CN1318646A - 编码伤寒沙门氏菌特异性和抗原性外膜蛋白的dna序列 - Google Patents
编码伤寒沙门氏菌特异性和抗原性外膜蛋白的dna序列 Download PDFInfo
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- CN1318646A CN1318646A CN01117058A CN01117058A CN1318646A CN 1318646 A CN1318646 A CN 1318646A CN 01117058 A CN01117058 A CN 01117058A CN 01117058 A CN01117058 A CN 01117058A CN 1318646 A CN1318646 A CN 1318646A
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- 101710116435 Outer membrane protein Proteins 0.000 title claims abstract description 44
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 title claims abstract description 40
- 108091028043 Nucleic acid sequence Proteins 0.000 title claims description 19
- 230000000890 antigenic effect Effects 0.000 title description 3
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 79
- 208000037386 Typhoid Diseases 0.000 claims abstract description 24
- 201000008297 typhoid fever Diseases 0.000 claims abstract description 24
- 229960005486 vaccine Drugs 0.000 claims abstract description 11
- 108020004414 DNA Proteins 0.000 claims description 39
- 150000001413 amino acids Chemical group 0.000 claims description 36
- 239000002773 nucleotide Substances 0.000 claims description 24
- 125000003729 nucleotide group Chemical group 0.000 claims description 24
- 230000008521 reorganization Effects 0.000 claims description 16
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 14
- 108020004511 Recombinant DNA Proteins 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- 108091034117 Oligonucleotide Proteins 0.000 claims description 7
- 244000005700 microbiome Species 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 5
- 238000011238 DNA vaccination Methods 0.000 claims description 3
- 108091033319 polynucleotide Proteins 0.000 claims description 3
- 102000040430 polynucleotide Human genes 0.000 claims description 3
- 239000002157 polynucleotide Substances 0.000 claims description 3
- 241001465754 Metazoa Species 0.000 claims description 2
- BYXHQQCXAJARLQ-ZLUOBGJFSA-N Ala-Ala-Ala Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O BYXHQQCXAJARLQ-ZLUOBGJFSA-N 0.000 claims 30
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Abstract
已经分离出编码一种伤寒沙门氏菌的特异外膜蛋白(OMP)的遗传物质,并对其特性进行了描述。这种遗传物质(ST50)可生产应用于伤寒沙门氏菌的诊断、检测的特异的蛋白/肽/DNA/RNA,或用于生产伤寒疫苗。
Description
本发明涉及的领域是与伤寒沙门氏菌(Salmonella typhi)的特异性外膜蛋白(OMP)的编码基因(ST50)相关的基因工程学、诊断学和疫苗学。此蛋白的分子量估计为50kDa。通过编码这种蛋白的基因的基因工程，可以获得这种蛋白。
在大多数发展中国家，伤寒仍是一个公众健康的难题。现有的诊断此病常用方法由于太慢而不能使临床快速作出决定，所以仍然不能令人满意。培养方法虽然具有特异性，但它也缺乏灵敏度和速度。它在2-7天内才能得出结果，而且充分认识到有培养阴性的伤寒的病例。尽管已经广泛地应用了抗体检测试验(肥达氏试验)，但它也缺乏速度、灵敏度和特异性。该试验的有意义的解释必需在10-14天以后急性期和恢复期血清之间的抗体效价出现4倍的升高才能得出。理想的诊断伤寒的试验应当是快速而方便的，既具灵敏性又具特异性。上面所述的方法都不符合这些标准。因此需要开发一种快速且特异的试验。这种试验与灵敏的诊断结合，可对伤寒提供快速、有效和确切的处理方法。依快速诊断伤寒，随着50kDa OMP的发现，我们在伤寒的诊断方面已经取得了显著的突破。这种50kDa OMP对于病原伤寒沙门氏菌来说是特异的(Ismail A等。1991．《生物化学生物物理学研究通讯》27:301-5)。伤寒沙门氏菌外膜的特异性50kDa抗原的发现已经在马来西亚获得了专利(马来西亚专利第MY-106708-A号)。应用这种分离的OMP，我们成功地开发了一种快速斑点酶免疫测定法(TYPHIDOTTM)，以检测该细菌特异性IgM和IgG抗体的存在。此试验的灵敏度大于95％，具有一种较高的阴性预测值，并且可在1-3小时内得出结果。单独检测到IgM或同时检测到IgM和IgG提示为急性伤寒，而检测到IgG仅提出几种解释，诸如为恢复期或可能为再感染。由于对伤寒缺乏有效的免疫，在高流行地区的患者通常存在再感染。在目前再感染的条件下，会产生继发免疫反应，其中IgG超过IgM的显著的“加强免疫”效应，从而造成后者可能检测不到。解决这个难题的可能的策略是除去全部的IgG，而“揭示出”IgM的存在。这种3小时IgM检测试验(TYPHIDOTTM)的开发对高流行地区是非常有用的，因为它可区分出新病例和恢复期病例。研究各种试验的灵敏度、特异性和优势，由所得数据可以看出，与常用方法相比，这种试验能可靠地替代肥达氏试验。
为了大规模地生产这种蛋白，我们已经纯化并测定了50kDa OMP的氨基酸序列。基于这种发现，我们已经分离、克隆和测序了编码50kDa OMP的DNA。另外我们还通过表位定位确定了该蛋白的免疫原性表位。这个表位已经使50kDa OMP成功地应用于伤寒的诊断。
本发明提供了编码伤寒沙门氏菌50kDa OMP的遗传物质。此遗传物质可用于生产在诊断伤寒的方法中所用的蛋白。另外，这种遗传物质可用作检测伤寒沙门氏菌的一种探针。来源于此蛋白的完整蛋白或表位可用于伤寒的诊断和疫苗开发。更进一步的应用包括伤寒DNA/RNA疫苗的开发。
伤寒沙门氏菌50kDa OMP已经被插入两种不同的表达载体：pST50-USM1(附图1)和pST50-USM2(附图2)。这些克隆已经于2000年10月26日保藏在一个获批准的国际保藏单位，澳大利亚政府分析实验室，并且克隆pST50-USM1的登记号为NM00/16074,pST50-USm2的登记号为NM00/16075。
附图1是在TOP02.1克隆载体中的ST50克隆的质粒图。在图中标明了抗生素标记物氨苄青霉素(AmpR)、卡那霉素(KanR)、复制起始位点COLE1和F1ORI,以及ST50基因。此克隆的大小为5384bp。
附图2是在pRSETB表达载体中的ST50克隆的质粒图。在图中标明了抗生素标记物氨苄青霉素(AmpR)、复制起始位点FIORI和ST50基因。此克隆的大小为4401bp。
本发明人已经识别并首次获得了编码伤寒沙门氏菌特异性OMP的遗传物质，它先前只能以有限量获得。因为50kDa OMP是检测特异性IgM和IgG抗体及其它抗体种类的关键，以纯形式大量获得这种蛋白允许设计出伤寒的特异性诊断方法。而且，这种特异性遗传物质的识别和分离允许生产出新的伤寒疫苗。它还可成为特异性核酸探针的来源，这种探针可用于直接检测伤寒沙门氏菌的杂交技术。预测的起始密码子的第一残基标示为核苷酸7，表1描述的是其整个序列。表1伤寒沙门氏菌ST50基因DNA和氨基酸序列：
