。     

Rapid purification method for the 26S proteasome from the filamentous fungus Trichoderma reesei

We have developed a fast and simple two column chromatographic method for the purification of the 26S proteasome from the filamentous fungus Trichoderma reesei that simplifies the overall procedure and reduces the purification time from 5 to 2.5 days. The combination of only the anionic exchange POROS^® HQ column (Applied Biosystems) together with a size exclusion column has not been used previously for proteasome purification. The purified complex was analysed further by two-dimensional electrophoresis (2DE) and examined by transmission electron microscopy (TEM). A total of 102 spots separated by 2DE were identified by mass spectrometry using cross-species identification (CSI) or an in-house custom-made protein database derived from the T. reesei sequencing project. Fifty-one spots out of 102 represented unique proteins. Among them, 30 were from the 20S particle and eight were from the 19S particle. In addition, seven proteasome-interacting proteins as well as several non-proteasome related proteins were identified. Co-purification of the 19S regulatory particle was confirmed by TEM and Western blotting. The rapidity of the purification procedure and largely intact nature of the complex suggest that similar procedure may be applicable to the isolation and purification of the other protein complexes.     

Refolding and purification of recombinant human (Pro)renin receptor from Escherichia coli by ion exchange chromatography

Purification of the recombinant human renin receptor (rhRnR) is a major aspect of its biological or biophysical analysis, as well as structural research. A simple and efficient method for the refolding and purification of rhRnR expressed in Escherichia coli with weak anion‐exchange chromatography (WAX) was presented in this work. The solution containing denatured rhRnR in 8.0 mol/L urea extracted from the inclusion bodies was directly injected into the WAX column. The aggregation was prevented and the soluble form of renatured rhRnR in aqueous solution was obtained after desorption from the column. Effects of the extracting solutions, the pH values and urea concentrations in the mobile phase, as well as the sample size on the refolding and purification of rhRnR were investigated, indicating that the above mentioned factors had remarkable influences on the efficiency of refolding, purification and mass recovery of rhRnR. Under the optimal conditions, rhRnR was successfully refolded and purified simultaneously by WAX in one step within only 30 min. The result was satisfactory with mass recovery of 71.8% and purity of 94.8%, which was further tested by western blotting. The specific binding of the purified rhRnR to recombinant human renin was also determined using surface plasmon resonance (SPR). The association constant of rhRnR to recombinant human renin was calculated to be 3.25 × 10⁸ L/mol, which demonstrated that rhRnR was already renatured and simultaneously purified in one step using WAX. All of the above demonstrate that protein folding liquid chromatography (PFLC) should be a powerful tool for the purification and renaturation of rhRnR. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:864–871, 2014     

S100a0 (αα) Protein: Distribution in Muscle Tissues of Various Animals and Purification from Human Pectoral Muscle

When the concentrations of alpha-S100 (alpha subunit of S100 protein) and beta-S100 (beta subunit) proteins in various tissues of human and rat were determined by the immunoassay method, immunoreactive beta-S100 was present at high levels in the CNS, adipose tissue, and cartilaginous tissue. In contrast, the alpha-S100 was found in the heart and skeletal muscles at concentrations much higher than in the CNS. The concentration of alpha-S100 protein was also high in the heart and skeletal muscles of bovine, porcine, canine, and mouse. Since beta-S100 protein levels in those tissues were low, it was suggested that S100 protein in the muscle tissues is present mainly as the alpha alpha form (S100a0 protein). To confirm the above findings, immunoreactive alpha-S100 protein was purified from human pectoral muscle by employing column chromatographies with butyl-Sepharose, diethylaminoethyl (DEAE)-Sepharose, Sephadex G-75, and finally with an anion-exchange Mono Q column in a HPLC system. The elution profile of alpha-S100 protein from the Mono Q column suggested some heterogeneity of the final preparation. However, each of these fractions traveled with a single band at a position similar to that of bovine S100a0 protein on slab-gel electrophoresis. The amino acid composition of the final preparation was very similar to the composition of bovine S100a0 protein. The purified alpha-S100 protein was eluted from a gel-filtration column (Superose 12) in the same fraction as bovine S100a0 protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for direct interaction of Gs alpha with the Ca2+ channel of skeletal muscle.

