甜菜坏死黄脉病毒内蒙分离物RNA3序列分析及编码25kD蛋白基因在大肠杆菌中的表达

以甜菜坏死黄脉病毒（ＢＮＹＶＶ）内蒙分离物的总ＲＮＡ为模板,通过反转录－ＰＣＲ扩增获得ＢＮＹＶＶＲＮＡ３全长ｃＤＮＡ。将其克隆到ｐＧＥＭ－７Ｚｆ（＋）上,得到重组质粒ｐＧＢＹ５６。序列分析结果表明,内蒙分离物ＲＮＡ３基因组全长为１７７５ｎｔ,其中包含３个开放阅读框架,分别编码２５ｋＤ蛋白、４．６ｋＤ蛋白和一种由５９个氨基酸组成的Ｎ蛋白。与法国Ｆ２分离物、德国Ｇ１分离物和日本Ｓ分离物相比,其核苷酸序列的同源性分别为９６．４％、９６．８％和９７．３％。将２５ｋＤ蛋白编码基因克隆到ｐＪＷ２上,构建了该基因的原核表达载体。ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ分析结果表明,２５ｋＤ蛋白基因在Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）中经温度（４２℃）诱导后,可特异地表达２５ｋＤ蛋白     

水稻富硫10kD醇溶谷蛋白基因的克隆和序列分析比较

用ＰＣＲ技术从我国水稻品种“广陆矮”（Ｏｒｙｚａ ｓａｔｉｖａ ｖａｒ． ｉｎｄｉｃａ）和“中花８ 号”（Ｏ． ｓａｔｉｖａｖａｒ． ｊａｐｏｎｉｃａ）中特异地扩增并克隆测序了富硫１０ ｋＤ醇溶谷蛋白基因,它共有５２５ 个核苷酸,编码１３４ 个氨基酸。经分析,克隆的基因与水稻属其它种或品种的同类基因同源率为９６．６％ 到１００％ ,与玉米１０ ｋＤ醇溶蛋白基因同源率为３４．２％ ;与某些双子叶植物的贮藏蛋白也有一定的同源性,例如和巴西豆富硫水溶蛋白同源率达３１．２％ 。水稻１０ ｋＤ醇溶谷蛋白Ｎ 端有一段信号肽含２４ 个氨基酸,经分析,发现这段信号肽与禾谷类玉米、高粱和燕麦的贮藏蛋白信号肽同源率很高,分别为６５．０％ 、６５．０％ 和６２．５％ ,而在双子叶植物的贮藏蛋白中未发现有相似序列。所测定的“广陆矮”和“中华８ 号”１０ ｋＤ醇溶谷蛋白基因序列已被ＥＭＢＬ数据库接受,收录号分别为Ｌ３６６０４ 和Ｌ３６６０５     

人白细胞介素-17基因的克隆、表达及其表达产物的分离纯化

利用ＲＴ－ＰＣＲ方法成功地从ＰＩ（ＰＭＡ,Ｉｏｎｏｍｙｃｉｎ）、ＰＨＡ活化的人Ｔ细胞中扩增出ｈＩＬ－１７编码区ｃＤＮＡ（４６８ｂｐ）,克隆测序证实得到该基因．用特殊设计的引物扩增ｈＩＬ－１７成熟肽编码区（４１４ｂｐ）,接入表达载体ｐＥＴ３０ａ质粒中．ｐＥＴ３０ａ－ｍｈＩＬ－１７在大肠杆菌ＢＬ２１（ＤＥ３）中获得高效表达,目的蛋白占总菌体蛋白５０％左右．经凝胶过滤和阴离子交换层析两步分离纯化和复性,得到单体型ｒｍｈＩＬ－１７,纯度达９８％以上,Ｎ端１６个氨基酸序列分析,结果与文献报道一致．ＳＤＳ－ＰＡＧＥ法测定分子量为１６ｋＤ,薄层丙烯酰胺凝胶等电聚焦分析该蛋白等电点为ｐＨ８．８～８．９,３ＨＴｄＲ参入法测定ｒｍｈＩＬ－１７单体的体外活性,实验结果表明ｒｍｈＩＬ－１７对ＰＨＡ活化人的ＰＢＭＣ细胞具有抑制作用,结果同可溶型ＩＬ－１７Ｒ性质相似     

