Nucleotide sequence of Escherichia coli tyrosine transfer ribonucleic acid

The nucleotide sequence of one of the Escherichia coli tyrosine-transfer ribonucleic acids was determined in order to compare its sequence with that of yeast tyrosine-transfer ribonucleic acid. Forty-four positions of both transfer ribonucleic acids are occupied by the same nucleotides if they are arranged in the manner shown here. The information obtained suggests that the conformation of transfer ribonucleic acid molecules may be a greater contributing factor than a specific nucleotide sequence in the interaction of transfer ribonucleic acid with its corresponding aminoacyl-transfer ribonucleic acid synthetase.     

Herpes simplex virus type-1 glycoprotein D gene: nucleotide sequence and expression in Escherichia coli

The protein coding region of the herpes simplex virus type-1 glycoprotein D (gD) gene was mapped, and the nucleotide sequence was determined. The predicted amino acid sequence of the gD polypeptide was found to contain a number of features in common with other virus glycoproteins. Insertion of this protein coding region into a bacterial expressor plasmid enabled synthesis in Escherichia coli of an immunoreactive gD-related polypeptide. The potential of this system for preparation of a type-common herpes simplex virus vaccine is discussed.     

鸡Prnp基因原核表达载体的构建及其在大肠埃希菌中的表达

试验旨在构建鸡Prnp基因原核表达载体,并在大肠埃希菌中进行表达,为制备鸡朊蛋白单克隆抗体提供材料。根据GenBank已报道的鸡Prnp基因组序列和pET-28a质粒多克隆位点设计引物,以健康的鸡全血基因组DNA为材料,采用PCR的方法扩增鸡的Prnp基因,将目的基因片段与pET-28a载体连接,构建重组原核表达载体。重组菌转化到E. coli BL21(DE3)感受态细胞中,并用异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG)进行诱导表达,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定表达的重组蛋白。结果表明,重组蛋白在诱导剂的终浓度为0.08 mmol·L^-1,16 ℃,220 r·min^-1诱导7 h,蛋白表达量最高。综上所述,本研究成功构建了pET-28a-ChPrnp重组表达菌株,为Prnp朊蛋白的结构、生理功能和致病机制研究提供方法。     

Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution

The sequence of the entire RNA genome of the type flavivirus, yellow fever virus, has been obtained. Inspection of this sequence reveals a single long open reading frame of 10,233 nucleotides, which could encode a polypeptide of 3411 amino acids. The structural proteins are found within the amino-terminal 780 residues of this polyprotein; the remainder of the open reading frame consists of nonstructural viral polypeptides. This genome organization implies that mature viral proteins are produced by posttranslational cleavage of a polyprotein precursor and has implications for flavivirus RNA replication and for the evolutionary relation of this virus family to other RNA viruses.     

蚯蚓MT基因的克隆及其在大肠杆菌中的表达

为了研究蚯蚓金属硫蛋白的生物学功能,采用RT-PCR方法从镉诱导后的赤子爱胜蚓(Eisenia foetida)中克隆到蚯蚓金属硫蛋白基因cDNA。将其克隆入原核表达载体pET-DsbA中,然后转化至大肠杆菌表达受体菌BL21(DE3)中进行表达。产物经SDS-PAGE进行分析,可见约32.4 kD的融合蛋白。Zn2+抗性测定表明含pET-DsbA-efMT的重组菌较含空载体pET-DsbA的对照菌重金属抗性有所提高。     

杜仲EuCHIT1基因表达载体构建及在大肠杆菌中的表达

构建了T7启动子驱动杜仲几丁质酶基因EuCHIT1的原核表达载体pET-EuCHIT1和pMCSG-EuCHIT1,其表达产物分别含6个组氨酸(6His)标签和6个组氨酸连接的麦芽糖结合蛋白(his6-tag–maltose-binding protein,MBP)标签,分别将重组载体遗传转化大肠杆菌细胞BL21(DE3),在37℃、150 rpm条件下培养至菌液OD值为0.4~0.8后,转到16℃、150 rpm条件下以1 m MIPTG诱导培养12 h,表达产物经SDS-PAGE分析,p ET-EuCHIT1/BL21(DE3)表达以包涵体形式存在36.03 kD融合蛋白,pMCSG-EuCHIT1/BL21(DE3)成功表达可溶形式存在的77.21 kD融合蛋白。故可以使用pMCSG-EuCHIT1/BL21(DE3)获得可溶的融合蛋白,为之后的EuCHIT1的多克隆抗体的制备和及功能研究奠定基础。     

