CN104745574B - 用于在细菌表面呈递目的蛋白的dna分子及其应用 - Google Patents
用于在细菌表面呈递目的蛋白的dna分子及其应用 Download PDFInfo
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- CN104745574B CN104745574B CN201310750736.6A CN201310750736A CN104745574B CN 104745574 B CN104745574 B CN 104745574B CN 201310750736 A CN201310750736 A CN 201310750736A CN 104745574 B CN104745574 B CN 104745574B
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Abstract
本发明公开了一种用于在细菌表面呈递目的蛋白的DNA分子及其应用。本发明提供的DNA分子，自上游至下游依次包括N端信号肽的编码序列、Passenger结构域的编码序列和C端跨膜转运结构域的编码序列；所述N端信号肽如序列表的序列2所示；所述Passenger结构域如序列表的序列3所示；所述C端跨膜转运结构域如序列表的序列4所示。本发明为进一步开发应用于工业生产的全细胞生物催化剂打下了坚实的基础。本发明在全细胞生物催化、环境生物吸附剂的开发、抗体制备等众多领域具有潜在的应用价值。
Description
技术领域
本发明涉及一种用于在细菌表面呈递目的蛋白的DNA分子及其应用。
背景技术
细菌表面呈递技术是运用DNA重组技术使外源的蛋白质或多肽以融合蛋白的形式展示在细菌的表面，被展示的目标蛋白质或多肽可以保持相对独立的空间结构和生物活性，因此重组细菌就具有了该蛋白质和多肽的功能。更为重要的是，利用细菌表面呈递技术可以省去目标蛋白的提取纯化等一系操作，直接用细胞进行后续研究和应用，方便快捷、经济实用。细菌表面展示系统呈现载体多样性，有细菌外膜蛋白和脂蛋白系统，冰晶核蛋白系统和自体运输蛋白系统等。
发明内容
本发明的目的是提供一种用于在细菌表面呈递目的蛋白的DNA分子及其应用。
本发明提供的DNA分子，自上游至下游依次包括N端信号肽的编码序列、Passenger结构域的编码序列和C端跨膜转运结构域的编码序列；所述N端信号肽如序列表的序列2所示；所述Passenger结构域如序列表的序列3所示；所述C端跨膜转运结构域如序列表的序列4所示。
所述N端信号肽的编码序列可如序列表的序列1自5’末端第4-129位核苷酸所示。所述Passenger结构域的编码序列可如序列表的序列1自5’末端第250-810位核苷酸所示。所述C端跨膜转运结构域的编码序列可如序列表的序列1自5’末端第811-1650位核苷酸所示。
所述DNA分子中，在所述N端信号肽的编码序列和所述Passenger结构域的编码序列之间，还可具有多克隆位点序列。所述多克隆位点序列可如序列表的序列1自5’末端第199-249位核苷酸所示。
所述DNA分子中，在所述N端信号肽的编码序列和所述多克隆位点序列之间，还可具有蛋白标签的编码序列。所述蛋白标签具体可为His标签。所述蛋白标签的编码序列具体可如序列表的序列1自5’末端第181-198位核苷酸所示。
所述DNA分子具体如序列表的序列1所示。
含有以上任一所述DNA分子的重组质粒、转基因细胞系或重组菌均属于本发明的保护范围。
所述重组质粒可为将以上任一所述DNA分子插入表达载体的多克隆位点得到的重组质粒。所述重组质粒具体可为将以上任一所述DNA分子插入pET-22b(+)载体的多克隆位点（如NdeI和Bpu1102I酶切位点之间）得到的重组质粒pAutoDisplay。
所述重组菌具体可为将以上任一所述重组质粒（如重组质粒pAutoDisplay）导入宿主菌得到的重组菌。所述宿主菌可为大肠杆菌，具体可为大肠杆菌C41(DE3)。
本发明还保护以上任一所述的DNA分子或以上任一所述重组质粒的应用；所述应用为在细菌表面呈递目的蛋白。所述细菌可为大肠杆菌，具体可为大肠杆菌C41(DE3)。所述目的蛋白具体可为GFP蛋白或CotA蛋白。
本发明还保护一种在细菌表面呈递目的蛋白的方法，包括如下步骤：