用带有它们的位置号的三联体描述这些核苷酸序列。开放阅读框(ORF)起始于7bp核苷酸位置，其起始密码子为“ATG”，终止于1474bp核苷酸位置的终止密码子“TGA”。通过DNA序列编码的氨基酸描述在DNA序列下面，并用它们的位置号表示。ST50蛋白的信号序列是一个16氨基酸肽，它起始于第3氨基酸位置，并终止于第18氨基酸位置。1 ATG CAA ATG AAG AAA TTG CTC CCC ATC CTT ATC GGC CTG AGC CTG 451 Met Gln Met Lys Lys Leu Leu Pro Ile Leu Ile Gly Leu Ser Leu 1546 TCG GGG TTC AGC ACA CTA AGC CAG GCA GAG AAC CTG ATG CAA GTT 9016 Ser Gly Phe Ser Thr Leu Ser Gln Ala Glu Asn Leu Met Gln Val 3091 TAT CAG CAA GCA CGC CTG AGC AAC CCG GAA TTG CGT AAA TCC GCT 13531 Tyr Gln Gln Ala Arg Leu Ser Asn Pro Glu Leu Arg Lys Ser Ala 45136 GCC GAT CGC GAT GCT GCA TTC GAA AAA ATT AAC GAA GCG CGT AGT 18046 Ala Asp Arg Asp Ala Ala Phe Glu Lys Ile Asn Glu Ala Arg Ser 60181 CCT TTA CTG CCG CAA CTG GGT TTA GGT GCC GAC TAC ACC TAC AGC 22561 Pro Leu Leu Pro Gln Leu Gly Leu Gly Ala Asp Tyr Thr Tyr Ser 75226 AAC GGT TAT CGC GAT GCG AAC GGT ATC AAC TCC AAT GAA ACC AGC 27076 Asn Gly Tyr Arg Asp Ala Asn Gly Ile Asn Ser Asn Glu Thr Ser 90271 GCT TCT CTG CAA TTA ACG CAG ACG CTA TTT GAT ATG TCG AAA TGG 31591 Ala Ser Leu Gln Leu Thr Gln Thr Leu Phe Asp Met Ser Lys Trp 105316 CGT GGG CTC ACC CTG CAA GAA AAA GCA GCA GGC ATT CAG GAT GTC 360106 Arg Gly Leu Thr Leu Gln Glu Lys Ala Ala Gly Ile Gln Asp Val 120361 ACC TAT CAG ACC GAT CAG CAG ACG CTG ATC CTC AAT ACC GCG AAC 405121 Thr Tyr Gln Thr Asp Gln Gln Thr Leu Ile Leu Asn Thr Ala Asn 135406 GCG TAT TTT AAG GTA TTG AAC GCT ATT GAT GTG CTT TCC TAT ACC 450136 Ala Tyr Phe Lys Val Leu Asn Ala Ile Asp Val Leu Ser Tyr Thr 150451 CAG GCG CAA AAA GAG GCT ATC TAC CGT CAG TTA GAT CAA ACG ACG 495151 Gln Ala Gln Lys Glu Ala Ile Tyr Arg Gln Leu Asp Gln Thr Thr 165496 CAA CGT TTT AAC GTG GGT CTG GTC GCC ATT ACC GAC GTG CAA AAC 540166 Gln Arg Phe Asn Val Gly Leu Val Ala Ile Thr Asp Val Gln Asn 180541 GCC CGT GCG CAA TAT GAT ACC GTA CTG GCG AAT GAA GTG ACC GCC 585181 Ala Arg Ala Gln Tyr Asp Thr Val Leu Ala Asn Glu Val Thr Ala 195586 CGC AAC AAC CTG GAT AAC GCG GTA GAA GAG CTG CGC CAG GTA ACC 630196 Arg Asn Asn Leu Asp Asn Ala Val Glu Glu Leu Arg Gln Val Thr 210631 GGC AAT TAT TAC CCG GAG CTG GCG TCG CTT AAC GTC GAG CAT TTT 675211 Gly Asn Tyr Tyr Pro Glu Leu Ala Ser Leu Asn Val Glu His Phe 225676 AAA ACC GAC AAA CCC AAA GCT GTT AAT GCG CTG TTG AAG GAA GCG 720226 Lys Thr Asp Lys Pro Lys Ala Val Asn Ala Leu Leu Lys Glu Ala 240721 GAA AAC CGT AAC CTG TCG CTG TTG CAG GCG CGT TTA AGT CAG GAT 765241 Glu Asn Arg Asn Leu Ser Leu Leu Gln Ala Arg Leu Ser Gln Asp 255766 CTG GCG CGC GAG CAA ATC CGT CAG GCG CAG GAT GGT CAC CTG CCG 810256 Leu Ala Arg Glu Gln Ile Arg Gln Ala Gln Asp Gly His Leu Pro 270811 ACG CTG AAT TTA ACG GCC TCA ACC GGC ATT TCT GAT ACC TCT TAT 855271 Thr Leu Asn Leu Thr Ala Ser Thr Gly Ile Ser Asp Thr Ser Tyr 285856 AGC GGT TCT AAA ACC AAC TCC ACC CAG TAC GAC GAT AGC AAC ATG 900286 Ser Gly Ser Lys Thr Asn Ser Thr Gln Tyr Asp Asp Ser Asn Met 300901 GGG CAG AAT AAA ATC GGC CTT AAC TTC TCC CTG CCG CTG TAT CAA 945301 Gly Gln Asn Lys Ile Gly Leu Asn Phe Ser Leu Pro Leu Tyr Gln 315946 GGT GGG ATG GTT AAC TCG CAG GTA AAA CAG GCG CAG TAT AAC TTC 990316 Gly Gly Met Val Asn Ser Gln Val Lys Gln Ala Gln Tyr Asn Phe 330991 GTC GGC GCA AGC GAA CAG CTG GAA AGC GCG CAC CGT AGC GTG GTG 1035331 Val Gly Ala Ser Glu Gln Leu Glu Ser Ala His Arg Ser Val Val 3451036 CAG ACC GTA CGT TCT TCC TTT AAC AAT ATT AAC GCC TCC ATC AGC 1080346 Gln Thr Val Arg Ser Ser Phe Asn Asn Ile Asn Ala Ser Ile Ser 3601081 AGC ATC AAC GCG TAT AAA CAG GCG GTC GTT TCC GCG CAA AGT TCT 1125361 Ser Ile Asn Ala Tyr Lys Gln Ala Val Val Ser Ala Gln Ser Ser 3751126 TTG GAT GCC ATG GAA GCC GGT TAC TCG GTC GGT ACA CGT ACC ATT 1170376 Leu Asp Ala Met Glu Ala Gly Tyr Ser Val Gly Thr Arg Thr Ile 3901171 GTT GAC GTA CTG GAT GCC ACC ACC ACT CTG TAT GAT GCC AAG CAG 1215391 Val Asp Val Leu Asp Ala Thr Thr Thr Leu Tyr Asp Ala Lys Gln 4051216 CAA CTG GCC AAC GCG CGT TAT ACC TAT TTG ATT AAT CAG TTA AAT 1260406 Gln Leu Ala Asn Ala Arg Tyr Thr Tyr Leu Ile Asn Gln Leu Asn 4201261 ATC AAA TAT GCG CTC GGT ACG CTG AAC GAG CAG GAT CTG CTC GCG 1305421 Ile Lys Tyr Ala Leu Gly Thr Leu Asn Glu Gln Asp Leu Leu Ala 4351306 CTT AAC AGT ACG TTG GGT AAA CCT ATC CCG ACG TCG CCG GAA AGC 1350436 Leu Asn Ser Thr Leu Gly Lys Pro Ile Pro Thr Ser Pro Glu Ser 4501351 GTA GCG CCG GAA ACG CCA GAT CAG GAT GCT GCC GCA GAC GGT TAT 1395451 Val Ala Pro Glu Thr Pro Asp Gln Asp Ala Ala Ala Asp Gly Tyr 4651396 AAT GCT CAT AGC GCC GCG CCA GCA GTA CAG CCG ACC GCC GCT CGC 1440466 Asn Ala His Ser Ala Ala Pro Ala Val Gln Pro Thr Ala Ala Arg 4801441 GCC AAC AGC AAT AAC GGC AAT CCA TTC CGG CAT TGA 1476481 Ala Asn Ser Asn Asn Gly Asn Pro Phe Arg His End 491