核盘菌EPSP合酶基因在大肠杆菌中的表达

不含有内含子的核盘菌arom基因已经被扩增、测序. 该基因编码五功能的AROM蛋白. 为了大量获得核盘菌AROM蛋白的结构域之一5烯醇丙酮酰莽草酸-3磷酸合酶（EPSPS）,将该菌arom基因编码脱氢奎尼酸合酶（DHQS）和EPSPS两结构域的DNA序列和只编码EPSPS结构域的DNA序列分别克隆入载体pGEX-4t-2和载体pET28b中,构建了4个表达载体pGEX-DE、pGEX-E、pET-DE和pET-E, 并将其转入大肠杆菌DH5α、大肠杆菌BL21（DE3）或大肠杆菌JM109中表达. 酶活测定和SDS-PAGE分析结果显示,上述编码序列在大肠杆菌细胞内获得了表达,含有表达载体pGEX-E、pET-DE和pET-E的大肠杆菌BL21（DE3）转化子具有EPSPS的催化活性,说明核盘菌arom基因的这些DNA片段可以被单独表达. 核盘菌EPSPS异源表达系统的 建立为该酶的抑制剂设计奠定了基础.      

茶树Mn-SOD基因在大肠杆菌中的高效表达

聚合酶链式反应(PCR)扩增茶树嫩叶锰超氧化物歧化酶基因,并与原核表达载体pET22b( )连接,构建重组质粒pET/msod,将该质粒转化至大肠杆菌Escherichia coliBL21(DE3),获得转基因工程菌BL21-pET/msod。在1 mmol.L-1异丙基硫代-β-半乳糖苷(IPTG)诱导下,重组蛋白得到高效表达,酶活性可达2×105U.L-1。SDS-PAGE检测表达蛋白分子量为25 kD,与通过核苷酸推测的分子量一致。     

大肠杆菌K88菌毛蛋白faeG亚基基因克隆及表达

利用PCR技术,从致仔猪黄痢的大肠杆菌中扩增不含信号肽序列的K88菌毛蛋白faeG亚基基因片段,将其克隆到表达载体pGEX-6P-1中,构建该基因的原核表达载体pGEX-FaeG,通过测序证明序列正确后,导入大肠杆菌BL21,得到工程菌株PGEX-FaeG。IPTG诱导表达,SDS-PAGE分析结果表明,融合蛋白（约53ku）在大肠杆菌BL21中得到高效表达,表达产物约占菌体蛋白的35％,免疫印记结果表明此融合蛋白与K88单抗反应。     

Nucleotide sequence of the transforming gene of avian myeloblastosis virus

Avian myeloblastosis virus is defective in reproductive capacity, requiring a helper virus to provide the viral proteins essential for synthesis of new infectious virus. This virus arose by recombination of the nondefective helper virus and host cellular sequences present within the normal avian genome. These latter sequences are essential for leukemogenic activity. The complete nucleotide sequence of this region is reported. Within the acquired cellular sequences there is an open reading frame of 795 nucleotides starting with the initiation codon ATG (adenine, thymine, guanine) and terminating with the triplet TAG. This open reading frame could code for the putative transforming protein of 265 amino acids with a molecular weight of approximately 30,000.     

Molecular cloning and expression of a human B-cell growth factor gene in Escherichia coli

A human B-cell growth factor (BCGF) (12 kilodaltons) supports the clonal proliferation of B lymphocytes. A clone was isolated that contained the proper structural sequence to encode biologically active, 12-kilodalton BCGF in Escherichia coli and to hybridize to a specific messenger RNA, identified by in vitro translation in Xenopus laevis oocytes. A relatively hydrophobic region of 18 amino acids was found at the amino terminal of the 124-amino acid-long polypeptide. The carboxyl terminal is composed of at least 32 amino acids that are derived from nucleotide sequences bearing significant homology to the Alu repeat family.     

