核定位信号筛选系统的构建

建立了一酵母克隆系统用于克隆含核定位信号 (NLS)的蛋白质的基因 .用表达转录因子GAL4 DNA结合域 - p53(GAL4- DBD- p53)融合蛋白的质粒转化酵母 HF7c,使 GAL4- DBD- p53可结合于报告基因的启动子但因无转录激活域而不能激活转录 .构建一酵母穿梭载体 ,可表达无NLS的 GAL4转录激活域 -大 T抗原 (GAL4- AD- LT)融合蛋白 .融合蛋白基因的下游插入一多克隆位点 .将 c DNA文库插入多克隆位点后 ,如果 c DNA片段可编码 NLS,则 GAL4- AD- LT分子可进入细胞核 ,并通过 LT与 p53的相互作用而使 GAL4- AD结合于启动子和激活报告基因的转录 .构建了这一克隆系统的各质粒 ,并用绿色荧光蛋白 (GFP)验证了其对核内蛋白和胞浆蛋白的甄别能力 .这一系统将有助于从 c DNA文库中筛选编码带有 NLS的蛋白质的基因     

利用核定位信号筛选系统初步筛选小鼠胚胎核定位蛋 …

在已建立的核定位信号（ｎｕｃｌｅａｒ ｌｏｃａｌｉｚａｔｉｏｎ ｓｉｇｎａｌ,ＮＬＳ）筛选系统的基础上,对这一系统进行了改进并对改进的系统进行了验证。将小鼠１１天胚胎ｃＤＮＡ文库插入改进后的筛选载体的多克隆位点,转化酵母宿主菌。然后将约１０＾４个酵母克隆接种于选择性平板上进行筛选,得到了２２个可在选择性培养基上生长的克隆。分析了其中１８个克隆的ＤＮＡ序列,见到１３个克隆含有以正确读框融合的编码ＮＬ     

NLS-RARα蛋白相互作用蛋白的筛选与验证

急性早幼粒细胞白血病(APL)具有特征性染色体易位,产生的PML-RARα融合基因在其发生发展中有重要作用．PML-RARα融合蛋白在细胞内被中性粒细胞弹性蛋白酶(neutrophil elastase,NE)切割为PML突变蛋白(核定位信号NLS缺失)和RARα突变体 (NLS-RARα,包含有PML的核定位信号),这两段蛋白质在APL的发生中可能具有重要作用．为进一步研究NLS-RARα的生物学功能,运用酵母双杂交技术在白血病cDNA文库中筛选与其作用的蛋白质．首先PCR技术扩增NLS-RARα编码序列,克隆至诱饵载体pGBKT7,测序鉴定后将其转化酵母AH109．免疫印迹检测到诱饵蛋白表达后,将含有诱饵载体的AH109与含有白血病cDNA文库的酵母Y187交配,在含有X-α-gal的营养缺陷性培养基上选择和筛选二倍体酵母．经回转实验和测序分析验证得到8个与NLS-RARα相互作用的蛋白质．为进一步验证这些相互作用,克隆其中的JTV-1蛋白,利用间接免疫荧光,GST pull-down和免疫共沉淀技术成功验证了它与NLS-RARα的相互作用．为进一步探讨APL的发生机制提供了新的线索．     

筛选G-box结合蛋白的酵母单杂交文库的构建

目的：筛选花青素合成中的关键基因查尔酮合成酶基因CHS启动子中G-box的结合蛋白,从而找到调节CHS表达的转录因子。方法：采用Matchmaker Gold Yeast One-Hybrid Library Screening System,将CHS启动子G-box序列串联后整合入酵母染色体,构建诱饵菌株;采用SMART技术合成芜菁幼苗下胚轴cDNA,将该cDNA与pGADT7-Rec表达载体共同转化诱饵菌株,通过同源重组在酵母细胞内同步进行cDNA文库的构建和筛选;用酵母菌落PCR法获得阳性克隆中的cDNA插入片段,测序后在NCBI网站进行Blast分析。结果：共筛选了2.52×106个酵母克隆,得到94个阳性克隆,菌落PCR获得了长度为0.4~2.0 kb的cDNA插入片段,并通过Blast推测了其编码蛋白。结论：实验结果证明酵母单杂交文库构建成功,初步筛选获得了G-box结合蛋白的候选蛋白,为研究CHS的表达调控奠定了基础。     

