用酵母双杂合系统筛选与人血小板生成素受体c—Mpl相互作用 …

血小板生成素（ｔｈｒｏｍｂｏｐｏｉｅｔｉｎ,ＴＰＯ）是调节血小板生成最主要的细胞因子,其生物学效应由其受体ｃ－Ｍｐｌ介导,利用酵母双杂合系统（ｔｗｏ－ｈｙｂｒｉｄ ｓｙｓｔｅｍ）筛选与ｃ－Ｍｐｌ相互作用的蛋白质因子,以Ｇａｌ ４ＢＤ融合ｃ－Ｍｐｌ膜内部分ｃＤＮＡ的ｐＡＳＭＭ为靶蛋白质粒,筛选了人胎盘ｃＤＮＡ文库,分离到人波形纤维蛋白部分编码序列,首次检测到波形纤维蛋白与ＴＰＯ受体之间的相互作用,这     

人血清中真核复制起始区DNA（Ors12DNA）结合蛋白的鉴定和纯化

利用含有真核细胞复制起始区（ＯｒｉｇｉｎｏｆＤＮＡＲｅｐｌｉｃａｔｉｏｎ）的Ｏｒｓ１２ＤＮＡ作为探针，检测了人血清中与Ｏｒｓ１２ＤＮＡ特异结合的蛋白质。凝胶电泳迁移率改变实验表明人血清中存在特异Ｏｒｓ１２ＤＮＡ结合蛋白。实验还对影响Ｏｒｓ１２ＤＮＡ与蛋白质最适宜结合的各种因素进行了研究。苯酚处理反应体系及限制性内切酶的酶切位点保护实验都证实了Ｏｒｓ１２ＤＮＡ－蛋白质复合物的存在。利用硫酸铵盐析及ＤＮＡ亲和层析，对血清中的Ｏｒｓ１２ＤＮＡ结合蛋白进行了部分纯化，ＳＤＳ－聚丙烯酰胺凝胶电泳结果显示，Ｏｒｓ１２ＤＮＡ结合蛋白为一组非均一的蛋白质，亚基分子量分别为６４ｋＤ、４４ｋＤ和２４ｋＤ。     

Bc1-2 基因加强表达对 OA 诱发的细胞编程死亡的效应

ＯＡ能诱发人神经母细胞瘤ＳＫ细胞进行编程死亡．死亡细胞缩小变圆，细胞质凝聚，ＤＮＡ有控降解成约２００ｂｐ左右的片段，且这一过程可由蛋白质合成抑制剂亚胺环己酮（ＣＨＸ）抑制．将编码Ｂｃ１－２全长蛋白质的ｃＤＮＡ植入ｐＸＪ４１ｎｅｏ载体中，使其表达由ＨＣＭＶ病毒启动子控制．形成的顺义（ｐＢｃｌ－２－Ｓ）及反义（ｐＢｃｌ－２－ＡＳ）表达质粒经转染导入ＳＫ细胞中获得稳定转染子，Ｗｅｓｔ－ｅｒｎ印迹表明顺义转染子表达较大量的２６ｋｄＢｃ１－２蛋白，而反义转染子则不表达．增强表达的Ｂｃ１－２基因产物对ＯＡ引ＳＫ细胞编程死亡无抑制效应．     

人血小板生成因子（TPO）的cDNA克隆及促血小板生成效应

用ＲＴ－ＰＣＲ方法，从人胎肝总ＲＮＡ中扩增得到约１．１ｋｂ人ＴＰＯｃＤＮＡ编码顺序，并进行分子克隆．将其中一个克隆（ｐＴＰＯ４７）亚克隆到Ｍ１３载体，测定全部ＤＮＡ顺序．它的碱基顺序与已报道的顺序完全相同，有一个１０５９ｂＰ的开放阅读框架可编码３５３个氨基酸．ｐＴＰＯ４７亚克隆到哺乳动物表达载体ｐＣＥＰ４，在哺乳动物２９３细胞系瞬时表达后，进行Ｎｏｒｔｈｅｒｎ杂交，结果显示有约１．４ｋｂ的转录产物．瞬时表达培养液腹腔注射小鼠后，可提高血小板计数４２．３％．血小板生成因子的克隆对进一步研究血小板生成调节以及该因子作用的分子机理具有重要意义．     

