首页> 外国专利> RECOMBINANT VECTORS, POLYNUCLEOTIDE ENCODING A 12KD POLYPEPTIDE PD428 OF MYCOBACTERIA BELONGING TO THE MYCOBACTERIUM TUBERCULOSIS COMPLEX AND APPLICATIONS TO DIAGNOSIS AND THE PREVENTION OF TUBERCULOSIS

RECOMBINANT VECTORS, POLYNUCLEOTIDE ENCODING A 12KD POLYPEPTIDE PD428 OF MYCOBACTERIA BELONGING TO THE MYCOBACTERIUM TUBERCULOSIS COMPLEX AND APPLICATIONS TO DIAGNOSIS AND THE PREVENTION OF TUBERCULOSIS

机译：重组载体，编码结核分枝杆菌的12KD多肽PD428的多核苷酸和结核分枝杆菌复合物及其在诊断和预防结核中的应用

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The following are claimed: (1) a recombinant screening, cloning and/or expression vector that replicates in mycobacteria, comprising: a replicon functional in mycobacteria; a selectable marker; and a reporter cassette comprising (a) a multiple cloning site (polylinker), (b) optionally a mycobacterial transcription terminator upstream of the polylinker, (c) a coding sequence from a gene coding for a protein expression, export and/or secretion marker, the coding sequence lacking its start codon and regulatory sequences, and (d) a coding sequence from a gene coding for a cis-acting promoter activity marker, the coding sequence having its start codon; (2) a recombinant vector as above containing a mycobacterial nucleic acid sequence in one of the cloning sites of the polylinker, where the mycobacterial nucleic acid sequence is one "in which one detects a polypeptide capable of being exported and/or secreted, and/or of being induced or repressed during infection by said mycobacterium or constitutively expressed or produced, as well as associated promoter and/or regulatory sequences capable of permitting or enhancing the export and/or secretion of said polypeptide, or all or part of a gene coding for said polypeptide"; (3) a method for screening mycobacterial nucleotide sequences to detect sequences coding for exported and/or secreted polypeptides that may be induced or repressed during infection, promoter and/or (other) regulatory sequences associated with such coding sequences, especially sequences that permit or enhance the export and/or secretion of such polypeptides, or all or part of genes coding for such polypeptides, using a vector as above; (4) a method as in (3) comprising: (a) physically or enzymatically fragmenting mycobacterial DNA sequences and recovering the resulting fragments; (b) inserting the fragments into a compatible cloning site in the vector's polylinker; (c) if necessary, amplifying the fragments contained in the vector, e.g. by replicating the vector in a cell, preferably of E. coli; (d) transforming host cells with the vector; (e) culturing the transformed cells in a medium permitting detection of the export and/or secretion marker and/or the promoter activity marker; (f) detecting positive colonies that express the export and/or secretion marker and/or the promoter activity marker; (g) isolating DNA from the positive colonies and inserting this DNA into a cell identical to that of step (c); (h) "the selection of insertions contained in the vector, permitting clones positive for the export and/or secretion marker, and/or for the promoter activity marker to be obtained"; and (i) isolating and characterising mycobacterial DNA sequences contained in the inserts and optionally sequencing selected inserts; (5) a mycobacterial genomic DNA or cDNA library obtained by the method of (3) and/or by a method comprising steps (a) and (b) or steps (a), (b) and (c) of the method of (4); (6) a mycobacterial nucleotide sequence selectable by the method of (3) or (4); (7) a polynucleotide which has a sequence complementary to the sequence of (6), or has a sequence that is at least 50% identical to the sequence of (6), or hybridises with the sequence of (6) under high-stringency conditions, or consists of a fragment of at least 8 consecutive nucleotides of the sequence of (6); (8) a polypeptide, "their fragments or biologically active fragments or their homologous polypeptides", that is encoded by a mycobacterial nucleotide sequence as in (6) or (7) and is capable of being exported and/or secreted and/or induced and/or repressed or constitutively expressed during infection; (9) a recombinant mycobacterium transformed with a recombinant vector as above; (10) a polypeptide encoded by a mycobacterial nucleotide sequence as in (6) or (7); (11) a polypeptide selected from: (a) a polypeptide having a sequence selected from SEQ ID NO:1 to SEQ ID NO:24C, SEQ ID NO:27A to SEQ ID NO:28 and SEQ ID NO:30 to SEQ ID NO:50F all defined sequences given in the specification, (b) a polypeptide homologous to the polypeptide of (a), (c) a fragment comprising at least 5 amino acids of the polypeptide of (a) or (b), and (d) a biologically active fragment of the polypeptide of (a), (b) or (c); (12) a recombinant cloning, expression and/or insertion vector containing a nucleotide sequence as in (6) or (7); (13) a host cell transformed with the vector of (12); (14) a process for preparing a polypeptide using the vector of (12); (15) a recombinant polypeptide obtainable by the process of (14); (16) a hybrid polypeptide comprising at least one polypeptide sequence as in (8), (11) or (15) and a sequence of a polypeptide capable of inducing an immune response in humans or other animals; (17) a polynucleotide encoding the hybrid polypeptide of (16); (18) mono- or polyclonal antibodies, antibody fragments or chimeric antibodies capable of specifically recognising the polypeptide of (8), (11) or (15); (19) a method of screening for molecules capable of inhibiting the growth or survival of mycobacteria in a host, characterised in that the molecules block the synthesis or function of polypeptides encoded by the nucleotide sequence of (6) or the polynucleotide of (7); (20) molecules capable of inhibiting the growth or survival of mycobacteria in a host, where the molecules are synthesised according to the structure of polypeptides encoded by the nucleotide sequence of (6) or the polynucleotide of (7).