本发明已经特别构思了每个及所有可能的多核苷酸变异，这些变异可通过选择基于表1和表2所示可能的密码子选择的组合而形成，并且所有这些变异将被认为被具体公开。优先选择适于宿主细胞的密码子，将在这些宿主细胞中生产此蛋白。选择可在异源宿主中最大限度地表达蛋白的密码子是一种已知的技术。表2遗传密码
圆括号中描述的是氨基酸和它们的三字母缩写和单字母缩写。密码子是3字母三联体，每个代表一个DNA的三核苷酸，它在左边有一个5’端，在右边有一个3’端。RNA密码也是同样的，除了U(尿嘧啶)取代了T。氨基酸 密码子丙氨酸(Ala,A) GCC,GCG,GCT精氨酸(Arg,R) AGG,CGA,CGC,CGG,CGT天冬酰胺(Asn,N) AAC,AAT天冬氨酸(Asp,D) GAC,GAT半胱氨酸(Cys,C) TGC,TGT谷氨酰胺(Gln,Q) CAA,CAG谷氨酸(Glu,E) GAA,GAG甘氨酸(Gly,G) GGA,GGC,GGG,GGT组氨酸(His,H) CAC,CAT异亮氨酸(Ile,I) ATA,ATC,ATT亮氨酸(Leu,L) CTA,CTC,CTG,CTT,TTA,TTG赖氨酸(Lys,K) AAA,AAG蛋氨酸(Met,M) ATG苯丙氨酸(Phe,F) TTC,TTT脯氨酸(Pro,P) CCA,CCC,CCG,CCT丝氨酸(Ser,S) AGC,AGT,TCA,TCC,TCG,TCT苏氨酸(Thr,T) ACA,ACC,ACG,ACT色氨酸(Trp,W) TGG酪氨酸(Tyr,Y) TAC,TAT缬氨酸(Val,V) GTA,GTC,GTG,GTT终止信号 TAA,TAG,TGA
由表2中列举的密码可容易地确定编码这些肽的其它DNA分子，并且也构思了表1的DNA序列的等同物。实际上因为在DNA密码和肽中的氨基酸之间存在着一种固定的关系，所以本申请中肽的替换或其它改变的任何讨论同样可应用于相应的DNA序列，或者可应用于该序列所在的DNA分子、重组载体、或转化微生物(并且反过来也是这样)。
另外，本发明表1中的特异核苷酸、DNA分子(或相应的RNA)可在那些特别列举的核苷酸之前或之后带有附加核苷酸。例如，一个短序列(如，短于20个核苷酸)可加至3’端上而提供一种末端序列，此序列与限制性内切核酸酶位点相对应；终止密码子可接在此肽序列后以终止翻译；如此等等。另外，还可生产含有此基因上游的一个启动子区或其它控制区的DNA分子。含有本发明序列的所有DNA分子至少可有一种用途，因为至少可将它们切成片段以产生寡核苷酸探针，并用于生物来源的DNA的分离或检测。
此说明书中的许多词除具有其常用含义外，还具有特定的含义。当涉及两个核苷酸序列时，“等同”指这两个核苷酸序列编码的氨基酸序列相同。当“等同”涉及两个肽时，它指这两个肽具有相同的性质(诸如抗原性，如上下文确定)。两种肽的性质不需要完全相同(例如：两种肽可表现出不同的抗原反应性)，但优选它们的性质基本上完全相同。当涉及两个核苷酸序列时，“互补”指这两个序列在相对的核苷酸之间可进行杂交，错配优选小于25％、更优选小于15％、甚至优选小于5％，最优选没有错配。在实施例中描述优选的杂交条件(它并不限制于特定的错配数)。名词“基本上”的含义随上下文而变化，它可被本领域的技术人员理解为不同的含义，但通常指至少70％，优选指至少80％，更优选指至少90％，最优选指至少95％。在此所用的名词“分离的”分别指从其它多种肽、DNA或RNA中分离的肽、DNA或RNA，并且可见在其生化溶液中正常存在的(如果有的话)溶剂、缓冲剂、离子或其它组分。“分离的”既不包含为天然状态的天然物质，也不包含已经分离为组分但既不为纯物质获得也不是作为溶液获得的天然物质(例如在丙烯酰胺凝胶中)。
因为已经鉴定了此基因的DNA序列，所以完全通过合成化学来生产DNA基因是可能的，然后可通过已知的重组DNA技术，将此基因插入任一可应用的DNA载体中。因此可以应用在本专利提交时属于公共领域的不需要遗传材料保藏的可自由利用的试剂、质粒和微生物完成本发明。
例如，在一种单一反应中应用40个破基的特异寡核苷酸可合成完整的1476bp的ST50基因，这种寡核苷酸横跨基因的两条链。可通过“装配PCR技术”进行这项工作，例如Stemmer等在《基因》164:49-53中对这种技术进行了详细地描述。
然后按照本文所述在一种宿主生物体中表达这些肽。而且还可利用自动设备完成这里公开的可方便应用的许多肽的合成，尤其是伤寒沙门氏菌的完整50kDa OMP的肽片段。不论是通过直接合成，还是通过一系列片段的合成，这种设备可使本发明的肽容易获得。在上面的一系列片段的合成中，所述片段可通过其它已知技术被偶联。
除了表1中所示的特定的多肽序列，基于这种序列和片段的肽片段和具有最小变异的全长序列将具有特异的伤寒沙门氏菌50kDa OMP的某些生物活性，并且因此可用于疫苗和诊断试剂的开发或其它研究。例如50kDa OMP序列的片段可容易地制备，可筛选用作诊断试剂或DNA疫苗开发的表位。可应用肽合成仪器制备小的多肽片段(例如小于100氨基酸)，或者可应用基因工程技术制备较大的片段。应用抗-50kDa OMP抗体通过抗体亲和层析法可衍生出免疫原性肽/表位。这种肽也可用作制备抗体的免疫原，或者成为检测的标准，这种检测是应用伤寒沙门氏菌50kDa OMP的抗体来判断伤寒感染存在与否的一种方法。
从较大的蛋白中制备和选择具有适宜免疫学反应性的肽片段的技术是一种公知的技术，它在许多出版物包括专利中都有详细的描述。例如，美国专利第4,629,783号描述了病毒蛋白的免疫学活性片段的制备，所述片段可象整个病毒蛋白一样与相同的抗体结合。
另外正如本领域技术人员所认为的那样，先前提到的肽和DNA分子的微小差异被视为与更详细说明的那些肽和DNA分子是等同的。例如，用异亮氨酸或缬氨酸替换亮氨酸、用谷氨酸替换天冬氨酸、用丝氨酸替换苏氨酸的孤立替换，或用结构相关的氨基酸替换一种氨基酸的相似的替换(即保守性替换)对所得分子的生物活性没有较大的影响，认为这种情况是合理的，尤其是如果这种替换未涉及结合位点或尤其是这样生物活性位点的氨基酸时。由于在种之间已知存在有明显的变异，所以ST50基因尤其是这样。而且在两个末端都可以有附加的氨基酸，或者在一个或两个末端都可无氨基酸，这在本领域是公知的。用同样的方法可容易地检测已经发生一个以上替换的肽。在任意20个氨基酸相邻组中，优选肽的不同不超过12个，更优选不超过5个氨基酸。当发生氨基酸的替换时，优选是在标准保守组之内。氨基酸标准保守组用单字母氨基酸码表示在圆括号中(表2)：非极性(A、V、L、I、P、M)；芳香族(F、T、W)；不带电极性(G、S、T、C、N、Q)；酸性(D、E)；碱性(K、R、H)。有时芳香族氨基酸被认为属于广义非极性(F、W)或不带电极性(T)组。但是这种修饰的蛋白在与伤寒沙门氏菌鉴定相关的特异性诊断检测中较少应用。
当这种肽存在于各种pH值的水性溶液中(或从其中分离出)时，将会自然产生在此所述的任何肽的盐。我们认为具有所说的生物活性的所有肽的盐是在本发明的范围内。实例包括羧酸基的碱金属、碱土金属和其它金属盐，氨基残基的酸加成盐(例如HCl)，以及在同一分子内由羧酸基和氨基之间反应而形成的两性离子。
既可通过直接合成，也可通过应用在此所述的一种克隆的基因或其片段，将本发明的肽首次制备为不含其它沙门氏菌物质的均匀制剂。先前可以获得一种粗匀浆化物形式的伤寒沙门氏菌50kDa OMP，其纯度小于0.1％。这种粗制剂并不是不含所有其它的伤寒沙门氏菌物质。尽管可通过上面所述的全部合成技术制备基因及其相应的蛋白，但在本发明的优选实施例中，是从天然来源获得遗传信息并按照在此所述的方法进行鉴定。应用各种大量的现有技术中任一种可以基因文库的形式首先获得遗传物质。首先随机剪切基因组DNA，并将这种剪切的物质插入表达载体中。如果产生足够多的重组子，很有可能在该群体中的至少一个重组子可表达50kDa OMP蛋白。
下面将详细论述，包括获得这个基因完整序列的实验细节。但是没有理由相信本发明人所制备的序列和特定基因工程化生物体将优于按照本说明书所述的方法制备的其它克隆。事实上，选择其它表达系统，将可能改善本文所述的50kDa OMP蛋白的表达。
因为已经检测了ST50基因的序列，所以不再需要进行这些步骤来获得本发明的遗传材料。现在可应用聚合酶链反应(PCR)从天然来源中分离基因，这种方法更简单更直接。这种PCR技术，包括它在诊断中的应用，公开在美国专利4,683,202中，在此将它作为参考。因为伤寒沙门氏菌标本容易从各种来源获得，诸如Rockville的美国典型培育物保藏中心，Md.，并且因为应用本说明书中的序列可制备PCR探针，所以应用PCR技术和市售的伤寒沙门氏菌基因组物质可获得在此所述序列的任何所需要的区段。下面将描述这种分离伤寒沙门氏菌染色体基因的技术的具体实例。
尽管上面已经描述了这些技术，当遗传工程领域的技术人员将其知识与前面所述的准则结合运用时，将能容易地分离所需要的基因，并将其应用于重组DNA载体中，这是因为有充足的信息可用于此基因的定位，但是达到同样目的的其它方法也是已知的，而且也可用于制备本发明的重组DNA载体。