GHRP—scu—PA—32K突变体的构建,表达及纯化产物的部分性 …

在ｓｃｕ－ＰＡ３２Ｋ的ｃＤＮＡ分子基础上经定点突变,在Ｎ端紧接Ｌｅｕ＾１以前引入编码ＧＨＲＰ四肽的寡核苷酸序列（ＧＧＴＣＡＴＡＧＧＣＣＴ）,构建了ＧＨＲＰ－ｓｃｕ－ＰＡ－３２Ｋ的突变体ｃＤＮＡ。将它克隆到表达载体ｐＣＭ－β－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ＾－细胞。筛选到的稳定表达株在无 清培养基的表达量为５８０ＩＲ／（１０＾６细胞．２４ｈ）。经锌离子螯合亲和柱纯化的产物,ＳＤＳ－ＰＡＧＥ显示为一蛋     

胞内型磷脂酶A2基因亚克隆与基因表达

目的 研究花生四稀酸在细胞中的释放及前列腺素的合成,构建与花生四稀酸释放相关的表达系统。即胞内型磷脂酶Ａ２（ｃｙｔｏｓｏｌｉｃ ｐｈｏｓｐｈｏｌｉｐａｓｅ ｃＰＬＡ２）ｃＤＮＡ－ｐＲＣ／ＣＭＶ。方法 从克隆载体ｐＭＴ２用Ｓａｌ１制备ｃＰＡＬ２ｃＤＮＡ;平末端连接ｃＰＡＬ２－ｐＲＣ／ＣＭＶ;转染鼠巨噬细胞及筛选正向阳性克隆;ｎｏｒｔｈｅｒｎ ｂｌｏｔ检测ｃＰＬＡ２ ｃＤＮＡ基因表达。结果 人ｃＰＬＡ     

多能硫杆菌RubisCO基因（rbcL－rbcS）的克隆及限制性内切酶图谱分析

以光合细菌圆球状红杆菌Ｒｈｏｄｏｂａｃｔｅｒｓｐｈａｅｒｏｉｏｄｅｓ的ｒｂｃＬ－ｒｂｃＳ基因为探针，与多能硫杆菌（Ｔｈｉｏｂａｃｉｌｌｕｓｖｅｒｓｕｔｕｓ）染色体ＤＮＡ酶切谱带进行Ｓｏｕｔｈｅｒｎ杂交，检测到了ｒｂｃＬ－ｒｂｃＳ基因的同源序列，又以ｐＵＣ９为载体，克隆了Ｔ．ｖｅｒｓｕｔｕｓ染色体ＤＮＡＰｓｔＩ酶切片段，构建成Ｔ．ｖｅｒｓｕｔｕｓ基因文库，并从这个基因文库中筛选到了含有ＲｕｂｉｓＣＯ基因的重组质粒，将其命名为ｐＳＤＬＳ－１０，进一步对ｐＳＤＬＳ－１０进行了限制性酶切分析，作出了ｐＳＤＬＳ－１０限制性内切酶图谱     