信号肽捕获系统的建立

细胞分泌蛋白的分泌有赖于蛋白质N端的信号肽的存在,利用酵母建立了从cDNA文库中筛选编码信号肽的基因片段的遗传系统,为此,用一步基因破坏法对酿酒酵母EGY48基因组中的suc2基因（编码酵母蔗糖转换酶）进行了定位突变,获得了无蔗糖转换酶表达的酵母株EGY48－suc。将无信号肽的suc 2成熟肽基因克隆于酵母乙 氢酶（ADHI）基因启动子下游,得到用于文库筛选的酵母真核表达工体,启动子与成熟肽基因之间为多克隆位 ,用于插入待筛选的CDNA文库,用此载体转化酵母EGY48－suc,所得克隆可以在葡萄糖为碳源的培养基上生长,但不能在以棉子糖为碳源的培养基上生长,在suc 2成熟肽基因前分别插入suc 2信号肽基因片段或人IL－2受体α链信号肽基因片段,然后转染EGY48－suc,所得克隆既能在以葡萄糖为碳源的培养基上生长,也能在以棉子糖为碳源的培养基上生长,表明构建的系统可用于筛选插篱多克隆位点cDNA片段是否具有编码信号肽的功能。     

酵母双杂交技术筛选hCLP46的相互作用蛋白

目的:利用酵母双杂交技术,筛选人白细胞cDNA文库中能与人类CD34 干/祖细胞异常表达蛋白hCLP46(human CAP10-like protein46)相互作用的未知蛋白.通过对其相互作用蛋白的筛选和研究,研究hCLP46基因在骨髓增生异常综合症MDS-AML的作用机制.为MDS-AML的临床治疗和诊断提供理论基础.方法:以pGBKT7-hCLP46为诱饵质粒筛选人白细胞cDNA文库,得到阳性克隆,并对验证后的阳性克隆的外源性片段进行测序及同源性分析.结果:经过两次筛选、验证,从人白细胞cDNA文库中得到5个阳性克隆,对验证后的阳性克隆的外源片段进行测序及同源性分析,最终得到4个不同的候选基因序列.结论:应用酵母双杂交系统,共筛选得到4个不同的基因,其编码蛋白与hCLP46有相互作用,可能与MDS-AML发病机制相关.     

酵母双杂交技术筛选与hCLP46蛋白相互作用的蛋白

目的：利用酵母双杂交技术,筛选人类白细胞cDNA文库中能与人类CD34＋干/祖细胞异常表达蛋白hCLP46（Human CAP10-Like Protein46）相互作用的未知蛋白。通过对其相互作用蛋白的筛选和研究,深入探讨hCLP46的功能,为骨髓增生异常综合症MDS-AML的病理提供线索。方法：以pGBKT7-hCLP46为诱饵质粒筛选人类白细胞cDNA 文库,得到阳性克隆,并对验证后的阳性克隆的外源性片段进行测序及同源性分析。结果：从人类白细胞cDNA文库中得到86个阳性克隆,经过验证,最终得到5个阳性克隆。对验证后的阳性克隆的外源片断进行测序及同源性分析,最终得到4个不同的候选基因序列。结论：应用酵母双杂交系统,共筛选得到4个不同的基因,其编码蛋白与hCLP46 有相互作用,可能与MDS-AML发病机制相关。     

