大鼠α-酰胺化酶(α-AE)在大肠杆菌中的表达、纯化及活性研究

α-酰胺化是神经和内分泌系统中许多生物活性肽重要的翻译后加工过程,C末端α酰胺基团的存在对于许多生物活性肽的生物活性极为重要。本研究通过PCR扩增获得了编码大鼠α-酰胺化酶的基因,以pET-30a为载体,重组质粒pET-A转化至 E.coli BL21成功表达了α-酰胺化酶。采用Ni2+-NTA亲和层析纯化重组蛋白。该蛋白具有催化三肽底物((Dns-Tyr-Val-Gly)成为酰胺化二肽 (Dns-Tyr-Val-NH2)的酶活性,这表明重组蛋白是α-酰胺化酶,有可能用于生物活性肽的酰胺化研究。     

Characterization of Rat N-Acetylglucosaminyltransferase I Expressed in Esherichia coli

The catalytic domain (sGnT-I) of rat liver N-acetylglucosaminyltransferase I (GnT-I) was expressed in Escherichia coli. Lysates from pETsGnT-I transformants contained a prominent protein species of 46 kDa with which a significant GnT-I activity was associated. To purify the relevant enzyme, we constructed cDNAs encoding sGnT-ICH and sGnT-INH, which had six additional histidine residues as an affinity tag at the C-terminal and the N-terminal of sGnT-I, respectively, and introduced them into E. coli cells for expression. sGnT-INH was purified and its enzymatic properties were examined.     

重组人SNX9蛋白在大肠杆菌中的表达、纯化及性质分析

SNX9是近年发现的一种蛋白分选与转运蛋白, 属于SNX家族。将原核表达载体pET-SNX9重组质粒转化大肠杆菌BL21(DE3),IPTG诱导表达可获得分子量为70 kD的融合蛋白,Western blotting结果证明此蛋白即为目的蛋白,经检测融合蛋白主要以可溶形式表达。表达产物通过亲和层析和分子筛层析两步纯化,可以获得纯度超过95％的SNX9融合蛋白。分子筛层析中蛋白的出峰体积显示SNX9融合蛋白是以二聚体形式存在。Native-PAGE和动态光散射实验均显示其具有良好的均一性。热稳定性实验表明SNX9蛋白在15 ℃以下基本稳定。这些信息为进一步研究SNX9 的结构和功能具有重要的指导意义。     

重组人MBD4蛋白在大肠杆菌中的表达、纯化及活性分析

为获得重组人MBD4蛋白,将编码MBD4的开放式阅读框（ORF）插入原核表达载体pGEX6P1 GST基因下游的多克隆位点（MCS）.将获得的表达质粒转化入大肠杆菌BL21(DE3) 菌株扩大培养并用IPTG诱导融合蛋白的表达.用谷胱甘肽琼脂糖凝胶 4B亲和介质从菌体裂解液中纯化了GST-MBD4融合蛋白.经过Prescision protease专一性裂解成功去除了融合蛋白上的GST标签.通过Mono Q阴离子交换层析获得了纯度达94%以上的MBD4蛋白,该蛋白具有甲基化DNA结合和糖苷酶生物活性.     

Bacillus Stearothermophilus IAM 11001 L-阿拉伯糖异构酶在大肠杆菌中的表达、纯化及活性研究

L-阿拉伯糖异构酶是生物法生产新型功能性因子D-塔格糖最为有效的酶。本文获得了一种新型耐热L-阿拉伯糖异构酶的编码基因araA,来源于Bacillus stearothermophilis IAM 11001,经NCBI Blastn分析,与GenBank中Thermus sp. IM6501 araA序列的同源性为95%,并将该新基因提交到GenBank,获得登陆号：EU394214。以pET-22b(+)为载体质粒,E. coli BL21(DE3)为宿主细胞,构建了基因重组菌,IPTG可诱导目的蛋白的过量表达;经亲和层析纯化的重组蛋白样品进行SDS-PAGE电泳分析,约在59 kDa处出现显著的特征蛋白条带;同时对重组L-AI的活性进行了初步研究,全细胞反应24小时D-塔格糖的转化率为39.8%。     

Characterization of Green Tissue-Specific Phytochrome Isolated Immunochemically from Pea Seedlings

Phytochrome was isolated and purified from light-grown pea (Pisumsativum) seedlings and compared with that from dark-grown seedlingsin terms of spectral and immunochemical properties. Approximately40% of phytochrome in the brushite eluate prepared from light-grownpea tissue bound with a monoclonal anti-pea phytochrome antibody(mAP3), but the remaining 60% did not. Both phytochrome fractionsshowed a typical photoreversible absorbance change after alternatered and far-red actinic irradiations, which was similar to thatof phytochrome from etiolated pea tissue. The peptide mappingof the mAP3-bound phytochrome from light-grown tissue was essentiallythe same as that of the mAP3-bound phytochrome from etiolatedtissue. However, the digestion pattern of the phytochrome thatwas prepared from light-grown tissue but which did not bindto mAP3 was obviously different from that of mAP3-bound phytochrome.Polyclonal anti-pea phytochrome antibodies and mAP5 and 10,however, bound to both the phytochromes. These results suggestthat light-grown tissue contains two phytochrome pools whichare distinct from each other with respect to the primary structureof the phytochrome polypeptide but which share a few commondeterminant sites. ¹ Permanent address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Fukazawa, Tokyo 158, Japan (H.A.), and Department of Botany, Faculty of Science, Universityof Tokyo, Hongo, Tokyo 113, Japan (M. F.).     