ORL1受体在SK-N-SH细胞中的功能性表达及其跨膜信号传导

一种新型的阿片受体ＯＲＬ１（ｏｐｉｏｉｄ－ｒｅｃｅｐｔｏｒ－ｌｉｋｅ１ｒｅｃｅｐｔｏｒ）及最近发现的ＯＲＬ１的内源性激动剂（ｎｏｃｉｃｅｐｔｉｎ）在痛觉的调制机制中扮演着某种重要角色．通过Ｎｏｒｔｈｅｒｎ杂交技术在人的神经母细胞ＳＫ－Ｎ－ＳＨ中检测到ＯＲＬ１受体ｍＲＮＡ的天然高量表达；ｃＡＭＰ放射免疫测定技术发现，在经ｎｏｃｉｃｅｐｔｉｎ处理的ＳＫ－Ｎ－ＳＨ细胞中，由环甘酸环化酶激活剂（Ｆｏｒｓｋｏｌｉｎ）引起的细胞内ｃＡＭＰ水平的升高受到抑制，其ＩＣ５０为６０±１５ｎｍｏｌ／Ｌ，最大抑制率为７０％．同时，用ｄｅｌｔａ阿片受体ＤＯＲ激动剂（ＤＰＤＰＥ）、Ｍｕ阿片受体ＭＯＲ激动剂（ＤＡＧＯ）分别处理ＳＫ－Ｎ－ＳＨ细胞，也产生了不同程度的抑制，但抑制率均不如ｎｏｃｉｃｅｐｔｉｎ．发现阿片受体的广谱拮抗剂（ｎａｌｏｘｏｎｅ），可使ｎｏｃｉｃｅｐｔｉｎ及ＤＰＤＰＥ的抑制作用反转；用ＤＯＲ的特异性拮抗剂（ｎａｌｔｒｉｎｄｏｌｅ）只能使ＤＰＤＰＥ的抑制反转，而不能影响ｎｏｃｉｃｅｐｔｉｎ的抑制作用．用５μｍｏｌ／Ｌ的ｎｏｃｉｃｅｐｔｉｎ对ＳＫ－Ｎ－ＳＨ细胞进行２０ｍｉｎ（３７℃）的预处理，可引起细胞对ｎｏｃｉｃｅｐｔｉ     

人血小板生成因子（TPO）的cDNA克隆在小板生成效应

用ＲＴ－ＰＣＲ方法，从人胎肝总ＲＮＡ中扩增得到约１．１ｋｂ人ＴＰＯ ｃＤＮＡ编码顺序，并进行分子克隆，将其中一个克隆（ｐＴＰＯ４７）亚克隆到Ｍ１３载体，测定全部ＤＮＡ顺序。它的碱基顺序与已报道的顺序完全相同，有一个１０５９ｂｐ的开放阅读框架可编码３５３个氨基酸。ｐＴＰＯ４７亚克隆到哺乳动物表达载体ｐＣＥＰ４，在哺乳动物２９３细胞系瞬时表达后，进行Ｎｏｒｔｈｅｒｎ杂交，结果显示有约１．４ｋｂ的转录产     

核酸的分子静电势（I）──C－DNA的分子静电势

对于序列聚（ｄＧ－ｎｒ）和聚（ｄＡ－ｄＴ）来说，本文介始了Ｃ－ＤＮＡ活性位置的分子静电势（ＭｏｌｅｃｕｌｅｒＥｌｅｃｔｒｏｓｔａｔｉｃＰｏｔｅｎｔｉａｌ）的计算方法和结果［１］．描述了双螺旋表面外壳（ＳｕｒｆａｃｅＥｎｖｅｌｏｐｅｓ）上势的分布，并将其与Ｂ－ＤＮＡ有关结果［２－４］作了比较．     

血小板生成素在枯草杆菌中的表达分泌及前肽作用

利用PCR方法扩增得到枯草杆菌本身的表达调控序列SP（含有信号肽）和Pro序列（包含信号肽和前肽）,分别与血小板生成素(TPO)结构基因及枯草杆菌载体片段连接,构建两个重组TPO枯草杆菌表达载体,转化受体菌,得到DB403（SP-TPO）和DB403（Pro-TPO）重组克隆,比较有无前肽对其表达分泌的作用.用ELISA和Western Blot检测TPO的表达分泌,发现DB403（Pro-TPO）能够表达分泌TPO,而DB403（SP-TPO）未发现TPO的分泌.对小鼠腹腔注射DB403（Pro-TPO）发酵浓缩液,发现能够明显增加血小板数目,与自身对照相比增加84.2％,与生理盐水对照比较增加99.7％.以上实验说明Pro-TPO能够表达分泌有明显活性的TPO蛋白,而且前肽对外源蛋白的分泌是必要的.     