酵母豆蔻酰辅酶A：蛋白质N端转酰基酶基因的克隆与表达

利用ＰＣＲ技术,从酵母染色体中扩增得到酵母豆蔻酰－ＣｏＡ：蛋白质Ｎ端转酰基酶（ＹＳＣＮＭＴ）基因,并克隆到ｐＢｌｕｅｓｃｒｉｐｔＫＳ＋载体中。由ＤＮＡ全序测定表明,获得了ＹＳＣＮＭＴ编码基因。进一步构建了Ｔ７Ｐｒｏｍｏｔｅｒ控制下的含上述完整ＹＳＣＮＭＴ编码基因的表达质粒ｐＭＦＴ７－５－ＮＭＴ,转化大肠杆菌ＢＬ２１（ＤＥ３）,进行ＩＰＴＧ诱导表达研究。通过ＳＤＳ－ＰＡＧＥ分析,观察到一与理论分子量一致的诱导条带（约５３ｋＤ）,占全菌蛋白的３９％左右,且可溶性部分约占上清液中全部蛋白的３４％。经一步Ｐ１１磷酸纤维素阳离子交换柱层析,将其纯化到纯度达９７％以上．纯化的表达产物经Ｎ端氨基酸序列分析,所测定的Ｎ端５个氨基酸的序列,与从克隆的ＹＳＣＮＭＴ基因推出的氨基酸序列完全一致（不含Ｎ端Ｍｅｔ）。对所得的ＹＳＣＮＭＴ进行酶活力鉴定,观察到了明显的活力。     

水稻10kD醇溶蛋白基因克隆,序列分析及对植物百脉根的转化

应用ＰＣＲ 技术,从水稻基因组中扩增１０ ｋＤ 水稻醇溶蛋白基因的编码区,得到一特异的０．５ ｋｂ 的片段。对该片段进行酶切分析和全序列测定,结果表明： 该片段与Ｍａｓｕｍ ｕｒａ Ｔ．等的报道相比, 其核苷酸序列及推测的氨基酸序列的同源率分别为９５％ 和９３％ 。就其分子数计算,甲硫氨酸及半胱氨酸含量分别占１８．２％ 和９％ , 含硫氨基酸总数为２７．２％ , 比同类的１０ ｋＤ玉米醇溶蛋白的含硫氨基酸总数还要高。将该基因置于ｒｂｃ Ｓ启动子调控下,动员入农杆菌中,转化豆科植物百脉根（ＬｏｔｕｓｃｏｒｎｉｃｕｌａｔｕｓＬ．）, 在含有卡那霉素的抗性培养基上筛选抗性植株。利用ＰＣＲ 方法检测１０ ｋＤ 醇溶蛋白基因整合到百脉根基因组中     

减蛋综合征病毒100K蛋白基因的克隆与序列分析

用常规方法提取减蛋综合征病毒（ＥＤＳＶ）中国分离株（ＡＡ２株）病毒ＤＮＡ,分别构建了限制性内切酶ＨｉｎｄⅢ、ＳｐｈⅠ、ＰｓｔⅠ水解片段的全基因文库,并对其中１００Ｋ蛋白基因的序列进行了分析。ＥＤＳＶ１００Ｋ蛋白基因位于减蛋综合征病毒基因组５５．７～６４．８物理图谱单位（ｍ．ｕ）,共２０９１个核苷酸（ｎｔ）,其编码产物由６９６个氨基酸（ａａ）组成,推测其分子量为７７．７ｋＤ。编码蛋白氨基酸同源性分析表明,ＥＤＳＶ１００Ｋ蛋白与人腺病毒（Ａｄ２、Ａｄ５、Ａｄ１２、Ａｄ４１）、Ⅰ群禽腺病毒（ＣＥＬＯ和ＦＡＶ１０）的同源性为３２．３～３４．４％之间,而与羊腺病毒（ＯＡＶ）的同源性达到５６．４％。     

兔阑尾中一种新的21kD的钙结合蛋白的纯化与鉴定

纯化与鉴定了Ｂ淋巴细胞中一种新的分子量为２１ｋＤ的钙结合蛋白（ＣａＢＰ２１）。兔阑尾淋巴细胞匀浆经热变性,Ｐｈｅｎｙｌ－Ｓｅｐｈａｒｏｓｅ与ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱层析,自每１ｋｇ细胞沉积物中获得ＳＤＳ－ＰＡＧＥ均一的ＣａＢＰ２１５．３ｍｇ。ＨＣｌ水解后的酸性氨基酸（Ａｓｐ＋Ｇｌｕ）含量为２６％。如同大多数钙结合蛋白一样,Ｎ末端封闭阻止其进行Ｅｄｍａｎ降解。ＣａＢＰ２１中疏水性氨基酸（计Ｇｌｙ,不计Ｔｒｐ）约占４６％,碱性氨基酸１０％,酸性氨基酸与极性氨基酸约４４％。ＣａＢＰ２１有较高的Ｓｅｒ、Ｔｙｒ含量。肽谱分析等确证ＣａＢＰ２１为２个相同或相似亚基二聚体。以ＡｒｓｅｎａｚｏⅢ作Ｃａ２＋结合分析表明每分子ＣａＢＰ２１可结合４分子Ｃａ２＋,对Ｃａ２＋的结合常数约为１０－５ｍｏｌ／Ｌ。各种性质表明ＣａＢＰ２１是一种不同于其他已知钙结合蛋白的新钙结合蛋白。     

