研究聚ADP核糖基化作用与细胞恶变关系的双基因真核表达重…

本文将聚ＡＤＰ核糖聚合酶基因１．４ｋｂ部分序列反向插入真核表达载体ｐＭＡＭｎｅｏ和ｐＳＭＧ中，同时１２位密码子突变的活化ｒａｓ癌基因也一同克隆到上述载体中，从而获得具有不同真核基因筛选标记的双基因真核表达重组ｐＭＡＭｎｅｏ－Ｃｌ．４－Ｔ２４，ｐＭＡＭｎｅｏ－Ｃｌ．４－ａｒＴ２４，及ｐＳＭＧ－Ｃｌ．４－Ｔ２４，上述质粒的构建成功为研究聚ＡＤＰ核糖基化作用与细胞恶变的关系提供了一种新的分子模型。     

小鼠金属硫蛋白基因—I在CHO细胞中稳定表达及其在医疗…

将小鼠的ＭＴ－Ｉ基因插入ｐＳＶ２－ｄｈｆｒ质粒的ＥｃｏＲＩ位点，构建了具有ＳＶ４０和ＭＴ双启动子并正向插入ＭＴ－Ｉ基因的表达载体。用改良的磷酸钙／ＤＮＡ沉淀法将表达载体导入ＣＨＯ－ｄｈｆｒ＾－细胞内，用不含次黄嘌呤（Ｈ）和胸腺嘧啶（Ｔ）的选择培养基筛选出稳定表达ＭＴ阳性克隆Ｄ－２２，经检测１０＾６细胞的ＭＴ表达量约为１．８μｇ，Ｄ－２２细胞对Ｃｄ＾２＋浓度抗性较之ＣＨＯ－ｄｈｆｒ＾－细胞高１４倍。     

人Cu,Zn-SOD在酵母系统中的表达

甲醇酵母（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）是一种理想的真核蛋白高水平表达系统。将人Ｃｕ，Ｚｎ－ＳＯＤ基因克隆到酵母整合型质粒载体ｐＰＩＣ３．５Ｋ，经转化ｈｉｓ４缺陷型酵母ＧＳ１１５，用ＭＤ培养基及ＰＣＲ方法筛选阳性转化子，并用含Ｇ４１８的ＹＰＤ培养基筛选多拷贝转化子，经甲醇诱导表达，ＳＤＳ－ＰＡＧＥ和蛋白质印迹杂交结果证实了表达产物为重组的人Ｃｕ，Ｚｎ－ＳＯＤ，经计算机扫描分析，表达量占酵母可溶性     

—3位碱基对HBVc基因在昆虫细胞中表达的影响

应用ＰＣＲ定点突变技术,分别扩增出－３位碱基是Ａ或Ｔ的乙肝病毒核心抗原基因ｃ１,ｃ２,平端克隆到自行构建便于ＰＣＲ产物克隆的通用载体ｐＢｌｕｅｏＳ中后,利用经ＰＣＲ引物在ｃ１及ｃ２末端引入的酶切位点,切出ｃ１,ｃ２基因,构建出ｐＸ３－ｃ１和ｐＸ３－ｃ２．在Ｓｆ９细胞中瞬时表达ｃ１,ｃ２基因,乙肝核心抗原放免检测结果表明,两种形式的ｃ基因的表达无明显差异．说明－３位是否为Ａ对ｃ基因在昆虫细胞中的表达量没有影响．     

用LBA4404／pcAMBIA系列转化水稻的最佳条件

用带有ｇｕｓ报告基因的农杆菌ＬＢＡ４４０４／ｐＣＡＭＢＩＡ１３０１转化水稻品种鄂宜１０５成熟胚诱导的愈伤组织,以ｇｕｓ基因瞬间表达活性为指标测定转化率频率,对于利用ｐＣＡＭＢＩＡ表达载体系列进行水稻遗传转化的最佳条件进行了初步的研究。结果表明：最适菌液浓度值为Ｄ（６００）＝１．０,最适共培养温度为２２－２８℃;共培养培养基的ｐＨ值为５．２－６．２这一较宽的范围内均可。     