通过在一个转化宿主中包含该基因的多重拷贝；选择一个已知在该宿主中可复制的载体，籍此由外源性插入DNA产生大量的所述蛋白；或者通过其它已知的增强肽表达的任何方法均可增强50kDa OMP的表达。一个常见的变化是将本发明的多肽制备为一种融合多肽的形式。这种肽的制备一般是通过应用带有已知待表达的基因的启动子区的质粒载体，将编码本发明氨基酸序列的整个或大部分的核苷酸插入此遗传序列中。这种融合蛋白的实例包括组氨酸标记、β-半乳糖苷酶、麦芽糖结合蛋白和绿荧光蛋白。如果需要，可设计此融合肽，以便蛋白水解酶(例如肠激酶)识别的位点存在于这两个融合蛋白的结合处。然后可应用此蛋白水解酶切割此表达的蛋白，以便能获得所需要的纯形式的伤寒沙门氏菌特异的50kDa OMP。
在所有情况中，当DNA序列功能性插入该载体时，将表达伤寒沙门氏菌50kDa OMP。“功能性插入”是指具有适当的阅读框架和方向，本领域的专业技术人员对此非常清楚。一般把基因插入启动子的下游，随后连接一个终止密码子，尽管如果需要，可应用一种杂合蛋白(可能随后经裂解)的产物。
除了上面所述的按照本发明可用于制备重组DNA分子，并在单细胞生物体中转化的常用方法外，其它的已知技术及其改良的方法也可用于进行本发明。特别是最近与遗传工程相关的技术得到了迅猛的发展。许多最近的美国专利公开了质粒、基因工程化微生物和进行基因工程的方法，这些均可应用于本发明中。例如，美国专利4,273,875公开了一种质粒和分离它的方法。美国专利4,304,863公开了通过基因工程生产细菌的一种方法，在此方法中构建一种杂合质粒，并且用它转化细菌宿主。美国专利4,419,450公开了一种质粒，它可充当一种用于重组DNA过程的克隆载体。美国专利4,362,867公开了重组eDNA的构建方法和由此生产的可用于克隆方法的杂合核苷酸。美国专利4,403,036公开了可生成含有多个DNA区段拷贝的质粒的遗传试剂。美国专利4,363,877公开了重组DNA转移载体。美国专利4,356,270公开了一种重组DNA克隆载体，并且这个公开的专利对那些在基因工程领域缺乏经验的人员是非常有用的，因为它定义了许多在基因工程中所用的名词和在其中所用的基本方法。美国专利4,336,336公开了一种融合基因和制备它的方法。美国专利4,349,629公开了质粒载体和它的生产方法和应用。美国专利4,332,901公开了一种可用于重组DNA的克隆载体。尽管这些专利中的某些所涉及的特殊基因产物并不在本发明的范围内，但基因工程领域的技术人员可将其中所描述的方法进行修改，而应用到本发明中。
本发明的影响是非常大的，这是因为本发明大量有用的伤寒沙门氏菌特异50kDa OMP和遗传物质可应用于杂交分析的开发，或者可用于其它类型的分析，在这些分析中可将这些物质用作一种在诊断、免疫、治疗和研究中所用的试剂。将ST50基因或其已经分离的一部分转移至其它的表达载体，这将产生一些构建体，所述构建体将改善伤寒沙门氏菌特异50kDa OMP在大肠杆菌中的表达，或者在其它宿主中表达多肽。
特别构思了的是，通过应用基于在此公开的基本核苷酸序列和变异核苷酸序列的寡核苷酸探针，从伤寒沙门氏菌的其它菌株中分离基因。这种探针可以明显短于整个序列，但其长度应当至少是10个核苷酰，优选至少是14个核苷酸。特别是长度为20至500，尤其是30至200的中等寡核苷酸可提供特异且快速反应的探针。较长的寡核苷酸也有用，直至该基因的全长。不论是RNA还是DNA探针都可使用。
在应用中，一般以一种可检测的方法(例如P32、S35、生物素、抗亲和素蛋白、荧光素或洋地黄毒甙)标记这种探针，并且与来自在其中筛选基因的生物体的单链DNA或RNA温育。通过可使已经分离的双链(杂交的)DNA(或DNA/RNA)变性(典型地应用硝化纤维素纸)的标记测定杂交。适于用寡核苷酸的杂交技术是公知的。
尽管正常情况下所应用的探针带有便于鉴别的可检测的标记，但也可应用非标记的寡核苷酸，它既可作为标记探针的前体，也可在直接检测双链DNA(或DNA/RNA)的方法中应用。因此名词“寡核苷酸探针”既可为标记形式，也可为非标记的形式。
应用已知的技术可生产重组ST50蛋白的单克隆或多克隆抗体。抗ST50重组蛋白的抗体可用于开发一种抗原检测免疫法，以检测伤寒沙门氏菌，或者与其它的技术一起应用。在这些技术中，抗体用作一种组分。另外，来源于ST50基因的部分DNA或者肽或蛋白可用作疫苗制剂中的一种免疫原，以预防伤寒沙门氏菌的感染。可将这种免疫原掺入脂质体，或者与在疫苗制剂中所用的多糖和/或其它聚合物偶联。此ST50基因的DNA序列也可用于开发一种DNA疫苗制剂。本领域的技术人员可应用许多方法将这种疫苗制剂引入人体。这些方法包括，但不限于，真皮内给药、肌内给药、腹膜内给药、静脉内给药、皮下给药、口服和鼻内给药。
总之，已经在大肠杆菌中分离、克隆、测序和表达了伤寒沙门氏菌特异ST50基因。此基因的开放阅读框支配了与伤寒沙门氏菌50kDa OMP有可鉴别的相似性的50,000道尔顿的蛋白合成。整个ST50基因的序列为1476个碱基对(bp)，GC与AT的比例分别是52.03％和47.97％。开放阅读框(ORF)开始于7bp核苷酸位置，其起始密码子为“ATG”，并且其终止密码子为“TGA”，位于1474bp核苷酸位置。预测的ST50基因的氨基酸由491个氨基酸组成。50kDa OMP的信号序列是一个16氨基酸肽，位于第3和第18氨基酸之间。已经大量地在大肠杆菌中表达了50kDa OMP，它对未来的诊断和疫苗研究提供了大量的纯蛋白。
通过IMAC色谱法已经将所表达的50kDa OMP纯化为均质。通过在IgM和IgG水平的Western印迹法和斑点免疫测定法已经评价了重组ST50蛋白的特异免疫反应性。利用可应用的DNA序列的信息，将此序列或部分序列用作诊断伤寒的DNA探针或PCR引物不再困难。现在已经对本发明进行了一般的描述，通过参照下面的实施例，将能更好地理解本发明。下面的实施例仅仅是为了举例说明，并不是要对本发明进行限制，除非进行特殊地说明。
实施例细菌菌株和培养基
从患有伤寒的病人分离出伤寒沙门氏菌(USM1)菌株，并从1987年起一直在我们实验室保存。使用血液琼脂和营养肉汁繁殖伤寒沙门氏菌菌株。在Luria肉汁上培养伤寒沙门氏菌用于分子生物学实验。外膜蛋白的分离
按照Schnaitman等人(1971),108:553-556中所述得到伤寒沙门氏菌部分纯化的OMP。简言之，伤寒沙门氏菌在营养肉汁中生长并在37℃下在摇床上培育18小时直至晚对数期(OD=0.8,660nm)。收集细胞并悬浮于0.01MHEPES缓冲液(pH 7.4)中。通过用玻璃珠(0.15mm直径)旋转1.5小时在冰上间隔1分钟直至95％破裂，使细菌细胞破裂，用连续革兰氏染色监测。吸出细胞裂解物，并用0.01M HEPES缓冲液冲洗玻璃珠。通过在4℃下以5,000g离心15分钟去掉细胞碎片和未破裂的细胞。以200,000g离心上清液1小时得到细胞包膜。用含有2％Triton X-100的0.01M HEPES去掉胞质膜，并在室温下放置10分钟。然后，通过在4℃下以200,000g离心1小时，将不溶性的外膜沉淀。用Bio-Rad蛋白测定染料试剂(Bio-Rad,Richmond,CA)通过比色微量测定确定OMP蛋白浓度，用牛血清白蛋白作为标准物。纯化伤寒沙门菌的50kDa OMP
用聚丙烯酰胺十二烷基硫酸钠凝胶电泳(SDS-PAGE)和电洗脱分离伤寒沙门氏菌的50kDa OMP。在还原条件下，使用不连续的缓冲系统(Lammli,1970)用垂直板电泳装置(Bio-Rad)完成SDS-PAGE。堆积胶和分离胶分别含有4.5％和9％丙烯酰胺。每个预制备凝胶上加样500μg OMP，并在4℃下以每板250mA的恒定电流跑板4小时。用考马斯蓝给分离的OMP染色，并用14.4-94kDa分子量标记物确定分子量。
为了N-末端氨基酸测序，将OMP在SDS-PAGE上电泳并电杂交到聚偏氟乙烯(PVDF)膜上。用考马斯蓝染色，仔细地将目的50kDa带移出，并送去氨基酸测序(MidWest Analytical，美国)。克隆ST50基因(ⅰ)引物设计
分析从ST50 OMP的N末端氨基酸序列中获得的氨基酸序列，根据分析，设计一对PCR引物STPCS1-ATG CAA ATG AAG AAA TTG CTC和STR1-TCA ATG CCGGAA TGG ATT GC用于扩增ST50基因的PCR。(ⅱ)聚合酶链反应(PCR)
用标准方法提取伤寒沙门氏菌(USM1)基因组DNA，并将50ng DNA用作用STPS1和STR1进行PCR扩增的模板。在下列条件下完成PCR扩增：200μM的每种dNTP、1×PCR缓冲液(50mM KCl、10mM Tris.Cl,pH 8.3)、2.5mM MgCl2、20pmol STPCS1和STR1引物和一个单位的TAQ DNA聚合酶。PCR在Perkin-Elmer 9600热循环仪上进行。用于引物对的最适宜退火温度是50℃。所用PCR参数如下：在95℃下进行初始变性5分钟，然后附加1分钟。分别在55℃和72℃下进行1分钟引物退火和延伸。这些步骤重复30次。最后包括一个72℃温度最终延伸5分钟。用琼脂糖凝胶电泳分析PCR产物，PCR产物的大小为1476bp。因为当大肠杆菌基因组DNA用作模板时引物没有扩增任何产物，因此，PCR产物具有对伤寒沙门氏菌的特异性。(ⅲ)ST50基因的克隆
如每一个生产者所教导，将ST50基因的扩增1476 bp PCR产物克隆于PCR克隆载体(TOPO 2.1,Invitrogen)。用内部引物STPR1:GGC CGT TAA ATT CAGCGT CG和M13反向引物：CAG GAA ACA GCT ATG AC筛选克隆。ST50克隆pST50-USM1的图谱在附图-1中描述。ST50基因的序列