人胰岛素原类似物(BKRA)基因的合成与表达

为了利用基因工程生产胰岛素,按照已知的人胰岛素Ａ、Ｂ链氨基酸序列和大肠杆菌偏爱的氨基酸密码子设计并合成了人胰岛素原类似物（ＢＫＲＡ）基因,其中以赖（Ｋ）－精（Ｒ）二肽编码区取代人胰岛素原Ｃ肽编码区．为了避免其编码蛋白在大肠杆菌中表达时被降解,通过人工接头将２个ＢＫＲＡ基因串联起来,接头部分氨基酸序列为Ａｒｇ－Ａｒｇ－Ａｓｎ－Ｓｅｒ．将串联的ＢＫＲＡ基因克隆到表达载体ｐＥＴ－２８ａ（＋）,实现了在大肠杆菌中的融合表达,表达产物以包含体形式存在,约占细菌总蛋白２４％．表达产物氨基末端具有六组氨酸肽段,以ＨｉＴｒａｐ凝胶进行亲和层析,一步纯化可达纯度９５％以上．放射免疫测定表明,纯化的融合蛋白具有胰岛素抗原活性．表明已构建成人胰岛素原类似物的高效表达菌株     

富硫蛋白基因对牧草百脉根的转化

豆科植物百脉根（ＬｏｔｕｓｃｏｒｎｉｃｏｆａｔｕｓＬ．）是一种优良的牧草。１０ｋＤ玉米醇溶蛋白是一种富硫蛋白,依分子数计算,含硫氨基酸占总氨基酸量的２５％。通过根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａｃｉｅｎｓ）的介导,将ｒｂｃＳ启动子及ＣａＭＶ３５Ｓ启动于调控下的１０ｋＤ玉米醇溶蛋白基因的嵌合质粒导入百脉根,得到转化的植株,其卡那霉素的抗性由ＢＮＰＴⅡ活性分析进一步得到证明。Ｓｏｕｔｈｅｒｎｂｌｏｔ分析表明,１０ｋＤ玉米醇溶蛋白基因已整合到百脉根的核基因组中。     

一种可识别神经元特异微管蛋白的抗体

通过人工合成大鼠巢蛋白Ｃ－末端的一段２０肽,制备得到了抗巢蛋白的抗体－Ａｎｔｉ－Ｎｅｓ－２。我们发现该抗体除了能识别小鼠２４０ｋＤ的巢蛋白条带外,还特异性识别另一分子量约为５０ｋＤ的蛋白条带,蛋白印迹反应提示,该５０ｋＤ蛋白可能与神经细胞的增殖和分化相关。经分离纯化和Ｎ－端氨基酸序隐测定证实：该５０ｋＤ蛋白为微管蛋白。     

传染性支气管炎病毒核衣壳蛋白基因克隆及在E.coli中的表达

核衣壳蛋白基因 (N基因 )是传染性支气管炎病毒的重要结构基因 .根据已报道的序列设计引物 ,利用RT PCR技术从病毒RNA中扩增和克隆到了N基因的cDNA ,并测定了核苷酸序列 .克隆的N基因片段ORF全长 12 30bp ,编码 4 0 9个氨基酸 .将该片段序列与其他IBV病毒株比较 ,核苷酸的同一性为 87 0 %～ 98 6 %,氨基酸的同一性为 91 0 %～ 98 1%.将该cDNA亚克隆到pBV2 2 0表达载体 ,转化大肠杆菌DH5α菌株 ,Western印迹检测 ,获得了分子量约 4 5kD表达蛋白     

球毛壳菌60S核糖体蛋白L10a基因克隆与特性分析

用粗糙脉孢菌（Neurospora crassa）XP_322380和赤霉菌（Gibberella zeag）PH-1（EAA76971）的60S核糖体蛋白L10a基因（60S ribosomal protein L10a,RPL10a）蛋白序列对球毛壳菌（Chaetomium globosum）ESTs序列数据库进行tBlastn检索,获得了球毛壳菌RPL10a cDNA序列。cDNA序列长765bp,开放阅读框654bp,编码217个氨基酸组成的多肽,蛋白分了量为23．9kD。BlastP分析表明该基因氨基酸序列与粗糙脉胞菌相似最高为89％;与玉蜀黍黑粉菌（Ustilago maydis）相似性最低为78％。cDNA序列及推测的氨基酸序列在GenBank登录（登录号分别为AY669070,AAT74578）。     

Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors.