点突变ras癌基因全cDNA真核表达重组体的构建及诱导NIH3T3细胞转化的研究

将活化癌基因Ｔ２４－ｒａｓ的全ｃＤＮＡ序列正向插入真核载体ｐＭＡＭｎｅｏ，构建成重组质粒ｐＭＡＭｎｅｏ－Ｔ２４－ｒａｓ．将该质粒转染ＮＩＨ３Ｔ３细胞，通过药物筛选，建成细胞系３Ｔ３（Ｔ２４）．Ｓｏｕｔｈｅｒｎ杂交证明外源Ｔ２４－ｒａｓ基因已整合于受体细胞染色体中．３Ｔ３（Ｔ２４）细胞表现出形态学方面的明显变化：具失去接触抑制能力，且在裸鼠体内致瘤等恶性行为．本文构建的Ｔ２４－ｒａｓ基因真核表达重组体和建立的转化细胞系可用于肿瘤的诊断、预防、治疗及抗肿瘤药物的筛选、评估等研究．     

红细胞生成素(EPO)在中国仓鼠卵巢细胞(CHO)中稳定表达

将ＥＰＯｃＤＮＡ分别插入真核表达质粒载体ｐＳＶ２ｄｈｆｒ和ｐｃＤＮＡ３的适当位点,用电脉冲法导入ＣＨＯ细胞,用ＥＬＩＳＡ法检测ＥＰＯ的表达量,用ＴＦ１细胞检测ＥＰＯ表达产物的生物活性．结果表明,用ｐＳＶ２ｄｈｆｒ作为载体,在抗ＭＴＸ阳性细胞克隆中,获得了表达ＥＰＯ（２５８ｎｇ／ｍＬ培养液）的ＣＨＯ细胞株,并且表达产物具有生物活性．进一步的传代和冻存试验表明,所得细胞株表达ＥＰＯ的水平不受传代和冻存的影响     

形成多角体家蚕重组病毒通用转移载体构建

从家蚕核型多角体病毒（ＢｍＮＰＶ）通用转称载体ｐＢＫ２８３和粉纹夜蛾核型多角体病毒（ＴｎＮＰＶ）转移载体ｐＳＸＩＶＶＩ＋Ｘ３系列出发，构建了一个７．２ｋｂ能形成多角体且可以用于克隆含不同读码框外源基因的ＢｍＮＰＶ通用转移载体系列ｐＢＭＸ３．ｐＢＭＸ３系列以ＢｍＮＰＶ多角体结构基因上游的１．９ｋｂ和下游１．３ｋｂ的片段作为与ＢｍＮＰＶ基因组进行体内同源重组的同源序列；ｐＳＸＩＶＶＩ＋Ｘ３系列的ＳＸＩＶ双启动子用于外源基因的表达；ＡｃＮＰＶ的多角体基因作为重组病毒形成多角体的基因．以ＢｍＮＰＶ－ＬａｃＺ（ｏｃｃ－ｇａｌ＋）为出发株病毒，ｐＢＭＸ３系列的重组病毒筛选有ｏｃｃ＋和ｇａｌ－两个遗传标记     

环状芽胞杆菌（Bacilluscirculans）的转化和中性蛋白酶基因的表达

用枯草芽胞杆菌质拉载体ｐＮＱ１２２和含有中性蛋白酶基因的重组质粒ｐＭＰＲ８转化环状芽胞杆菌Ｃ－２，转化频率为１．０×１０３转化子／μｇＤＮＡ．含有ｐＭＰＲ８的转化子能在酪蛋白平板上形成清晰的水解圈，其蛋白酶活性与相同质粒转化枯草杆菌ＤＢ１０４所得酶活性相近．Ｓｏｕｔｈｅｒｎ杂交结果证实了转化细胞中存在质粒ｐＮＱ１２２和ｐＭＰＲ８，两种质粒在该菌中的稳定性差别很大．     

美洲拟鲽(Pseudopleuronectes americanus)抗冻肽基因在大肠杆菌中的表达

将美洲拟鲽抗冻肽基因克隆于原核高效表达载体ｐＫＫ２２３－３中,经酶切分析、ＰＣＲ检测筛选出重组克隆ｐＭＫ１１,转化大肠杆菌ＪＭ１０９和ＤＨ５α,ＩＰＴＧ诱导２～３ｈ后,超声波裂解细菌产物,在ＳＤＳ－ＰＡＧＥ电泳图谱分子量９ｋＤａ处显示出一条明显的蛋白带,与美洲拟鲽抗冻肽大小一致,表明美洲拟鲽抗冻肽基因在大肠杆菌中表达,为进一步研究抗冻肽基因在大肠杆菌中的表达水平及条件打下基础．     

一种简便有效的PCR扩增片段直接回收克隆方法

作者提供一种简便快速的克隆方法，即直接对琼脂糖凝胶电泳分离的PCR产物进行克隆，可获得较高的阳性克隆率.结果显示，300～700 bp大小的片段阳性克隆率为70.72%，300 bp以下和大于700 bp的片段阳性克隆率分别达到60.38%和45.33%.该方法适用于含有大量不同大小DNA片段的PCR产物并需要同时对其进行回收和克隆的样品，不仅可以大大简化实验过程，也可以降低假阳性出现的机会.     