大肠杆菌表达的人重组IL-3的纯化

本文继大肠杆菌表达含凝血酶切点的人重组IL-3融合蛋白成功的基础上进一步探讨了天然型rhIL-3的纯化。超声破碎细菌细胞得包涵体,经洗涤处理可使包涵体纯度达90％,用8mol/L尿素变性溶解包涵体沉淀后直接稀释复性,再超滤浓缩、凝血酶消化,释放天然型rhIL-3。经DEAE Sepharose Fast Flow和Sepharcyl-100 HR层析得到天然型IL-3,纯度达96％,回收率20％以上,具有刺激正常人骨髓细胞形成集落的活性。本实验为大批量重组IL-3的生产创造了条件。     

大肠杆菌表达的重组白细胞介素-2的初步纯化

本文对大肠杆菌表达产生的重组白细胞介素-2进行了纯化研究。通过比较两种方法制备的rIL-2包含体的纯度,发现用4mol/L脲溶解可溶性细菌蛋白后可使rIL-2包含体纯度达70％;在高浓度变性剂条件下进行凝胶过滤,解决了rIL-2易聚合的问题;结合透析,利用空气氧化形成高活性氧化型rIL-2;经SephadexG-100凝胶过滤,DEAE离子交换等步骤纯化,得到了均一性rIL-2,纯度达98％,比活达4.3×10~6u/mg蛋白,得率为30.8％。     

三结构域重组FN多肽表达质粒的构建及其表达产物性质的初步鉴定

为探讨三结构域重组迁连蛋白（ＦＮ）在肿瘤治疗中的作用,构建了两个三结构域重组ＦＮ表达质粒ｐＦ９４－６２和ｐＦ９４－８２,它们分别编码两个重组多肽：ＣＨ６２（ＦＮＰｒｏ１２３９－Ｓｅｒ１５１５经Ｍｅｔ、Ａｌａ １６９０－Ｖａｌ２０４９相连）和ＣＨ８２（从ＣＨ６２中删除了ＨｅｒｐⅡＣ端和ＣｅｌｌⅡ结构域Ｎ端的Ｐｒｏ１９５３－Ｇｌｕ１９７８）。含表达质粒ｐＦ９４－８２的工程菌经３７℃培养,ＣＨ８２得到表     

人骨骼肌肌钙蛋白C在大肠杆菌中的表达及其抗肿瘤作用

为了探索重组人骨骼肌型肌钙蛋白C(rhTnC)的抗肿瘤作用,采用PCR技术,从人乳腺cDNA文库中获得编码TnC的cDNA序列,在大肠杆菌中进行了表达.经镍柱亲和层析纯化后,分别检测其在体外对人脐静脉内皮细胞生长的抑制作用与对小鼠异位移植肿瘤(S-180)生长的抑制作用.结果表明,rhTnC在大肠杆菌中呈可溶性表达.分离纯化后的rhTnC在体外对人脐静脉内皮细胞生长呈剂量依赖性抑制(IC50为7.5μg/ml);对小鼠异位移植肿瘤的生长也有明显的抑制作用,在20mg/(kg·d)剂量下对S-180的抑制率为64.4%.以上结果说明,rhTnC可抑制血管内皮细胞生长,并具有抗肿瘤作用.     

重组蛋白GST-OsCATB的表达特性研究

为了阐明水稻Catalase（CAT）的酶学功能,首先需要获得出足量的、活性的该酶蛋白。本研究克隆了水稻OsCAT_B基因(GenBank accession No.D26484),构建到原核表达载体pGEX-6p-3中形成重组蛋白,继而转入E.coli菌株BL21中进行表达特性研究。结果表明,GST-OsCAT_B融合蛋白在E.coli中进行了过量表达,表达受到诱导剂浓度、诱导时间、诱导温度和诱导体系等多因素影响;通过谷胱甘肽Sepharose-4B亲合层析,纯化出足量、活性的融合蛋白GST-OsCAT_B,每克表达细胞（干重）中得率为51 mg GST-OsCAT_B。     

Photomorphogenesis in Sinningia speciosa, cv. Queen Victoria I. Characterization of Phytochrome Control

The morphological development of Sinningia speciosa plants that were exposed to supplementary far red light was very different from that of plants receiving dark nights. After several nights of such irradiation, stems and petioles were elongated, petioles were angulated, leaf blade expansion was inhibited, plants were chlorotic and the accumulation of shoot dry weight was retarded.

Red reversibility of the morphological changes potentiated by far red light indicated control by the phytochrome system. A high P_FR level during the last half of the night inhibited stem elongation and promoted leaf blade expansion, but both of these processes were hardly affected by the P_FR level during the first half of the night. Thus sensitivity to P_FR was cyclic.