The alpha subunits of heterotrimeric GTP-binding (G) proteins act upon ion channels through both cytoplasmic and membrane-delimited pathways (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213). The membrane pathway may involve either a direct interaction between G protein and ion channel or an indirect interaction involving a membrane-delimited second messenger. To distinguish between the two possibilities, we tested whether a purified G protein could interact with a purified channel protein in a defined system to produce changes in channel currents. We selected the alpha subunit of Gs and the dihydropyridine (DHP)-sensitive Ca2+ channel of skeletal muscle T-tubules, the DHP binding protein (DHPBP), because: 1) a membrane-delimited interaction between the two has been shown (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213; Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895); and 2) at the present time, these Ca2+ channels are the only putative G protein channel effectors which, following purification, still retain channel function. We used a defined system in which purified components were studied by direct reconstitution in planar lipid bilayers. Just as we had found in crude skeletal muscle T-tubule membranes (Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895), alpha*s but not alpha*i-3 stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between Gs alpha and Ca2+ channels. To establish whether the two proteins were physically associated in the native T-tubule membrane, we examined the ability of either endogenous G proteins or exogenous alpha*s to purify with detergent-solubilized DHPBP through a wheat germ agglutinin affinity column and a sucrose gradient. Small amounts of a labeled G protein were found to co-purify with DHPBP. In addition, partially purified DHPBP increased the sedimentation rate of purified alpha*s but not alpha*i-3. G proteins were immunoprecipitated with an antibody to the alpha 1 subunit of the DHPBP, and, in addition, both alpha s and the beta subunit of Gs were detected in Western blots of the partially purified DHPBP. The results suggest that Gs and Ca2+ channels are closely associated in the T-tubule plasma membrane, and we conclude that skeletal muscle Ca2+ channels are direct effectors for Gs.     

Small molecular weight GTP-binding proteins in human erythrocyte ghosts

GTP-binding proteins (G proteins) were extracted from human erythrocyte ghosts by sodium cholate and purified by gel filtration on an Ultrogel AcA-44 column followed by hydroxyapatite column chromatography. At least two peaks of G proteins were separated by hydroxyapatite column chromatography. The second peak contained G proteins recognized by the antibodies against the respective alpha subunits of Gs and Gi, and the ras protein, while the G protein of the first peak was not recognized by any of these antibodies. The G protein of the first peak was purified further by Mono Q HR5/5 column chromatography. The purified G protein showed a molecular weight of about 22 kDa on SDS-polyacrylamide gel electrophoresis. This G protein (22K G) specifically bound guanosine 5'-(3-O-thio) triphosphate (GTP gamma S), GTP and GDP with a Kd value for GTP gamma S of about 50 nM. GTP gamma S-binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. The G proteins recognized by the antibodies against the alpha subunit of Go and the ADP-ribosylation factor for Gs, designated as ARF, were not detected in human erythrocyte ghosts. These results indicate that there are at least two species of small molecular weight G proteins in human erythrocyte ghosts: one is the ras protein and the other is a novel G protein of 22K G.     

人胱硫醚β-合酶及其截短型片段的表达、纯化和活性测定

将人胱硫醚β-合酶(CBS)基因克隆至质粒pGEX-4T-1中,获得的重组质粒pGEX-4T-1-CBS转入大肠杆菌E.coli Rosetta (DE3)菌株,构建了高效表达CBS的重组菌E.coli Rosetta (pGEX4T-1-CBS)。重组菌在0.1mmol/L的IPTG于30℃诱导16h,可溶性CBS表达量达到28mg/L培养基。将重组菌破碎后上清液经GSTrap Fast Flow亲和层析一步纯化得到CBS融合蛋白,在凝血酶柱上切割缓冲液中加入3%甘油和0.1%CHAPS可以有效抑制酶切后CBS聚沉,酶活性回收率为54.8%,蛋白质产率为15.2mg/L培养基,纯度达到95%,单位酶活为143U/mg,终浓度为1mmol/L的S-腺苷甲硫氨酸(AdoMet)可使CBS单位酶活提高5.1倍,达到735U/mg。同时构建了表达CBS_1-413(删除了CBS羧基端调控域138个氨基酸残基)的重组菌E.coli Rosetta (pETDuet-1-CBS_1-413),经过一步HisTrap Fast Flow亲和层析,酶活性回收率为74.3%,蛋白质产率为12.8mg/L培养基,纯度达到95%,单位酶活为965U/mg; 还表达和纯化了胱硫醚β-裂解酶(CBL),并在此基础上建立了一种新的CBL偶联的CBS酶活性测定方法。     

High-level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain

Botulinum neurotoxin light chain (BoNT LC, 50 kDa) is responsible for the zinc endopeptidase activity specific for proteins of neuroexocytosis apparatus. We describe the expression of recombinant type A BoNT LC in Escherichia coli as well as the purification and characterization of the recombinant protein. A high level of expression of BoNT/A LC was obtained by an extended postinduction time of 15 h at 30 degrees C. Recombinant BoNT/A LC was isolated from an Ni(2+) column. Due to its high pI ( approximately 8.7), purification was achieved by a single step of passing the protein through anion-exchange chromatography at pH 8.0 without the need of elution. The purified recombinant BoNT/A LC retained proteolytic activity and had a secondary structure similar to that of native LC determined by CD measurement.