血小板生成素在毕赤酵母中的表达

以人胎肝cDNA文库为模板,用PCR和DNA重组技术,将TPOcDNA克隆到pGEM     

Preliminary study on discovery of a novel molecule that interacts with CPP32 using two-hybrid system

Of ICE protease family, CPP32 (apopain or Yama) plays a central role in different apoptotic pathways. To study the molecules regulating CPP32, yeast two_hybrid system was used to identify proteins (peptides) that interact with CPP32. First, the CPP32 gene was cloned into plasmid vector pGBT9. The resulting recombinant plasmid was designated as pGBT9/CPP. The pGBT9 /CPP plasmid was transformed into the yeast strain HF7C, then the leukemia library was introduced. The transformation mixture was plated on medium lacking Trp, Leu and His in the initial screen. Colonies growing on the selection medium were further assayed for β galactosidase activity. Within 42 Trp +Leu +His + colonies only 5 turned blue in the presence of X_Gal. Plasmid DNA from 5 positive yeast colonies was prepared respectively and used to transform HB101 by electroporation. The transformation mixture was plated on medium lacking Leu to be selected for the library plasmid. Finally, only one library plasmid, designated as pY1, was determined to be truly positive by retransformation of pGBT9/CPP and the library plasmid into HF7C. The inserted cDNA of pY1 encodes a peptide of 15 amino acids, suggesting that it may be the domain interacting with CPP32.     

分离与克隆中国人TPO基因片段及序列分析

应用ＲＴＰＣＲ技术从中国人胎肝细胞中分离出１个５６６ｂｐ大小的基因片段,经过克隆、限制性内切酶鉴定和序列分析证实为ＴＰＯ的ｃＤＮＡ片段．与ＧｅｎＢａｎｋ中发表的人ＴＰＯｍＲＮＡ的序列比较同源性在８２％～９９％之间,仅有２个碱基不相同,在１７５位密码子（５２３～５２５位的碱基）分别是ＣＧＧ和ＣＡＡ,即精氨酸（Ｒ）的位置上,中国人是谷胺酰氨（Ｇ）．而与测得的韩国人序列比较,在相应位置的氨基酸是相同的     

c-Jun氨基末端激酶3结构域突变质粒的构建、鉴定与转染

为构建c-Jun氨基末端激酶3 (c-Jun N-terminal kinase3,JNK3)的p VP16-eGFP-Myc-JNK3活化环(activation-loop,A-loop)结构域突变型重组质粒,通过A-loop突变型JNK3的c DNA序列以及p VP16-eGFP-Myc的酶切位点设计引物,PCR扩增目的基因,使用限制性内切酶Mlu I与Xba I将基因克隆到p VP16-eGFP-Myc真核表达载体中,构建p VP16-eGFP-Myc-JNK3(A-loop)结构域突变型重组质粒。转化后,通过双酶切鉴定与DNA测序判断是否成功构建重组质粒,使用Trans IntroTMEL Transfection Reagent转染人胚肾(human emborynic kidney,HEK) 293T细胞,通过荧光显微镜下观察融合蛋白表达情况。结果表明:构建p VP16-eGFP-Myc-JNK3 (A-loop)结构域突变型重组质粒,双酶切鉴定和测序结果显示构建成功; p VP16-eGFP-MycJNK3 (A-loop)成功转染HEK293T细胞且表达。鼠源JNK3结构域突变型质粒构建成功,对进一步研究JNK3结构域在相关疾病的作用和机制提供实验工具。     

铁电相变的简谐子软模图像

在自由边界条件下计算了立方钛酸钡有限尺寸晶体中原子的简谐振动模 ,发现许多简谐子软模。用这些软模花样说明了晶体冷却时发生具有a畴和c畴结构的铁电相变。理论表明铁电相变过程涉及屏蔽电荷的激发及其在界面的缓慢扩散 ,以最后得出各个电畴内部的均匀自发极化。铁电相变过程的这些细节 ,都得到了实验的有力证明     

Cloning and expression of catalytic domain of Abl protein tyrosine kinase gene in E. coli