The interpretation of our experiments was complicated by quantitative morphological differences resulting from long, as compared to short, far red irradiations.

     

大肠杆菌表达重组人白细胞介素-9的纯化及部分性质

表达重组人白细胞介素－９（ｒｅｃｏｍｂｉｎａｎｔｈｕｍａｎｉｎｔｅｒｌｅｕｋｉｎ－９ｒｈＩＬ－９）的大肠杆菌经破碎、包涵体洗涤、裂解提取、凝胶过滤和离子交换色谱分离,得到了电泳纯的ｒｈＩＬ－９,回收率６６％,分子量与理论值相符,有刺激小鼠骨髓巨核系集落生成的活性．为ｒｈＩＬ－９的大规模制备及更深入的研究奠定了基础     

Changes in the Content of Phytochrome I and II Apoproteins in Embryonic Axes of Pea Seeds during Imbibition

The contents of phytochrome I and II in crude extracts fromembryonic axes of Pisum sativum cv. Alaska seeds were immunochemicallydetermined using purified pea phytochrome I and II as standards.We have produced and used three different types of mouse monoclonalanti-pea phytochrome antibodies (mAP) such as one reacting preferentiallywith phytochrome I, one with phytochrome II, and one with bothI and II. Phytochrome II was separated from I in the samplesusing immobilized column chromatography with mAPl. The amountsof two phytochrome species were quantitatively measured withwestern blotting and ELISA. Ca. 0.2 µg /axis of phytochromeI and ca. 0.05 µg /axis of phytochrome II were detectedby ELISA after imbibition for 12 h in the dark, though smallamounts of both were detected in dry axes. Ca. 0.05 µg/axis each of phytochrome I and II were detected by ELISA afterimbibition for 12 h in the light, and the results were confirmedby western blotting. This study showed that phytochrome II isnot green-tissue-specific, being also found in dark-imbibedembryonic axes, and that although light significantly lowersthe content of phytochrome I in the axis, it does not significantlyaffect that of phytochrome II. (Received June 10, 1987; Accepted August 27, 1987)     

Rhizoid Differentiation in Spirogyra: III. Intracellular Localization of Phytochrome

Localization of phytochrome which mediates rhizoid differentiation in Spirogyra was investigated. The red-absorbing form of phytochrome (Pr) seems to be distributed all over the cell periphery which remained in the centripetal end part after the centrifugation, as rhizoids formed equally well with red spotlight irradiation of three different parts of an end cell, i.e. distal end, middle, and proximal end, and with irradiation of centrifugal and centripetal end parts of a centrifuged end cell. The Pr distribution was confirmed with an experiment using far red irradiation over the entire cell, centrifugation, and red spotlight irradiation. The Pr-phytochrome molecules appeared to be mobile because no dichroic orientation was shown with polarized red spotlight irradiation. On the contrary, it is suggested that far red-absorbing form of phytochrome molecules are evacuated from the centripetal end part by the centrifugation in an experiment involving red irradiation over the entire cell-centrifugation-far red spot irradiation. Rhizoid formation was repressed markedly by far red irradiation of the centrifugal end part but not of the centripetal end part.     

Intracellular Distribution of Heat Shock Proteins in Pigeon Pea (Cajanus cajan)

The proteins synthesized In response to higher temperature In pigeon pea (Cajanus cajan) plants have been studied with respect to their Intracellular localization using root tissue. The heat shock proteins (hsps) of 18, 20, 22 and 24 kD were found to be associated with mitochondrial and membrane fractions, while the 60, 70 and 81 kD hsps were found In the soluble fraction. No evidence for the presence of hsps among the proteins synthesized in organello by isolated mitochondria could be obtained. Low molecular weight hsps (18, 20, 22 and 24 kD) were found associated with mitochondria Isolated from the heat shocked tissue suggesting that these hsps may have been transported post-translationally into mitochondria.     

大肠杆菌表达的戊型肝炎病毒ORF2多肽对恒河猴的免疫保护研究

在大肠杆菌中表达的一段戊型肝炎病毒（HEV）结构蛋白NE2,纯化后以弗氏佐剂,按0d,10d,30d的方案10μg/针的剂量免疫3只恒河猴,在第2周抗体阳转,第6周时1只滴度达1∶100 000,另2只滴度1∶20 000,此时以106 PCR滴度的HEV病毒粪悬液攻击。对照组3只均出现血清转氨酶（ALT）升高,抗体阳转,粪便持续排毒1月以上;疫苗组无一发病,未检出非疫苗来源的抗体,其中1只始终未检出粪便排毒,另2只仅出现短暂排毒。以一份NE2免疫后猴血清（滴度1∶20 000）与103 PCR滴度的病毒混匀后感染2只恒河猴,结果对照组2只均持续排毒3周以上,抗体阳转,1只ALT明显升高;而抗体中和组2只猴始终未检出粪便排毒,抗NE2抗体缓慢下降,ALT正常。这些结果表明NE2具有良好的免疫原性和免疫保护性,有可能成为有效的戊肝疫苗。