鼻咽癌组织cDNA文库的构建及抗原基因的筛选

构建人鼻咽癌组织cDNA文库,以SEREX方法从cDNA文库中筛选鼻咽癌抗原基因。采用确诊鼻咽癌患者新鲜活检癌组织构建cDNA文库,测定原始文库滴度,进行蓝白筛选以确定文库的重组率。以建库组织来源患者的自身血清,采用“一对一” 的血清学方法筛选所构建的cDNA文库,阳性克隆经PCR检测鉴定后进行序列分析。经测定原始文库滴度为7.28×10⁶pfu/mL,含3.64×10⁶个重组子,重组率为94%,扩增文库滴度为3.8×10⁹pfu/mL,cDNA插入片段大小在0.5～3.0kb之间。文库经三轮血清学筛选共获得23个阳性克隆,分别代表了16个独立的cDNA插入片段（抗原基因）。其中10个与已知基因高度同源,另外6个基因与GenBank中已知基因的部分同源,其中有3个是新基因。利用SMART技术构建了高质量的人鼻咽癌组织cDNA表达文库,有利于以cDNA文库为基础的进一步的实验研究。应用SEREX技术初步筛选鼻咽癌组织cDNA文库,共得到16个鼻咽癌相关抗原基因,其中有3个是新基因,可能为鼻咽癌的免疫学研究提供新的研究分子。     

Bax Inhibitor-1与Herp的相互作用

应用酵母双杂交技术筛选Herp的相互作用蛋白。构建编码Herp的基因HERPUD1真核表达载体HERPUD1plexA,应用MATCHMAKERLexA酵母双杂交系统筛选人胎脑cDNA文库,获得的阳性克隆的插入子为Herp的候选相互作用蛋白质,将Herp与筛选到的相互作用蛋白再一对一回复进行酵母双杂交实验,去除假阳性。对阳性克隆插入子的DNA序列测序,在GenBank中作匹配及生物信息学分析。结果得到其中1个阳性克隆的插入子序列与TEGT基因序列一致,编码蛋白为Baxinhibitor1。得出结论:Herp与Baxinhibitor1相互作用,Baxinhibitor1具有调节凋亡特性,提示Herp可能参与凋亡调节。     

用suc2信号肽捕获系统筛选小鼠胚胎cDNA文库基因

PCR扩增 1 1d小鼠胚胎cDNA文库插入片段 ,将 0 .5～ 2 0kb的扩增产物插入筛选载体的多克隆位点 ,转化suc2基因缺陷酵母宿主菌 .然后将约 1 0 5个酵母菌落接种于选择性平板上进行筛选 ,得到了 1 82个可在选择性培养基上生长的菌落 .PCR扩增显示 ,插入片段大小分布于 0 1～ 1 5kb之间 .对其中 1 4个阳性菌落的重组子进行序列测定 ,分别代表 6种不同的基因序列 ,与报告基因都有正确的读框内融合 .其中两种基因序列反复被筛到 ,分别命名为spt1、spt2 .spt1 [gi:2 772 876 6 ],可能以非编码RNA的身份参与蛋白质向细胞外分泌的过程 ,而spt2编码多个连续的赖氨酸 ,可能通过非经典途径介导蛋白质的分泌     

Use of an epitope-tagged cDNA library to isolate cDNAs encoding proteins with nuclear localization potential

We have combined epitope tagging with an expression cDNA library in order to isolate cDNAs encoding nuclear proteins. This system allows us to detect proteins expressed from the cDNA library by using antibodies against the epitope tag. As a tag, we used the 85-aa N-terminal peptide of the SV40 T antigen which lacks the nuclear localization signal (NLS). A strong expression vector, pEF204 [Kim et al., Gene 91 (1990) 217–223], was modified into an epitope-tagging vector, pTkim, by putting the tag-coding region and a cDNA cloning site immediately after its promoter. From cDNA libraries constructed using pTkim, we isolated eight cDNA clones whose tagged proteins were localized within the nuclei. From partial sequence analysis, two cDNAs were shown to code for the ribosomal (r-) proteins, simian L44 and human L21, and the others were shown to be new. Furthermore, six cDNAs including those encoding the r-proteins could direct a non-karyophilic T antigen [Fischer-Fantuzzi et al. Virology 153 (1986) 87–95] into nuclei, showing that they have NLSs. These results indicate that this system is useful for isolating new cDNAs which code for nuclear proteins.     