Construction of single-chromosome DNA library fromLilium regale Wilson

A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA fromLilium regale Wilson is described. Single-chromosome, microdissected by micromanipulator, was put into a 0.5 mL Eppendorf tube and digested with Sau3A, and then the Sau3A linker adaptors were ligated to the ends of DNA fragments. After 2 rounds of PCR amplification with one chain of linker adaptor as primer, the PCR products thus obtained have a length of 300–2500 base pairs (bp) with predominant fragments at about 1000 bp. Southern blot analysis confirmed that the PCR products originated from the genome ofLilium regale Wilson. By cloning the amplification products from the second round of PCR, single-chromosome DNA library was constructed, in which about as many as 100000 recombinant clones were produced. A total number of 84 clones were analysed, and it was revealed that the inserts ranged in size from 300 to 1800 bp, with an average of780 bp. Compared with the methods described in other literature, this protocol, eliminating the need for enzymatic digestion and ligating micromanipulation of chromosomal DNA in nanoliter volumes, permits the efficient amplification of single chromosome (not tens of chromosomes as reported before) and the fragments (780 bp in average) cloned in this study are longer than those reported before (650 bp in average).     

Promoter activities of genes cpcT2 and cpcS2 in Nostoc PCC 7120

To study the promoter activities of genes cpcT 2 and cpcS 2,several upstream DNA fragments of these two genes with different lengths were selected.These fragments were fused with promoterless gfp(reporter gene) for constructing five recombinant plasmids.Then,these recombinant plasmids were transferred into Nostoc PCC 7120 by conjugation.The promoter activities of genes cpcT 2 and cpcS 2 were determined by observing the fluorescence of green fluorescent protein(GFP) with a fluorescence microscope.The result showed that the 1 300 bp(-1 300 to 0 bp) and 2 600 bp(-2 600 to 0 bp) upstream fragments of cpcT 2 had strong promoter activity and the promoter activity of the 680 bp(-680 to 0 bp) fragment was weaker than that of two above fragments of cpcT 2.The 2 000 bp(-2 000 to 0 bp) upstream fragment of cpcS 2 revealed weak promoter activity.This showed that a strong promoter of cpcT 2 was located in the upstream fragment between-680 and-1 300 bp.     

罗汉果性别相关的RAPD标记

应用随机扩增多态性DNA(RAPD)技术寻找与罗汉果性别相关的分子标记,筛选了130个10 bp的随机引物,发现有4个引物(S60、S90、S100、S343)能在雌、雄株DNA间扩增出5条差异性片段,大小在300~1 300 bp之间,表明这些特异带可被用来作为性别鉴别的特异性分子遗传标记。     

从福尔马林固定的癌组织中提取RNA的方法

目的:寻求从福尔马林固定的癌组织中提取RNA的合理方法.方法:运用Trizol法从福尔马林固定的胃癌组织中提取RNA,用分光光度计测定RNA的产量和纯度;通过RT-PCR对不同长度的管家基因-actin进行扩增.结果:100 bp和300 bp的-actin均能顺利扩增出特异性条带,500 bp的-actin未能扩增.结论:用Trizol法从福尔马林固定的癌组织中提取RNA是可行的;进行RT-PCR时应尽量设计小片段引物(100~300 bp)以获得更好的扩增效果.     

悬臂式调谐质量阻尼器抑制管道振动研究

基于调谐质量阻尼器(TMD)的结构振动控制机理,将调谐质量阻尼器应用到管道的振动控制中。针对传统弹簧结构无法满足高频振动下调谐质量阻尼器刚度设计要求的问题,设计了一种便于安装的圆形截面悬臂式调谐质量阻尼器,其周向对称布置式结构可以对管道各个方向下的振动进行控制。理论推导并对比了传统悬臂梁和悬臂式TMD固有频率的计算公式,计算得到300Hz对应的悬臂式TMD结构参数,并利用Ansys模态分析对设计参数的准确性进行了验证。最后建立了激振器-TMD高频振动实验台装置,对悬臂式TMD在300Hz左右的减振特性进行了实验,结果表明设计的TMD能够有效抑制300Hz左右的高频振动,且有较宽的减振频带范围。     