机译：要求保护以下内容：（1）在分枝杆菌中复制的重组筛选，克隆和/或表达载体，包括：在分枝杆菌中有功能的复制子;和可选标记;以及报告盒，其包含（a）多克隆位点（多接头），（b）任选地在多接头上游的分枝杆菌转录终止子，（c）来自编码蛋白质表达，输出和/或分泌标记的基因的编码序列，该编码序列缺乏其起始密码子和调控序列，和（d）来自编码顺式作用启动子活性标记的基因的编码序列，该编码序列具有其起始密码子; （2）如上所述的重组载体，其在多接头的克隆位点之一中包含分枝杆菌核酸序列，其中该分枝杆菌核酸序列为“其中一个检测到能够输出和/或分泌的多肽，和/或在感染过程中被所述分枝杆菌诱导或抑制或组成性表达或产生，以及相关的启动子和/或调节序列，其能够允许或增强所述多肽或编码基因的全部或部分的输出和/或分泌对于所述多肽”; （3）一种筛选分枝杆菌核苷酸序列以检测编码在感染过程中可被诱导或抑制的输出和/或分泌的多肽的序列，启动子和/或（其他）与这些编码序列相关的调控序列，特别是允许或抑制序列的序列的方法。使用上述载体，增强此类多肽或编码此类多肽的全部或部分基因的输出和/或分泌; （4）如（3）所述的方法，其包括：（a）物理或酶促断裂分枝杆菌DNA序列并回收所得片段; （b）将片段插入载体多接头的相容克隆位点; （c）如果需要，扩增载体中包含的片段，例如通过在载体，优选大肠杆菌的细胞中复制载体; （d）用载体转化宿主细胞; （e）在允许检测出口和/或分泌标记和/或启动子活性标记的培养基中培养转化的细胞; （f）检测表达出口和/或分泌标记和/或启动子活性标记的阳性菌落; （g）从阳性菌落中分离DNA，并将该DNA插入与步骤（c）相同的细胞中; （h）“选择载体中所含的插入物，从而获得对出口和/或分泌标记和/或启动子活性标记呈阳性的克隆”; （i）分离和鉴定插入物中包含的分枝杆菌DNA序列，并任选地对选定的插入物进行测序; （5）通过（3）的方法和/或通过包括步骤（a）和（b）或步骤（a），（b）和（c）的方法获得的分枝杆菌基因组DNA或cDNA文库（4）; （6）通过（3）或（4）的方法选择的分枝杆菌核苷酸序列。（7）具有与（6）的序列互补的序列或与（6）的序列至少50％相同的序列，或在高严紧度下与（6）的序列杂交的多核苷酸。条件，或由（6）的序列的至少8个连续核苷酸的片段组成; （8）由如（6）或（7）中的分枝杆菌核苷酸序列编码并且能够被输出和/或分泌和/或诱导的多肽，“它们的片段或生物活性片段或它们的同源多肽”。和/或在感染过程中被抑制或组成型表达; （9）用上述重组载体转化的重组分枝杆菌。（10）由（6）或（7）中的分枝杆菌核苷酸序列编码的多肽; （11）选自以下的多肽：（a）具有选自SEQ ID NO：1至SEQ ID NO：24C，SEQ ID NO：27A至SEQ ID NO：28和SEQ ID NO：30至SEQ ID的序列的多肽NO：50F本说明书中给出的所有定义的序列，（b）与（a）多肽同源的多肽，（c）包含（a）或（b）多肽至少5个氨基酸的片段，和（ d）（a），（b）或（c）的多肽的生物活性片段; （12）重组克隆，表达和/或插入载体，其含有如（6）或（7）中所述的核苷酸序列; （13）用（12）的载体转化的宿主细胞。（14）一种使用（12）的载体制备多肽的方法; （15）通过（14）的方法得到的重组多肽。（16）杂合多肽，其包含至少一个如（8），（11）或（15）中的多肽序列和能够在人或其他动物中诱导免疫应答的多肽序列; （17）编码（16）的杂合多肽的多核苷酸。（18）能特异性识别（8）的多肽的单克隆或多克隆抗体，抗体片段或嵌合抗体，（11）或（15）; （19）一种筛选能够抑制宿主中分枝杆菌的生长或存活的分子的方法，其特征在于，所述分子阻断由（6）的核苷酸序列或（7）的多核苷酸编码的多肽的合成或功能。 ; （20）能够抑制宿主中分枝杆菌的生长或存活的分子，其中根据（6）的核苷酸序列或（7）的多核苷酸编码的多肽的结构合成分子。

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公开/公告号FR2767336B1

专利类型
公开/公告日2001-05-18

原文格式PDF
申请/专利权人 INSTITUT PASTEUR;
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申请/专利号FR19970010404
发明设计人 BRIGITTE GICQUEL;DENIS PORTNOI;ENG MONG LIM;VLADIMIR PELICIC;
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申请日1997-08-14
分类号C12N15/70;C12N15/31;C12N1/21;C12Q1/68;C07K14/35;C07K19/00;G01N33/68;G01N33/569;A61K39/04;A61K48/00;C12R1:32;
国家 FR
入库时间 2022-08-22 01:08:05

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