A cDNA clone, NA-PI-II, encoding a protein with partial identity to proteinase inhibitor (PI) II of potato and tomato has been isolated from a cDNA library constructed from Nicotiana alata stigma and style mRNA. The cDNA encodes a polypeptide of 397 amino acids with a putative signal peptide of 29 amino acids and six repeated domains, each with a potential reactive site. Domains 1 and 2 have chymotrypsin-specific sites and domains 3, 4, 5, and 6 have sites specific for trypsin. In situ hybridization experiments demonstrated that expression of the gene is restricted to the stigma of both immature and mature pistils. Peptides with inhibitory activity toward chymotrypsin and trypsin have been isolated from stigmas of N. alata. The N-terminal amino acid sequence obtained from this protein preparation corresponds to six regions in the cDNA clone NA-PI-II. The purified PI protein preparation is likely to be composed of a mixture of up to five similar peptides of approximately 6 kD, produced in vivo by proteolytic processing of a 42-kD precursor. The PI may function to protect the reproductive tissue against potential pathogens.     

从云南烟草上检测到的黄瓜花叶病毒亚组Ⅱ分离物*

在对云南省烟草病毒病的研究中,分离到一种直径约２６￣３０ｎｍ的球形病毒。提纯病毒进行的ＳＤＳ－ＰＡＧＥ发现一条５５ｋＤ蛋白带。５５ｋＤ蛋白Ｎ－端１０个氨基酸与ＣＭＶ亚组Ⅱ的Ｑ株系外壳蛋白Ｎ－氨基酸同源性为１００％。以ＣＭＶ－Ｑ抗血清对５５ｋＤ蛋白进行了Ｗｅｓｔｅｒｎ ｂｌｏｔ检测,发现５５ｋＤ蛋白与ＣＭＶ Ｑ株系抗血清有血清学反应。根据已报道的ＣＭＶ亚组Ⅱ外壳蛋白基因序列合成引物,采用ＲＴ－ＰＣＲ     

Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

意大利蜜蜂丙糖磷酸异构酶基因的克隆及其原核表达与纯化

从意大利蜜蜂Apis mellifera ligustica的肌肉组织中提取总RNA,采用RT-PCR的方法克隆蜜蜂第16号染色体上的丙糖磷酸异构酶基因的cDNA序列,将测序结果(GenBank登录号EU76098)与推导的氨基酸序列分别与GenBank中的其他物种进行同源比对分析。结果表明,该基因全长744bp,为完整的阅读框,编码247个氨基酸,成熟蛋白的理论分子量为26.89kD。比对结果显示AmTPI与家蚕、德国小镰、黄粉虫、丽蝇蛹集金小蜂、水稻等物种的基因相似性达69%以上,蛋白相似性达59%以上。将目的基因克隆到pGEX-4T-2融合表达载体上,并在大肠杆菌中得到成功表达,4h的表达量为总蛋白的42.1%。为了进一步探讨产物的酶学特性,实验还对表达产物进行纯化与浓缩。实验还构建增强型荧光真核表达质粒,为进一步研究AmTPI在真核细胞中的表达情况奠定基础。     

烟实夜蛾信息素结合蛋白3 cDNA的克隆、序列分析与原核表达

利用RT-PCR技术从烟实夜蛾Helicoverpa assulta (Hass) 雄虫触角中扩增得到了信息素结合蛋白3(Hass PBP3)。克隆和测序结果表明,该基因核苷酸序列全长495 bp,编码164个氨基酸残基,预测分子量18.5 kD。并预测N-末端疏水区包含由22个氨基酸组成的信号肽。因此,成熟蛋白应包括142个氨基酸,预测分子量为16.1 kD,等电点为5.44。经氨基酸序列同源性分析发现,此序列与已知昆虫PBP3有较高的同源性,而且具有气味结合蛋白的典型特征。将该基因重组到表达载体pGEX-4T-2中进行原核表达。经IPTG诱导、SDS-PAGE分析和Western印迹检测,结果表明烟实夜蛾PBP3基因能在大肠杆菌BL21中表达,电泳检测到一条大约42 kD的外源蛋白,与预测的融合蛋白分子量相符。     