菠菜胆碱单氧化酶(CMO)基因的克隆及在大肠杆菌中的诱导表达

甘氨酸甜菜碱是一种非毒性的渗透调节物质 ,在植物体内是以胆碱为底物 ,经两步氧化而合成的 .菠菜中 ,催化第一步反应的酶为胆碱单氧化酶 (CholineMonooxygenase ,CMO) .为了研究胆碱单氧化酶基因的功能以及转基因植物的抗逆能力 ,在 30 0mmol L高盐浓度 (n(NaCl)∶n(CaCl2 ) =5 7∶1)的条件下 ,作者分离纯化了菠菜mRNA ,经RT PCR得到全长 (1.3kb)胆碱单氧化酶 (CMO)cDNA ,与已经报道的基因序列相比较 ,同源性为 99% .根据其核苷酸序列推导得到了氨基酸序列 .将PCR纯化产物与pET 30a+ 连接 ,构建了重组表达载体pETCMO ,并转化到大肠杆菌BL2 1(DE3) ,经IPTG诱导获得高效表达 .     

传染性支气管炎病毒核衣壳蛋白基因变异及在大场杆菌中的表达

核衣壳蛋白(N)是传染性支气管炎病毒的主要结构蛋白之一. 以前的研究结果显示IBV-ZJ971毒株以引起腺胃肿大为特征,IBV-X和N毒株以引起肾炎为特征.为了探讨不同组织嗜性传染性支气管炎病毒N基因的变异,我们依据已发表的IBV- Beaudette 毒株N基因的序列设计和合成引物,RT-PCR扩增IBV-ZJ971、N、X和H52的核衣壳蛋白基因,并测序、分析和在E.coli中进行表达.结果显示:IBV-ZJ971、N、X和H52毒株的N基因的ORF由1230 bp组成,编码409个氨基酸.与来源于GenBank中的IBV毒株比较,IBV-ZJ971、N、X和H52与其它IBV毒株的核苷酸同源性分别是87.0-98.6%、86.6-99.7%、86.3-99.7%、87.2%-98.5%,氨基酸的同源性分别是90.0-97.8%、 90.5-98.8%、 90.0-98.8%、 91-98%.IBV-ZJ971毒株的N蛋白不同于其他IBV的独特变异是111,117, 142, 171 和 401位点上的氨基酸改变,IBV-N和X毒株的N蛋白不同与其它IBV的独特变异是46, 48, 189,190, 220, 223, 236, 237, 240, 286, 299, 301 334, 335, 336 and 351位点上的氨基酸改变.说明IBV嗜腺胃毒株(ZJ971)和嗜肾毒株(N和X)的N基因以散在的点突变为特征.将IBV-ZJ971毒株的N基因在E.coli中表达,并用western-blotting检测,发现IBV-ZJ971的N蛋白是一分子量约为45 kD的蛋白.     

阿拉伯糖苷酶基因的克隆、表达及表达产物的酶稳定性

阿拉伯糖苷酶／木糖苷酶是木聚糖类半纤维素生物降解和转化所必需的酶类。本文在国内首次报道对该酶的研究：通过PCR从产乙醇热厌氧杆菌Thermoanaerobacter ethanolicus JW200克隆出编码高度热稳定性阿拉伯糖苷酶／木糖苷酶的基因，与组氨酸标签融合，以高拷贝质粒pAlter-Exl在大肠杆菌中得到高效表达；基因表达产物通过热处理和亲和层析柱纯化后，酶纯度达电泳均一。纯化重组酶稳定性检测表明，得到的阿拉伯呋喃糖苷酶在pH4．2～8．2之间酶活力稳定，75℃的半衰期为1h；β-木糖苷酶在pH5．0～8．2之间有较高的稳定性，酶1h半衰期温度为84℃。     