Protein tyrosine kinases (PTKs) regulate cell proliferation, differentiation and are involved in signal transduction. Uncontrolled signaling from receptor tyrosine kinases to intracellular tyrosine kinases can lead to inflamma tory responses and diseases such as cancer and atherosclerosis. Thus, inhibitors that block the activity of tyrosine kinases or the signaling pathways of PTKs activation could be assumed as the potential candidate for drug development. On this assumption, we cloned and expressed the Abl PTK gene in E. coli, and purified the PTK, which was used to screen the PTK inhibitors from the extracts of Chinese herbs. The catalytic domain sequence of PTK gene was amplified by PCR us ing the cDNA of abl from Abelson murine leukemia virus as template. The amplified fragment was then cloned into the GST-tagged expression vector pGEX2T. The recombinant plasmid was transformed into host cell E. coli DH5α and was induced to express PTK protein. The expression of the protein was detected using SDS-PAGE. The result showed that a specific protein was induced to express after 12 min induction, and reached peak level about 40% of the host total pro tein after 4 h induction. The molecular weight of the fusion protein was about 58 kD. The purified GST-PTK fusion pro tein presented higher activity for tyrosine phosphorylation.     

Expression and antigen activity determination of human thyroid peroxidase in silkworm and yeast

In this study, we report the expression of human thyroid peroxidase (TPO) in silkworm larvae and Pichia pastoris GS115. Recombinant TPO is sequentially purified from the hemolymph of infected silkworm larvae and yeast using a Ni-NTA resin kit. The concentration of yield of recombinant TPO is 4.87 mg per thousand larvae and 40.83 mg per liter yeast culture. However, the recombinant TPO produced in silkworm show similar binding ability with the specific anti-TPO serum to standard human TPO purified from insect cells. The lower antigen activity indicates the TPO expressed in yeast is not suitable to be used as the coating antigen in enzyme linked immunosorbent assay (ELISA). The cost of TPO expressed in B. mori is about 1/4 that of in insect cells, and the cost of TPO purified from silkworm for ELISA is only 1/8 that of TPO produced from Sf9 cells. It indicates the BmNPV-silkworm expression system is a cost-effective platform for producing TPO with high antigen activity.     

猪脑心肌炎病毒VR-129 B株VP2重组蛋白的构建表达及其免疫活性分析

为获得大量猪脑心肌炎VP2基因及蛋白研究的细胞模型,构建pDC315-EMCV-VP2真核表达载体,且将其转染到293T细胞,筛选出阳性质粒进行克隆。EMCV VR-129B株VP2基因序列参照GenBank(登录号:X74312),利用RT—PCR方法扩增VP2的全基因序列,将其与质粒pDC315经NheI和XhoI双酶切后连接,构建pDC315-EMCV-VP2重组表达质粒,并将其转染至293T细胞。使用荧光定量PCR方法观察pDC315-EMCV-VP2真核表达载体在细胞中的表达情况。双酶切PCR结果获得大小为780 bp的基因片段,测序结果显示与GenBank上已经公布的同名基因(登录号:X74312)序列片段同源性为100%,重组质粒构建成功。实时荧光定量PCR(QPCR)结果显示,重组质粒转染组与空质粒组之间相比,EMCV-VP2基因的表达量显著上调(P0.001);在荧光显微镜下观察转染细胞,转染成功部分出现较亮绿色荧光,EMCV-VP2基因表达稳定。从转染细胞中提取重组蛋白与EMCV阳性血清和阴性血清进行ELisa反应,具有较好免疫活性。     

SGK and 14-3-3 protein are involved in the serine/ threonine phosphorylation mechanism for TPO/MPL signal transduction

Thrombopioetin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Yeast two-hybrid screening was performed to isolate the proteins interacting with the cytoplasmic domain of c-Mpl. 48 positive clones were isolated from 5 × 10⁶ independent transformants. The results of sequence analysis demonstrate that they represent 13 different protein encoding sequences. Among them there are a partial coding sequence of serine/threonine protein kinase SGK (serum and glucocorticoid-inducible kinase) and 14-3-3 theta protein partial coding sequence. GST-pull-down assay and co-immunoprecipitation in mammal cells have confirmed the interaction between these two proteins and c-Mpl. By constructing a series of deleted c-Mpl cytoplasmic domain, the interaction region in c-Mpl cytoplasmic tail was localized in amino acids 523–554. At the same time, the directed interaction between SGK and 14-3-3 proteins also has been verified by yeast two-hybrid assay. The present note is the first time to report that two proteins act with c-Mpl at the same time and put forward that SGK and 14-3-3 protein may be involved in the serine/threonine phosphorylation mechanism for signal transduction.