通过商业DNA测序公司(ACGT Inc，美国)，用pST50-USM1和M13-20正向CTG GCC GTC GTT TTA C和M13反向引物CAG GAA ACA GCT ATG AC进行ST50基因的DNA测序。整个ST50基因的序列为具有52.03％和47.97％GC和AT比例的1476碱基对(bp)。开放阅读框架(ORF)开始于具有一个起始密码子“ATG”的7bp核苷酸位置，其终止密码子“TGA”位于1474bp核苷酸位置。ST基因编码491氨基酸，蛋白的预期大小为53682道尔顿。ST50蛋白的信号序列为在第3和第18氨基酸位置延伸的16氨基酸肽。DNA序列和预期氨基酸序列在表1中给出。信号序列的存在表明蛋白位于周质区。ST50基因在表达载体的克隆
为了表达ST50基因，我们选择基于T7启动子的表达载体系统，pRSETB载体(Invitrogen)。pRSETB载体的优点是，它含有对T7 RNA聚合酶具有很高特异性的T7启动子。通过T7聚合酶的转录具有选择性，与大肠杆菌RNA聚合酶相比快5倍，并由此带来在T7启动子下克隆的基因的更高表达。载体还含有一个编码金属结合结构域的核苷酸序列，金属结合结构域是表达为与目的蛋白的N-末端融合的连续6个组氨酸。融合肽上的金属结合结构域(6个标记的组氨酸组成成分)对二价离子(像镍、铜和钴)具有高的亲和力，并且能够促进用(IMAC)固定金属亲和层析进行的蛋白的一步纯化。
用EcoR1限制酶将ST50基因从TOPO载体中切出。将切出的ST50kDa基因连接到pRSETB载体的EcoR1位点。用内部引物STPR1:GGC CGT TAA ATT CAGCGT CG和T7终止引物：GCT AGT TAT TGC TCA GCG G筛选克隆。ST50基因在pRSETB载体(pST50-USM2)的图谱在附图2中描述。通过用EcoR1 限制酶进行的酶切分析证实了ST50基因在pRSETB载体上的存在。蛋白表达
在BL21(DE3)大肠杆菌宿主中进行来自pST50-USM2克隆的ST50蛋白的蛋白表达。大肠杆菌BL21(DE3)含有在lac UV5启动子控制下的T7 RNA聚合酶基因的染色体拷贝，因此，在T7启动子下克隆的基因表达可以用诸如IPTG的义务诱导物诱导。而且，BL21(DE3)作为一种离子蛋白酶缺乏菌株，它可以预防表达的蛋白被蛋白酶酶解裂解。在用IPTG诱导之后，ST50蛋白在大肠杆菌BL21(DE3)中表达。简言之，为重组蛋白的表达，进行如下步骤：(为下列实验，用100μg/ml氨苄青霉素补加Luria肉汁)
(a)用(Sambrook等人1989)分子克隆-实验室手册(第二版)’，ColdSpring Harbor实验室，Cold Spring Harbor，纽约中提到的方法，用pST50-USM2克隆进行大肠杆菌BL21(DE3)的转化。
(b)将新鲜转化体的单个菌落接种入1.5ml LB，并在37℃、静止状态生长过夜(o/n)。
(c)将50ul过夜的培养物接种入含有10ml LB的100ml圆底烧瓶中，并以150rpm摇动，在37℃下生长，直至培养物的OD600达到0.6。
(d)加入IPTG至最终浓度为1mM，并以150rpm摇动，在37℃下生长3小时。
(e)以10,000g将培养物离心5分钟。去掉上清液，并将含有重组蛋白的大肠杆菌沉淀在-20℃下贮藏。
用SDS-PAGE分析重组的ST50蛋白。重组的ST50蛋白的大小为59kDa，由于来源于pRSETB载体的另外的52个氨基酸的存在，其分子量大于天然50kDa OMP。重组ST50蛋白的纯化
通过固定化金属亲和层析(IMAC)完成重组ST50蛋白的纯化。IMAC是一种特殊形式的亲和层析，其中一种固定金属离子例如铜、锌或一种过渡金属离子例如钴或镍用于通过与组氨酸残基的咪唑基团反应选择性地结合蛋白(Potath等人，1975)。通过降低pH值，由此使蛋白-金属复合物不稳定，或通过使用竞争性配体像咪唑，或退火使用络合剂像EDTA完成蛋白的洗脱。
在克隆pST50-USM2中，重组ST50蛋白以包含体的形式表达。因此，重组ST50蛋白用8M尿素增溶，并在变性条件下纯化蛋白。
使用下列层析方法，以纯化重组ST50蛋白：
从(Qiagen)购得NiNTA基质，并用于重组ST50蛋白的纯化。依生产者说明书所述制备柱，开始时用起始柱缓冲液(0.1M磷酸盐缓冲液pH8.0、含有8M尿素的0.01M Tris pH8.0)平衡基质。在4℃下，将重组ST50蛋白用裂解缓冲液(0.1M磷酸盐缓冲液pH8.0、0.01M Tris pH8.0和8M尿素)溶解8小时，并以12K旋转15分钟，去掉碎片。将含有溶解的重组蛋白的上清液通过柱，并使之结合4小时。通过用5至10柱容积的起始缓冲液(含有8M尿素的0.1M磷酸盐缓冲液pH8.0)冲洗柱，直至级分在A280显示为零。通过含有8M尿素的0.1M磷酸盐缓冲液pH4.5完成蛋白的洗脱。收集洗脱的部分，并通过测定在280nm的吸收将蛋白浓度定量。合并含有蛋白的洗脱部分，并通过分步透析去掉尿素。用洗涤物和洗脱的级分进行SDS-PAGE分析以核查洗脱蛋白的纯度。重组ST50蛋白的免疫反应性(ⅰ)蛋白印迹
通过蛋白印迹分析重组ST50蛋白的免疫反应性。简言之，将重组ST50蛋白在8％SDS-PAGE上电泳。电泳后，将凝胶在转移缓冲液(25mM Tris,192mM甘氨酸，20％甲醇)中培育10分钟。将切至分离凝胶准确大小的硝化纤维素膜(NCP)在转移缓冲液中培育10分钟。在不产生气泡的情况下，将NCP覆盖在凝胶上，并夹在滤纸和scotch brite垫之间。用Transblot(Bio Rad,USA)电印迹仪，电泳转移在冷室中以200mA进行3小时。转移后，切下分子量标记泳道，并用酰胺黑染色(含有100mg酰胺黑的45％甲醇，10％乙酸)。剩余的NCP用Ponceau S(含有0.2％Ponceau S,Sigma,USA的0.3％三氯乙酸和0.3％磺基水杨酸)染色，以保证蛋白的转移。用PBS冲洗膜，并在4℃下，用含有5％脱脂奶粉的PBS封阻过夜。
NCP用冲洗缓冲液(PBS)冲洗三次，每次5分钟，然后，用合并的正常血清、合并的伤寒血清、以及取自如下范围中常见的发热例如登革热、肝炎、丛林斑疹伤寒、副伤寒甲、乙和丙的病人血清的1∶200的稀释液培育过夜。合并的正常血清从5名健康个体中获得，合并的伤寒血清从血样培养阳性的伤寒病人中获得。用冲洗缓冲液冲洗后，膜用过氧化酶偶联的抗人IgM和IgG培育2小时。广泛冲洗后，用H2O2和4-氯-1-萘酚试剂将印迹展开15分钟，并将印迹用蒸馏水漂洗以终止反应。将重组ST50蛋白特别与沙门氏伤寒病人的血清在IgG和IgM抗体水平反应，但不与从其他感染中获得的血清反应。(ⅱ)斑点酶免疫测定
将0.45μ孔大小的硝化纤维膜(微滤系统，CA,USA)用于本测定。用微量注射器将1μl重组ST50蛋白即蛋白打点于硝化纤维素上并使其干燥。将小条浸入封阻缓冲液(3％脱脂牛奶，0.9％NaCl,10mM Tris·HCl,pH7.4)中,并在室温下放于摇动式平台上30分钟。用NETG缓冲液(150mM NaCl、50mMTris-HCl、50mM EDTA和0.25％明胶)将封阻条漂洗3次15分钟，使之干燥并在4℃下保存直至使用。然后，用1ml不同血清样本的1∶100稀释液探查小条，并在室温下在摇动式平台上培育1小时。用1M NETG缓冲液冲洗小条3次15分钟，并进一步用过氧化酶偶联的抗人IgG(Dakopatts,Glostrup，丹麦)的1∶1,600稀释液或抗人IgM(Dakopatts,Glostrup，丹麦)的1∶800稀释液在摇动式平台上培育。充分冲洗后，用H2O2和4-氯-1-萘酚试剂将印迹展开15分钟，并将印迹用蒸馏水漂洗以终止反应。重组ST50蛋白显示出对沙门氏伤寒病人的血清在IgG和IgM抗体水平的特异反应性。但是没有显示出与从其他感染中获得的血清的反应性。
本发明分离出的DNA或RNA分子(ST50)可以作为针对例如动物(例如哺乳动物、例如人)伤寒的疾病的免疫保护源，特别是基于能够诱导强免疫反应的疫苗。适宜的剂量和给药条件是本领域中已知的。
说明书中提及的所有出版物和专利申请对本发明涉及领域的普通技术人员是具有指导性的。如同每单个出版物或专利申请被特别地和独立地说明收编于此作为参考，所有出版物和专利申请均作为同等程度的参考收编于此。
由于本发明的充分说明，在不背离所附的权利要求的精神或范围内对本发明所作的许多变化和修改，对本领域的普通技术人员来说，将是显而易见的。
Claims (15)
1．一种分离的DNA或RNA分子(ST50)，它含有编码伤寒沙门氏菌特异性外膜蛋白OMP的核苷酸序列，估计其分子量为50kDa。