鲎抗菌肽polyphemusinⅡ基因的改造、克隆及其在大肠杆菌中的表达

中国鲎(Tachypleus trideutatus)血细胞产生的抗菌肽(Antibacterial peptide)—鲎素,具有广谱的杀菌、抑病毒和抗肿瘤细胞的作用。实验根据已报道的美洲鲎的抗菌肽polyphemusinⅡ氨基酸序列,设计了一对引物,并对原抗菌肽基因进行改造,在基因开放阅读框末端添加了Asn密码子,使抗菌肽羧基末端酰胺化,旨在提高其稳定性和抗菌活性。PCR扩增获得改造过的目的片段,连接于载体pMD18-T,测序后,与pEI-28c(+)连接,构建表达质粒pET28c-rr,测序鉴定正确后,转化大肠杆菌E.coli.BK21(DE3)。表达菌株经终浓度为1mmol/L的IPTG诱导后以包涵体的形式表达,在体外表现出明显的抑菌活性。     

鸡致病性大肠杆菌O78菌株iss基因的克隆与测序

对1株O78血清型鸡源致病性大肠杆菌采用PCR方法检测iss基因的存在。结果显示,鸡E.coliO78菌株携带iss基因,但采用目前通用的PCR方法并不能检测出iss基因,必须经套式PCR扩增才能检测出来。O78菌株所携带iss基因的序列与已知禽大肠杆菌iss基因序列同源性为99.4%,与人源大肠杆菌iss基因同源性为90.3%,与λ噬菌体bor基因序列同源性为87.7%,显示了该基因的保守性。     

Fitness of an Escherichia coli mutator gene

Competition experiments between Escherichia coli mutT1 and mut(+) populations show that the mutator gene confers selective advantage on the strain that carries it. The observed increase in fitness varies, with an average increase in mutator growth rate of 1.4 percent when mutator and wild-type strains are grown together in chemostats.     

Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2)

The nucleotide sequence of molecular clones of DNA from a retrovirus, ARV-2, associated with the acquired immune deficiency syndrome (AIDS) was determined. Proviral DNA of ARV-2 (9737 base pairs) has long terminal repeat structures (636 base pairs) and long open reading frames encoding gag (506 codons), pol (1003 codons), and env (863 codons) genes. Two additional open reading frames were identified. Significant amino acid homology with several other retroviruses was noted in the predicted product of gag and pol, but ARV-2 was as closely related to murine and avian retroviruses as it was to human T-cell leukemia viruses (HTLV-I and HTLV-II). By means of an SV-40 vector in transfected simian cells, the cloned gag and env genes of ARV-2 were shown to express viral proteins.     

稻褐飞虱羧酸酯酶基因的克隆与表达

以稻褐飞虱的cDNA为模板,进行PCR扩增,获得约1 900 bp的DNA片段.通过T-A克隆,将PCR产物插入pMD18-T载体中.再以此重组载体为模板,以羧酸酯酶成熟蛋白基因(cae-M)的特异性引物进行PCR扩增.将cae-M扩增产物和pET28a分别用EcoRⅠ和HindⅢ双酶切,酶切产物纯化后在T4连接酶作用连接成重组表达载体pET28a-cae-M.将pET28a-cae-M转化BL21(DE3),经IPTG诱导和SDS-PAGE分析,可见约62 kD外源蛋白带.蛋白质印迹分析表明,该外源蛋白与6×His融合表达.     

鸡γ-干扰素基因在大肠杆菌中的表达

构建鸡γ-干扰素基因(lFN-γ)的融合表达质粒,并在原核系统表达。根据GenBank发表的鸡γ-干扰素核苷酸序列,使用prim er5设计一对特异性引物,通过RT-PCR技术从ConA诱导培养的鸡脾脏淋巴细胞中克隆出鸡γ-干扰素基因并对其进行测序。测序结果表明,鸡γ-干扰素基因全长495bp,具有一个完整的开放阅读框,编码164个氨基酸,与国外发表的序列同源性为100%。将序列连接到原核表达载体pET28 a(+)上,转化大肠杆菌BL21(DE3),经IPTG诱导后进行SDS-PAGE电泳分析。结果表明鸡γ-干扰素表达蛋白大小为20.8KD,并以包涵体形式表达。为鸡γ-干扰素生物制剂或疫苗佐剂的开发奠定基础。