（1）在以上任一所述重组质粒中插入目的蛋白的编码基因，插入位点为所述N端信号肽的编码序列和所述Passenger结构域的编码序列之间；
（2）将步骤（1）得到的质粒导入宿主细菌；
（3）培养步骤（2）得到的细菌，从而在细菌表面呈递所述目的蛋白。
所述步骤（1）中，所述插入位点具体可位于所述DNA分子的多克隆位点序列中。
所述步骤（2）中，所述宿主细菌可为大肠杆菌，具体可为大肠杆菌C41(DE3)。
所示步骤（3）中，所述培养中包括IPTG诱导的步骤。所示步骤（3）中，所述培养的方法具体如下：将步骤（2）得到的细菌接种至LB液体培养基，37℃培养至OD600nm=0.8-1.0，然后加入IPTG并使其浓度为0.2mM，继续37℃、220rpm振荡培养4小时。
所述目的蛋白具体可为GFP蛋白或CotA蛋白。所述目的蛋白的编码基因具体可为序列表的序列5所示的GFP基因或序列表的序列6所示的CotA基因。
N端信号肽通过Sec(Secretion)依赖途径运输融合蛋白通过细胞内膜到达周质腔,信号肽被切掉。C端跨膜转运结构域在外膜上组装折叠成桶状结构。Passenger结构域通过桶状结构形成的通道被输送到细菌表面。蛋白标签的作用是便于检测目的蛋白是否呈递到细菌表面。多克隆位点序列便于不同异源蛋白表达载体的构建，以使操作过程简洁方便。
本发明的有益效果及优点如下：
（1）本发明提供的DNA分子实现将目的蛋白运输至细胞外膜和通过外膜分泌所需要的所有信息都集中在其本身,不需要其它蛋白的辅助；
（2）本发明提供的DNA分子可用于各种目的蛋白的细菌表面呈递，具有非常良好的通用性。
采用本发明提供的DNA分子或方法将目的蛋白呈递到细菌表面，可用于全细胞生物催化，例如将CotA蛋白呈递到大肠杆菌表面，可以作为具有降解多环染料功能的全细胞生物催化剂。本发明为进一步开发应用于工业生产的全细胞生物催化剂打下了坚实的基础。本发明在全细胞生物催化、环境生物吸附剂的开发、抗体制备等众多领域具有潜在的应用价值。
附图说明
图1为重组质粒pAutoDisplay的结构示意图。
图2为实施例2中的Western blot结果。
图3为实施例2中的荧光照片。
图4为实施例3中的Western blot结果。
图5为实施例3中的酶活检测结果。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
ABTS，英文全称为2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt，中文全称为2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐：Sigma，HPLC级纯度。pET-22b(+)载体：Merck Novagen，69744。大肠杆菌C41(DE3)：Lucigen，60442。Trypsin：Merck，108367。
实施例1、重组质粒（细菌表面呈递载体）的构建
1、合成序列表的序列1所示的双链DNA分子。
序列表的序列1中，自5’末端第4-129位核苷酸为N端信号肽的编码序列，第181-198位核苷酸为His标签（由六个组氨酸残基组成）的编码序列，第199-249位核苷酸为多克隆位点序列，第250-810位核苷酸为Passenger结构域的编码序列，第811-1650位核苷酸为C端跨膜转运结构域的编码序列。
2、将步骤1合成的双链DNA分子插入pET-22b(+)载体的NdeI和Bpu1102I酶切位点之间，得到重组质粒pAutoDisplay。重组质粒pAutoDisplay的结构示意图见图1。
实施例2、在细菌表面呈递绿色荧光蛋白（GFP蛋白）
1、将序列表的序列5所示的GFP基因插入重组质粒pAutoDisplay的BamHI和XhoI酶切位点之间，得到重组质粒pAutoDisplay-GFP。
2、将步骤1得到的重组质粒导入大肠杆菌C41(DE3)，得到重组菌。
3、将步骤2得到的重组菌接种至LB液体培养基，37℃培养至OD600nm=0.8-1.0，然后加入IPTG并使其浓度为0.2mM，继续37℃、220rpm振荡培养4小时。
4、完成步骤3后，取两份菌液（每份400μl），4℃、2000g离心10分钟，收集菌体，每份菌体用200μl PBS缓冲液（pH7.4）重悬。