2．如权利要求1所述的分子，其中所述分子包含下列伤寒沙门氏菌特异性OMP蛋白编码序列：1 ATG CAA ATG AAG AAA TTG CTC CCC ATC CTT ATC GGC CTG AGC CTG 451 Met Gln Met Lys Lys Leu Leu Pro Ile Leu Ile Gly Leu Ser Leu 1546 TCG GGG TTC AGC ACA CTA AGC CAG GCA GAG AAC CTG ATG CAA GTT 9016 Ser Gly Phe Ser Thr Leu Ser Gln Ala Glu Asn Leu Met Gln Val 3091 TAT CAG CAA GCA CGC CTG AGC AAC CCG GAA TTG CGT AAA TCC GCT 13531 Tyr Gln Gln Ala Arg Leu Ser Asn Pro Glu Leu Arg Lys Ser Ala 45136 GCC GAT CGC GAT GCT GCA TTC GAA AAA ATT AAC GAA GCG CGT AGT 18046 Ala Asp Arg Asp Ala Ala Phe Glu Lys Ile Asn Glu Ala Arg Ser 60181 CCT TTA CTG CCG CAA CTG GGT TTA GGT GCC GAC TAC ACC TAC AGC 22561 Pro Leu Leu Pro Gln Leu Gly Leu Gly Ala Asp Tyr Thr Tyr Ser 75226 AAC GGT TAT CGC GAT GCG AAC GGT ATC AAC TCC AAT GAA ACC AGC 27076 Asn Gly Tyr Arg Asp Ala Asn Gly Ile Asn Ser Asn Glu Thr Ser 90271 GCT TCT CTG CAA TTA ACG CAG ACG CTA TTT GAT ATG TCG AAA TGG 31591 Ala Ser Leu Gln Leu Thr Gln Thr Leu Phe Asp Met Ser Lys Trp 105316 CGT GGG CTC ACC CTG CAA GAA AAA GCA GCA GGC ATT CAG GAT GTC 360106 Arg Gly Leu Thr Leu Gln Glu Lys Ala Ala Gly Ile Gln Asp Val 120361 ACC TAT CAG ACC GAT CAG CAG ACG CTG ATC CTC AAT ACC GCG AAC 405121 Thr Tyr Gln Thr Asp Gln Gln Thr Leu Ile Leu Asn Thr Ala Asn 135406 GCG TAT TTT AAG GTA TTG AAC GCT ATT GAT GTG CTT TCC TAT ACC 450136 Ala Tyr Phe Lys Val Leu Asn Ala Ile Asp Val Leu Ser Tyr Thr 150451 CAG GCG CAA AAA GAG GCT ATC TAC CGT CAG TTA GAT CAA ACG ACG 495151 Gln Ala Gln Lys Glu Ala Ile Tyr Arg Gln Leu Asp Gln Thr Thr 165496 CAA CGT TTT AAC GTG GGT CTG GTC GCC ATT ACC GAC GTG CAA AAC 540166 Gln Arg Phe Asn Val Gly Leu Val Ala Ile Thr Asp Val Gln Asn 180541 GCC CGT GCG CAA TAT GAT ACC GTA CTG GCG AAT GAA GTG ACC GCC 585181 Ala Arg Ala Gln Tyr Asp Thr Val Leu Ala Asn Glu Val Thr Ala 195586 CGC AAC AAC CTG GAT AAC GCG GTA GAA GAG CTG CGC CAG GTA ACC 630196 Arg Asn Asn Leu Asp Asn Ala Val Glu Glu Leu Arg Gln Val Thr 210631 GGC AAT TAT TAC CCG GAG CTG GCG TCG CTT AAC GTC GAG CAT TTT 675211 Gly Asn Tyr Tyr Pro Glu Leu Ala Ser Leu Asn Val Glu His Phe 225676 AAA ACC GAC AAA CCC AAA GCT GTT AAT GCG CTG TTG AAG GAA GCG 720226 Lys Thr Asp Lys Pro Lys Ala Val ASn Ala Leu Leu Lys Glu Ala 240721 GAA AAC CGT AAC CTG TCG CTG TTG CAG GCG CGT TTA AGT CAG GAT 765241 Glu Asn Arg Asn Leu Ser Leu Leu Gln Ala Arg Leu Ser Gln Asp 255766 CTG GCG CGC GAG CAA ATC CGT CAG GCG CAG GAT GGT CAC CTG CCG 810256 Leu Ala Arg Glu Gln Ile Arg Gln Ala Gln Asp Gly His Leu Pro 270811 ACG CTG AAT TTA ACG GCC TCA ACC GGC ATT TCT GAT ACC TCT TAT 855271 Thr Leu Asn Leu Thr Ala Ser Thr Gly Ile Ser Asp Thr Ser Tyr 285856 AGC GGT TCT AAA ACC AAC TCC ACC CAG TAC GAC GAT AGC AAC ATG 900286 Ser Gly Ser Lys Thr Asn Ser Thr Gln Tyr Asp Asp Ser Asn Met 300901 GGG CAG AAT AAA ATC GGC CTT AAC TTC TCC CTG CCG CTG TAT CAA 945301 Gly Gln Asn Lys Ile Gly Leu Asn Phe Ser Leu Pro Leu Tyr Gln 315946 GGT GGG ATG GTT AAC TCG CAG GTA AAA CAG GCG CAG TAT AAC TTC 990316 Gly Gly Met Val Asn Ser Gln Val Lys Gln Ala Gln Tyr Asn Phe 330991 GTC GGC GCA AGC GAA CAG CTG GAA AGC GCG CAC CGT AGC GTG GTG 1035331 Val Gly Ala Ser Glu Gln Leu Glu Ser Ala His Arg Ser Val Val 3451036 CAG ACC GTA CGT TCT TCC TTT AAC AAT ATT AAC GCC TCC ATC AGC 1080346 Gln Thr Val Arg Ser Ser Phe Asn Asn Ile ASn Ala Ser Ile Ser 3601081 AGC ATC AAC GCG TAT AAA CAG GCG GTC GTT TCC GCG CAA AGT TCT 1125361 Ser Ile Asn Ala Tyr Lys Gln Ala Val Val Ser Ala Gln Ser Ser 3751126 TTG GAT GCC ATG GAA GCC GGT TAC TCG GTC GGT ACA CGT ACC ATT 1170376 Leu Asp Ala Met Glu Ala Gly Tyr Ser Val Gly Thr Arg Thr Ile 3901171 GTT GAC GTA CTG GAT GCC ACC ACC ACT CTG TAT GAT GCC AAG CAG 1215391 Val Asp Val Leu Asp Ala Thr Thr Thr Leu Tyr Asp Ala Lys Gln 4051216 CAA CTG GCC AAC GCG CGT TAT ACC TAT TTG ATT AAT CAG TTA AAT 1260406 Gln Leu Ala Asn Ala Arg Tyr Thr Tyr Leu Ile Asn Gln Leu Asn 4201261 ATC AAA TAT GCG CTC GGT ACG CTG AAC GAG CAG GAT CTG CTC GCG 1305421 Ile Lys Tyr Ala Leu Gly Thr Leu Asn Glu Gln Asp Leu Leu Ala 4351306 CTT AAC AGT ACG TTG GGT AAA CCT ATC CCG ACG TCG CCG GAA AGC 1350436 Leu Asn Ser Thr Leu Gly Lys Pro Ile Pro Thr Ser Pro Glu Ser 4501351 GTA GCG CCG GAA ACG CCA GAT CAG GAT GCT GCC GCA GAC GGT TAT 1395451 Val Ala Pro Glu Thr Pro Asp Gln Asp Ala Ala Ala Asp Gly Tyr 4651396 AAT GCT CAT AGC GCC GCG CCA GCA GTA CAG CCG ACC GCC GCT CGC 1440466 Asn Ala His Ser Ala Ala Pro Ala Val Gln Pro Thr Ala Ala Arg 4801441 GCC AAC AGC AAT AAC GGC AAT CCA TTC CGG CAT TGA 1476481 Ala Asn Ser Asn Asn Gly Asn Pro Phe Arg His End 491；或者一种DNA或一种RNA序列，并且编码与所述编码序列相同的氨基酸序列；或者编码与所述编码序列互补的在相对的核苷酸之间没有错配的一个DNA或RNA序列。
3．如权利要求2所述的分子，其中所述分子为DNA。
4．如权利要求3所述的分子，其中所述分子含有氨基酸序列。
5．如权利要求2所述的分子，其中所述分子为RNA，并且含有与所述ST50基因序列相对应或互补的序列。
6．如权利要求1所述的分子，其中所述序列之前为位于该序列5’侧的一种功能性启动子序列。
7．如权利要求6所述的分子，其中至少有一个所述序列的拷贝存在于功能性重组DNA或RNA载体中。
8．一种基因工程化微生物，其中所述的微生物含有权利要求7所述的载体。
9．如权利要求8的微生物，其中所述的微生物来源为细菌、真菌或病毒。