Characterization of a cDNA encoding rat sterol carrier protein2

Sterol carrier protein2 (SCP2) is a 13.2-kD protein that is thought to be involved in the intracellular transport of cholesterol. Using synthetic oligonucleotides based on the protein sequence of SCP2, a clone (SP43) was isolated from a rat liver cDNA library. The DNA sequence revealed that the cDNA could encode a polypeptide of 273 amino acids (28.9 kD) or 143 amino acids (15.3 kD) in which the carboxy-terminal 123 amino acids are identical to the SCP2 protein. RNA blot hybridization revealed that a variety of rat tissues contain a homologous RNA of a size similar to SP43 (approximately 1.5 kb). Levels of SCP2 mRNA increased in parallel with cytochrome P450scc mRNA in the immature gonadotropin-primed rat ovary. The isolation of a cDNA clone encoding SCP2 will facilitate studies on its role in cholesterol metabolism.     

鲤鱼(Cyprinus carpio)生长激素基因克隆及原核表达

采用逆转录—聚合酶链式反应（ＲＴ－ＰＣＲ）方法,从鲤鱼脑垂体总ＲＮＡ中扩增出编码鲤鱼生长激素（ＧＨ）成熟肽基因序列．定向克隆至质粒ｐＵＣ１８,克隆的鲤鱼ＧＨｃＤＮＡ不含信号肽序列并以新的起始密码子ＡＴＧ取代鲤鱼ＧＨｃＤＮＡ第１个密码子ＴＣＡ．序列分析表明,与Ｋｏｒｅｎ报道的鲤鱼ＧＨｃＤＮＡ相比有两个碱基差异,但推断的氨基酸序列完全一致．将鲤鱼ＧＨｃＤＮＡ定向克隆至原核表达载体ｐＢＶ２２０,构建成重组鲤鱼ＧＨ基因表达载体ｐＢＶｃＧＨ８．ＳＤＳ－ＰＡＧＥ和薄层扫描分析表明：经４２℃诱导,ｐＢＶｃＧＨ８在大肠杆菌中可表达一分子量约２２０００的特异蛋白,表达量占细胞总蛋白的２９．２％．该基因重组的鲤鱼ＧＨ添加到饲料中投喂罗非鱼,证实有明显的促进生长作用     

CoA-dependent methylmalonate-semialdehyde dehydrogenase, a unique member of the aldehyde dehydrogenase superfamily. cDNA cloning, evolutionary relationships, and tissue distribution.

Three overlapping cDNA clones encoding methylmalonate-semialdehyde dehydrogenase (MMSDH; 2-methyl-3-oxopropanoate:NAD+ oxidoreductase (CoA-propanoylating); EC 1.2.1.27) have been isolated by screening a rat liver lambda gt 11 library with nondegenerate oligonucleotide probes synthesized according to polymerase chain reaction-amplified portions coding for the N-terminal amino acid sequence of rat liver MMSDH. The three clones cover a total of 1942 base pairs of cDNA, with an open reading frame of 1569 base pairs. The authenticity of the composite cDNA was confirmed by a perfect match of 43 amino acids known from protein sequencing. The composite cDNA predicts a 503 amino acid mature protein with M(r) = 55,330, consistent with previous estimates. Polymerase chain reaction was used to obtain the sequence of the 32 amino acids corresponding to the mitochondrial entry peptide. Northern blot analysis of total RNA from several rat tissues showed a single mRNA band of 3.8 kilobases. Relative mRNA levels were: kidney greater than liver greater than heart greater than muscle greater than brain, which differed somewhat from relative MMSDH protein levels determined by Western blot analysis: liver = kidney greater than heart greater than muscle greater than brain. A 1423-base pair cDNA clone encoding human MMSDH was isolated from a human liver lambda gt 11 library. The human MMSDH cDNA contains an open reading frame of 1293 base pairs that encodes the protein from Leu-74 to the C terminus. Human and rat MMSDH share 89.6 and 97.7% identity in nucleotide and protein sequence, respectively. MMSDH clearly belongs to a superfamily of aldehyde dehydrogenases and is closely related to betaine aldehyde dehydrogenase, 2-hydroxymuconic semialdehyde dehydrogenase, and class 1 and 2 aldehyde dehydrogenases.