5、取一份步骤4得到的菌液，加入4μl10mg/ml Trypsin溶液然后37℃水浴10分钟，然后迅速置于4℃并加入20μl胎牛血清以终止反应，然后2000g离心5分钟收集菌体，然后用含10%FBS的PBS缓冲液（pH7.4）洗涤3遍，然后用PBS缓冲液（pH7.4）洗涤一遍，然后用200μlPBS缓冲液（pH7.4）重悬。
6、取另一份步骤4得到的菌液，加入4μl PBS缓冲液（pH7.4）然后37℃水浴消化10分钟，然后迅速置于4℃并加入20μl胎牛血清以终止反应，然后2000g离心5分钟收集菌体，然后用含10%FBS的PBS缓冲液（pH7.4）洗涤3遍，然后用PBS缓冲液（pH7.4）洗涤一遍，然后用200μl PBS缓冲液（pH7.4）重悬。
7、分别取步骤5得到的菌液（实验组）和步骤6得到的菌液（对照组），进行12%SDS-PAGE和western blot。采用的一抗为1:3000倍稀释的鼠源His抗体（Sigma，H1029)，采用的二抗为1:5000倍稀释的辣根过氧化氢酶标记的羊抗鼠二抗。
Trypsin消化的原理：在不破坏整个细胞的情况下，Trypsin可以将分泌至细菌表面的蛋白消化，通过实验组和对照组的比较，可以判断该蛋白是否分泌到细菌表面，实验组细菌表面的蛋白会被消化，Western blot图谱中不显示目的条带，对照组细菌表面的蛋白没有被消化，在Western blot图谱中显示目的条带。
序列1编码的融合蛋白中的C端跨膜转运结构域在外膜上组装折叠成桶状结构，Passenger结构域上游的片段（含Passenger结构域）通过桶装结构被输送到细胞表面，在细胞自身的作用下，N端信号肽被切割掉，Passenger结构域的最后一个氨基酸残基与C端跨膜转运结构域的第一个氨基酸残基的连接被切断，目的片段（具有GPF蛋白的片段，预期分子量约为49.3KD）粘附于细胞表面，加入Trypsin后粘附于细胞表面的目的片段会被消化。如果序列1编码的融合蛋白中Passenger结构域的最后一个氨基酸残基与C端跨膜转运结构域的第一个氨基酸残基尚未发生切割（预期分子量约为79.7KD），即使加入Trypsin也不会被消化。
Western blot结果见图2（泳道1为分子量标记，泳道2为实验组，泳道3为对照组，箭头标注目的片段）。对照组样本（未进行过Trypsin消化）显示约49.3KD的阳性条带，实验组样本（进行过Trypsin消化）没有对应的条带。结果表明，GFP蛋白成功展示到细菌表面。
8、取步骤3得到的菌液，在荧光显微镜下进行观察，照片见图3。可以观察到菌表面具有荧光。
进行五次重复实验，结果一致。
对比例、
1、将序列表的序列5所示的GFP基因插入pET-22b(+)载体的NdeI和XhoI酶切位点之间，得到对照质粒。
2、将步骤1得到的重组质粒导入大肠杆菌C41(DE3)，得到重组菌。
3、将步骤2得到的重组菌接种至LB液体培养基，37℃培养至OD600nm=0.8-1.0，然后加入IPTG并使其浓度为0.2mM，继续37℃、220rpm振荡培养4小时。
4、完成步骤3后，取两份菌液（每份400μl），4℃、2000g离心10分钟，收集菌体，每份菌体用200μl PBS缓冲液（pH7.4）重悬。
5、取一份步骤4得到的菌液，加入4μl10mg/ml Trypsin溶液然后37℃水浴10分钟，然后迅速置于4℃并加入20μl胎牛血清以终止反应，然后2000g离心5分钟收集菌体，然后用含10%FBS的PBS缓冲液（pH7.4）洗涤3遍，然后用PBS缓冲液（pH7.4）洗涤一遍，然后用200μlPBS缓冲液（pH7.4）重悬。
6、取另一份步骤4得到的菌液，加入4μl PBS缓冲液（pH7.4）然后37℃水浴消化10分钟，然后迅速置于4℃并加入20μl胎牛血清以终止反应，然后2000g离心5分钟收集菌体，然后用含10%FBS的PBS缓冲液（pH7.4）洗涤3遍，然后用PBS缓冲液（pH7.4）洗涤一遍，然后用200μl PBS缓冲液（pH7.4）重悬。
7、分别取步骤5得到的菌液（实验组）和步骤6得到的菌液（对照组），进行12%SDS-PAGE和western blot。采用的一抗为1:3000倍稀释的鼠源GFP抗体，采用的二抗为1:5000倍稀释的辣根过氧化氢酶标记的羊抗鼠二抗。