10．一种基因工程化细胞系，其中所述的细胞系含有权利要求7的载体。
11．如权利要求10所述的细胞系，其中所述的细胞系为动物和昆虫来源。
12．一种分离的大于5个连续核苷酸的寡核苷酸，其选自下列第一DNA序列：1 ATG CAA ATG AAG AAA TTG CTC CCC ATC CTT ATC GGC CTG AGC CTG 451 Met Gln Met Lys Lys Leu Leu Pro Ile Leu Ile Gly Leu Ser Leu 1546 TCG GGG TTC AGC ACA CTA AGC CAG GCA GAG AAC CTG ATG CAA GTT 9016 Ser Gly Phe Ser Thr Leu Ser Gln Ala Glu Asn Leu Met Gln Val 3091 TAT CAG CAA GCA CGC CTG AGC AAC CCG GAA TTG CGT AAA TCC GCT 13531 Tyr Gln Gln Ala Arg Leu Ser Asn Pro Glu Leu Arg Lys Ser Ala 45136 GCC GAT CGC GAT GCT GCA TTC GAA AAA ATT AAC GAA GCG CGT AGT 18046 Ala Asp Arg Asp Ala Ala Phe Glu Lys Ile Asn Glu Ala Arg Ser 60181 CCT TTA CTG CCG CAA CTG GGT TTA GGT GCC GAC TAC ACC TAC AGC 22561 Pro Leu Leu Pro Gln Leu Gly Leu Gly Ala Asp Tyr Thr Tyr Ser 75226 AAC GGT TAT CGC GAT GCG AAC GGT ATC AAC TCC AAT GAA ACC AGC 27076 Asn Gly Tyr Arg Asp Ala Asn Gly Ile Asn Ser Asn Glu Thr Ser 90271 GCT TCT CTG CAA TTA ACG CAG ACG CTA TTT GAT ATG TCG AAA TGG 31591 Ala Ser Leu Gln Leu Thr Gln Thr Leu Phe Asp Met Ser Lys Trp 105316 CGT GGG CTC ACC CTG CAA GAA AAA GCA GCA GGC ATT CAG GAT GTC 360106 Arg Gly Leu Thr Leu Gln Glu Lys Ala Ala Gly Ile Gln Asp Val 120361 ACC TAT CAG ACC GAT CAG CAG ACG CTG ATC CTC AAT ACC GCG AAC 405121 Thr Tyr Gln Thr Asp Gln Gln Thr Leu Ile Leu Asn Thr Ala Asn 135406 GCG TAT TTT AAG GTA TTG AAC GCT ATT GAT GTG CTT TCC TAT ACC 450136 Ala Tyr Phe Lys Val Leu Asn Ala Ile Asp Val Leu Ser Tyr Thr 150451 CAG GCG CAA AAA GAG GCT ATC TAC CGT CAG TTA GAT CAA ACG ACG 495151 Gln Ala Gln Lys Glu Ala Ile Tyr Arg Gln Leu Asp Gln Thr Thr 165496 CAA CGT TTT AAC GTG GGT CTG GTC GCC ATT ACC GAC GTG CAA AAC 540166 Gln Arg Phe Asn Val Gly Leu Val Ala Ile Thr Asp Val Gln Asn 180541 GCC CGT GCG CAA TAT GAT ACC GTA CTG GCG AAT GAA GTG ACC GCC 585181 Ala Arg Ala Gln Tyr Asp Thr Val Leu Ala Asn Glu Val Thr Ala 195586 CGC AAC AAC CTG GAT AAC GCG GTA GAA GAG CTG CGC CAG GTA ACC 630196 Arg Asn Asn Leu Asp Asn Ala Val Glu Glu Leu Arg Gln Val Thr 210631 GGC AAT TAT TAC CCG GAG CTG GCG TCG CTT AAC GTC GAG CAT TTT 675211 Gly Asn Tyr Tyr Pro Glu Leu Ala Ser Leu Asn Val Glu His Phe 225676 AAA ACC GAC AAA CCC AAA GCT GTT AAT GCG CTG TTG AAG GAA GCG 720226 Lys Thr Asp Lys Pro Lys Ala Val Asn Ala Leu Leu Lys Glu Ala 240721 GAA AAC CGT AAC CTG TCG CTG TTG CAG GCG CGT TTA AGT CAG GAT 765241 Glu Asn Arg Asn Leu Ser Leu Leu Gln Ala Arg Leu Ser Gln Asp 255766 CTG GCG CGC GAG CAA ATC CGT CAG GCG CAG GAT GGT CAC CTG CCG 810256 Leu Ala Arg Glu Gln Ile Arg Gln Ala Gln Asp Gly His Leu Pro 270811 ACG CTG AAT TTA ACG GCC TCA ACC GGC ATT TCT GAT ACC TCT TAT 855271 Thr Leu Asn Leu Thr Ala Ser Thr Gly Ile Ser Asp Thr Ser Tyr 285856 AGC GGT TCT AAA ACC AAC TCC ACC CAG TAC GAC GAT AGC AAC ATG 900286 Ser Gly Ser Lys Thr Asn Ser Thr Gln Tyr Asp Asp Ser Asn Met 300901 GGG CAG AAT AAA ATC GGC CTT AAC TTC TCC CTG CCG CTG TAT CAA 945301 Gly Gln Asn Lys Ile Gly Leu Asn Phe Ser Leu Pro Leu Tyr Gln 315946 GGT GGG ATG GTT AAC TCG CAG GTA AAA CAG GCG CAG TAT AAC TTC 990316 Gly Gly Met Val Asn Ser Gln Val Lys Gln Ala Gln Tyr Asn Phe 330991 GTC GGC GCA AGC GAA CAG CTG GAA AGC GCG CAC CGT AGC GTG GTG 1035331 Val Gly Ala Ser Glu Gln Leu Glu Ser Ala His Arg Ser Val Val 3451036 CAG ACC GTA CGT TCT TCC TTT AAC AAT ATT AAC GCC TCC ATC AGC 1080346 Gln Thr Val Arg Ser Ser Phe Asn Asn Ile Asn Ala Ser Ile Ser 3601081 AGC ATC AAC GCG TAT AAA CAG GCG GTC GTT TCC GCG CAA AGT TCT 1125361 Ser Ile Asn Ala Tyr Lys Gln Ala Val Val Ser Ala Gln Ser Ser 3751126 TTG GAT GCC ATG GAA GCC GGT TAC TCG GTC GGT ACA CGT ACC ATT 1170376 Leu Asp Ala Met Glu Ala Gly Tyr Ser Val Gly Thr Arg Thr Ile 3901171 GTT GAC GTA CTG GAT GCC ACC ACC ACT CTG TAT GAT GCC AAG CAG 1215391 Val Asp Val Leu Asp Ala Thr Thr Thr Leu Tyr Asp Ala Lys Gln 4051216 CAA CTG GCC AAC GCG CGT TAT ACC TAT TTG ATT AAT CAG TTA AAT 1260406 Gln Leu Ala Ash Ala Arg Tyr Thr Tyr Leu Ile Asn Gln Leu Asn 4201261 ATC AAA TAT GCG CTC GGT ACG CTG AAC GAG CAG GAT CTG CTC GCG 1305421 Ile Lys Tyr Ala Leu Gly Thr Leu Asn Glu Gln Asp Leu Leu Ala 4351306 CTT AAC AGT ACG TTG GGT AAA CCT ATC CCG ACG TCG CCG GAA AGC 1350436 Leu Asn Ser Thr Leu Gly Lys Pro Ile Pro Thr Ser Pro Glu Ser 4501351 GTA GCG CCG GAA ACG CCA GAT CAG GAT GCT GCC GCA GAC GGT TAT 1395451 Val Ala Pro Glu Thr Pro Asp Gln Asp Ala Ala Ala Asp Gly Tyr 4651396 AAT GCT CAT AGC GCC GCG CCA GCA GTA CAG CCG ACC GCC GCT CGC 1440466 Asn Ala His Ser Ala Ala Pro Ala Val Gln Pro Thr Ala Ala Arg 4801441 GCC AAC AGC AAT AAC GGC AAT CCA TTC CGG CAT TGA 1476481 Ala Asn Ser Asn Asn Gly Asn Pro Phe Arg His End 491，编码重组ST50蛋白相同序列的DNA和RNA序列，以及与所述第一序列在相对的核苷酸之间没有错配的互补的DNA和RNA序列，所组成的核苷酸序列。