Identification of seed-specific promoter nap300 and its comparison with 7S promoter

By fusing seed-specific promoter nap300 with β-glucuronidase gene, it was found that this about 300bp DNA fragment was sufficient to direct seed-specific gene expression. The substitution mutation in both distB and proxB elements had a little effect on the expression efficiency and almost no effect on the organ-specific expression pattern. In the experiment designed to compare nap300 with 7S promoter, the result showed that tissue specificity for nap300 was higher than that for 7S, and its expression level was lower than 7S's. There was no big difference in their expression pattern, and the maximal activity stage for the two promoters was identical, which indicated they could be used simultaneously for expressing different foreign genes in seeds.     

脱落酸受体及其基因的分子免疫学研究(英文)

发展了研究脱落酸（ＡＢＡ）结合蛋白的两类免疫探针。其一,用ＡＢＡ－Ｃ１－ＢＳＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析柱纯化出ＡＢＡ结合蛋白,聚丙烯酰胺凝胶电泳显示该蛋白分子量为５６ＫＤ的一条带,它具有特异结合ＡＢＡ的能力（Ｋｄ＝２．０×１０－９ｍｏｌ／Ｌ）。当用蛋白水解酶Ｋ水解该蛋白,并用１％琼脂糖凝胶电泳时,发现其中存在约３００个核苷酸的ｒＲＮＡ分子。用识别ＡＢＡ结合蛋白的抗体筛选ｃＤＮＡ表达文库,从２００,０００个独立噬斑中获得１２０个编码玉米１７ｓＲＮＡ的ｃＤＮＡ克隆和１个ｃＤＮＡ编码结合蛋白的ｃＤＮＡ克隆（２４ｃＤＮＡ）。２４ｃＤＮＡ有１０７５个碱基对,含编码２５４个氨基酸的开放阅读框架。其二,制备了识别抗ＡＢＡ单克隆抗体的独特型抗体（ａｎｔｉ－Ｉｄ）,它具有模拟并竞争ＡＢＡ的能力。用上述两类免疫探针定位了植物细胞中的ＡＢＡ结合蛋白。发展了研究脱落酸（ＡＢＡ）结合蛋白的两类免疫探针。其一,用ＡＢＡ－Ｃ１－ＢＳＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析柱纯化出ＡＢＡ结合蛋白,聚丙烯酰胺凝胶电泳显示该蛋白分子量为５６ＫＤ的一条带,它具有特异结合ＡＢＡ的能力（Ｋｄ＝２．０×１０－９ｍｏｌ／Ｌ）。当用蛋白水解酶Ｋ水解该蛋?     

干旱胁迫下开花期玉米消减文库的构建及分析

分别以1d干旱和7d干旱处理的开花期玉米顶叶cDNA为tester，正常生长的玉米顸叶cDNA为driver，利用抑制性差减杂交技术构建了两个干旱胁迫下开花期玉米消减文库．两个文库的重组率均高于95％，插入片段集中在300—600bp之间．对两个文库部分克隆进行测序发现，文库中含有脱水素、蔗糖合成酶、甜菜碱醛脱氢酶。DRE结合因子等大量的抗旱相关基因，说明两个干旱胁迫下开花期玉米抑制性消减文库已经构建成功。且具有重要意义．     

Evaluation and characterization of an endosperm-specific sbeⅡa promoter in wheat

Starch,the main component of the wheat grain,is the product of a complex biochemical pathway. The sbeⅡα gene plays a key role in controlling the synthesis of starch, in particular, the biosynthesis of amylopectin,in maturing wheat grain.To investigate its regulatory mechanisms and endosperm-specific expression pattern, the sbeⅡα promoter (3094 bp in length) was cloned using APCR and sequenced.The effect of a series of deletions was studied using a GUS transient assay system. Results showed that the 3094 bp sequence (sbe.g construct) exhibited full stable promoting activity and that the activities of 5′ or 3′ deletions reduced levels of GUS expression. Some constructs with internal deletions showed only weak activity, however,sbe.e, with a deletion from -1579—--1210 bp resulted in higher levels of expression than the full-length promoter sequence, sbe.g. This indicates that motifs such as the -300 bp element, G-box and/or P-box act as positive elements and are necessary in determining the promoter‘s endosperm-specific pattern and that negative repressor elements or motifs may also be present within the -1579—-1210 bp sequence. The age of wheate ndosperm tissue used in the GUS-transient assay system is shown to be of significant importance.