Gateway技术构建交链孢菌JH505 cDNA文库

Gateway(R)技术构建Cdna文库,利用λ噬菌体的位点特异性重组,避免使用限制性内酶切割Cdna,能够解决常规方法构建Cdna文库的技术缺陷.首次应用Gateway(R)技术构建交链孢菌Cdna文库,经检测Cdna入门文库的滴度达到1×107cfu/Ml,文库总容量为9×107cfu,平均插入片段为1510bp.通过LR重组把入门文库转换为表达文库,表达文库的滴度为1.58×106cfu/Ml,文库总容量为6.32×106cfu,平均插入片段大小为1680bp.表达文库的构建为进一步克隆植物激活蛋白基因打下了基础.     

Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits

We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.     

Identification of putative parasitism genes expressed in the esophageal gland cells of the soybean cyst nematode Heterodera glycines

Cloning parasitism genes encoding secretory proteins expressed in the esophageal gland cells is the key to understanding the molecular basis of nematode parasitism of plants. Suppression subtractive hybridization (SSH) with the microaspirated contents from Heterodera glycines esophageal gland cells and intestinal region was used to isolate genes expressed preferentially in the gland cells of parasitic stages. Twenty-three unique cDNA sequences from a SSH cDNA library were identified and hybridized to the genomic DNA of H. glycines in Southern blots. Full-length cDNAs of 21 clones were obtained by screening a gland-cell long-distance polymerase chain reaction cDNA library. Deduced proteins of 10 clones were preceded by a signal peptide for secretion, and PSORT II computer analysis predicted eight proteins as extracellular, one as nuclear, and one as plasmalemma localized. In situ hybridization showed that four of the predicted extracellular clones were expressed specifically in the dorsal gland cell, one in the subventral gland cells, and three in the intestine in H. glycines. The predicted nuclear clone and the plasmalemma-localized clone were expressed in the subventral gland cells and the dorsal gland cell, respectively. SSH is an efficient method for cloning putative parasitism genes encoding esophageal gland cell secretory proteins that may have a role in H. glycines parasitism of soybean.     

Construction and characterization of a normalized yeast two-hybrid library derived from a human protein-coding clone collection

The nuclear yeast two-hybrid (Y2H) system is the most widely used technology for detecting interactions between proteins. A common approach is to screen specific test proteins (baits) against large compilations of randomly cloned proteins (prey libraries). For eukaryotic organisms, libraries have traditionally been generated using messenger RNA (mRNA) extracted from various tissues and cells. Here we present a library construction strategy made possible by ongoing public efforts to establish collections of full-length protein encoding clones. Our approach generates libraries that are essentially normalized and contain both randomly fragmented as well as full-length inserts. We refer to this type of protein-coding clone-derived library as random and full-length (RAFL) Y2H library. The library described here is based on clones from the Mammalian Gene Collection, but our strategy is compatible with the use of any protein-coding clone collection from any organism in any vector and does not require inserts to be devoid of untranslated regions. We tested our prototype human RAFL library against a set of baits that had previously been searched against multiple cDNA libraries. These Y2H searches yielded a combination of novel as well as expected interactions, indicating that the RAFL library constitutes a valuable complement to Y2H cDNA libraries.     

A new recombinant DNA strategy for the molecular cloning of rare membrane proteins.

We have constructed a cDNA library in the plasmid expression vector pUEX enriched in sequences encoding membrane proteins. The procedure involved positive selection of sequences common to two different rat tissues (thus excluding tissue-specific mRNA) followed by positive selection between this material and RNA extracted from membrane bound polysomes (thus excluding cytoplasmic proteins). The resultant library prepared from rat kidney cDNA hybridized with rat liver poly(A)+ RNA, contained 30,000 clones and was shown to be enriched in cDNAs encoding membrane proteins. Seventeen clones selected because they encode large fusion proteins were shown to be single copy in the library, and not present in nucleotide data banks. Thus the strategy is particularly suitable for cloning low abundance cDNAs encoding membrane proteins.