13．一种多核苷酸序列，它由一个或多个基因组成，此基因编码一种或多种重组ST50蛋白或其结构性和/或功能性的等同物，一旦将其引入脊椎动物组织，便可诱导抗伤寒抗体或保护性免疫反应。
14．一种疫苗，它包含诱导抗伤寒免疫反应的重组ST50蛋白或其部分。
15．一种DNA疫苗，它包含权利要求13的多核苷酸序列，带有或不带有一种药物可接受的载体，它可在脊椎动物中诱导针对伤寒的保护。
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| Application Number | Priority Date | Filing Date | Title |
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| MYPI20000765A MY126769A (en) | 2000-02-28 | 2000-02-28 | Dna sequence encoding the specific and antigenic outer membrane protein of salmonella typhi |
| MYPI20000765 | 2000-02-28 |
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| CN1318646A true CN1318646A (zh) | 2001-10-24 |
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| CN01117058A Pending CN1318646A (zh) | 2000-02-28 | 2001-02-27 | 编码伤寒沙门氏菌特异性和抗原性外膜蛋白的dna序列 |
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| US (1) | US20020012668A1 (zh) |
| EP (1) | EP1132398A3 (zh) |
| JP (1) | JP2001292786A (zh) |
| KR (1) | KR100425884B1 (zh) |
| CN (1) | CN1318646A (zh) |
| AP (1) | AP1702A (zh) |
| AU (1) | AU783201B2 (zh) |
| BR (1) | BRPI0100837B8 (zh) |
| CA (1) | CA2333930A1 (zh) |
| ID (1) | ID29371A (zh) |
| MX (1) | MXPA01002178A (zh) |
| MY (1) | MY126769A (zh) |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102209724A (zh) * | 2008-09-09 | 2011-10-05 | 伯明翰大学 | 非伤寒类沙门氏菌疫苗 |
| CN102212120A (zh) * | 2011-03-30 | 2011-10-12 | 中国食品药品检定研究院 | 甲型副伤寒沙门氏菌PagC亚单位疫苗及其制备方法 |
| CN109371051A (zh) * | 2018-11-15 | 2019-02-22 | 华南农业大学 | 一种适于获得大量高纯度沙门氏菌外膜蛋白的提取方法与应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BR0316271A (pt) * | 2002-11-14 | 2005-10-11 | Inst Finlay Ct De Investigacio | Método para a obtenção de preparados vacinais conjugados multivalentes, composição vacinal multivalente e uso da mesma |
| WO2015185982A2 (en) * | 2014-06-05 | 2015-12-10 | Graphic Era University | Primers, probes and test kit for the detection of salmonella |
-
2000
- 2000-02-28 MY MYPI20000765A patent/MY126769A/en unknown
- 2000-11-14 AU AU71561/00A patent/AU783201B2/en not_active Ceased
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2001
- 2001-02-05 AP APAP/P/2001/002026A patent/AP1702A/en active
- 2001-02-14 KR KR10-2001-0007350A patent/KR100425884B1/ko active IP Right Grant
- 2001-02-14 ID IDP20010131D patent/ID29371A/id unknown
- 2001-02-20 ZA ZA200101406A patent/ZA200101406B/xx unknown
- 2001-02-21 CA CA002333930A patent/CA2333930A1/en not_active Abandoned
- 2001-02-23 BR BRPI0100837A patent/BRPI0100837B8/pt unknown
- 2001-02-26 EP EP01301728A patent/EP1132398A3/en not_active Withdrawn
- 2001-02-27 CN CN01117058A patent/CN1318646A/zh active Pending
- 2001-02-28 US US09/794,098 patent/US20020012668A1/en not_active Abandoned
- 2001-02-28 MX MXPA01002178A patent/MXPA01002178A/es not_active Application Discontinuation
- 2001-02-28 JP JP2001054570A patent/JP2001292786A/ja not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102209724A (zh) * | 2008-09-09 | 2011-10-05 | 伯明翰大学 | 非伤寒类沙门氏菌疫苗 |
| CN102212120A (zh) * | 2011-03-30 | 2011-10-12 | 中国食品药品检定研究院 | 甲型副伤寒沙门氏菌PagC亚单位疫苗及其制备方法 |
| CN109371051A (zh) * | 2018-11-15 | 2019-02-22 | 华南农业大学 | 一种适于获得大量高纯度沙门氏菌外膜蛋白的提取方法与应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| ZA200101406B (en) | 2001-09-20 |
| JP2001292786A (ja) | 2001-10-23 |
| AP2001002026A0 (en) | 2001-03-31 |
| BRPI0100837B1 (pt) | 2019-04-02 |
| US20020012668A1 (en) | 2002-01-31 |
| KR100425884B1 (ko) | 2004-04-03 |
| ID29371A (id) | 2001-08-30 |
| EP1132398A3 (en) | 2001-10-04 |
| AP1702A (en) | 2007-01-02 |
| MXPA01002178A (es) | 2004-02-17 |
| BRPI0100837B8 (pt) | 2021-05-25 |
| CA2333930A1 (en) | 2001-08-28 |
| AU7156100A (en) | 2001-08-30 |
| MY126769A (en) | 2006-10-31 |
| KR20010085387A (ko) | 2001-09-07 |
| EP1132398A2 (en) | 2001-09-12 |
| AU783201B2 (en) | 2005-10-06 |
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