结果表明，实验组和对照组均显示约28.1KD的目的带，即GFP蛋白存在于细菌内部，没有被Trypsin消化掉。
实施例3、在细菌表面呈递枯草杆菌漆酶（CotA蛋白）
漆酶属于多铜氧化酶，每个单体含有四个铜离子，单核铜离子中心位于底物结合位点,三核铜离子中心位于O2结合位点,漆酶吸收电子氧化底物,电子传递给O2,生成水。漆酶能够通过自由基-催化反应机制直接氧化芳香族化合物底物，或者在介体的介导作用下氧化木质素等大分子物质,介体系统的发展极大的扩展了漆酶的应用范围，使漆酶成为具有广泛应用前景的重要工业酶。目前,漆酶已经用于纸浆造纸、纺织工业、食品工程、药物有机合成、污染环境生物修复以及生物质能源开发等领域。
一、实验组的检测
1、将序列表的序列6所示的CotA基因插入重组质粒pAutoDisplay的BamHI和XhoI酶切位点之间，得到重组质粒pAutoDisplay-CotA。
2、将步骤1得到的重组质粒pAutoDisplay-CotA导入大肠杆菌C41（DE3)，得到重组菌甲。
3、将步骤2得到的重组菌甲接种至LB液体培养基，37℃培养至OD600nm=0.8-1.0，然后加入IPTG并使其浓度为0.2mM，继续37℃、220rpm振荡培养4小时。
4、完成步骤3后，取两份菌液（每份400μl），4℃、2000g离心10分钟，收集菌体，每份菌体用200μl PBS缓冲液（pH7.4）重悬。
5、取一份步骤4得到的菌液，加入4μl10mg/ml Trypsin溶液然后37℃水浴10分钟，然后迅速置于4℃并加入20μl胎牛血清以终止反应，然后2000g离心5分钟收集菌体，然后用含10%FBS的PBS缓冲液（pH7.4）洗涤3遍，然后用PBS缓冲液（pH7.4）洗涤一遍，然后用200μlPBS缓冲液（pH7.4）重悬。
6、取另一份步骤4得到的菌液，加入4μl PBS缓冲液（pH7.4）然后37℃水浴消化10分钟，然后迅速置于4℃并加入20μl胎牛血清以终止反应，然后2000g离心5分钟收集菌体，然后用含10%FBS的PBS缓冲液（pH7.4）洗涤3遍，然后用PBS缓冲液（pH7.4）洗涤一遍，然后用200μl PBS缓冲液（pH7.4）重悬。
7、分别取步骤5得到的菌液（实验组）和步骤6得到的菌液（对照组），进行12%SDS-PAGE和western blot。采用的一抗为1:3000倍稀释的鼠源His抗体（Sigma，H1029)，采用的二抗为1:5000倍稀释的辣根过氧化氢酶标记的羊抗鼠二抗。
目的片段的预期分子量约80.8KD。
Western blot结果见图4（泳道1为分子量标记，泳道2为实验组，泳道3为对照组，箭头标注目的片段）。对照组样本（未进行过Trypsin消化）显示约80.8KD的阳性条带，实验组样本（进行过Trypsin消化）没有对应的条带。结果表明，CotA蛋白成功展示到细菌表面。
8、枯草杆菌漆酶CotA的酶活性检测
漆酶活性检测试验以ABTS为底物，一个活力单位（1U）定义为：在40℃和pH5.0的条件下，一分钟内转化1μmol底物所需的酶量。
（1）取步骤3得到的菌液，4℃、2000g离心10分钟，收集菌体。
（2）用PBS缓冲液（pH6.8）悬浮步骤（1）得到的菌体，得到不同浓度的菌液。
（3）将50μl步骤（2）得到的菌液、900μl柠檬酸钠-磷酸二氢钠缓冲液（pH5.0、100mM）和50μl ABTS水溶液混合，得到反应体系，ABTS在反应体系中的初始浓度为1mM；在40℃水浴中反应3min。
（4）完成步骤（3）后，检测OD420nm吸光值的变化。
△OD420为OD420nm吸光值的变化值；t为反应时间（单位min）；V为反应体系的体积（单位ml）；εABTS为ABTS在420nm处的摩尔吸光系数，36/（μmol/ml×cm）；d为光程（单位cm）。
二、对照组的检测
将重组质粒pAutoDisplay导入大肠杆菌C41（DE3)，得到重组菌乙；将重组菌乙接种至LB液体培养基，37℃培养至OD600nm=0.8-1.0，然后加入IPTG并使其浓度为0.2mM，继续37℃、220rpm振荡培养4小时，4℃、2000g离心10分钟，收集菌体；用PBS缓冲液（pH6.8）悬浮菌体，得到不同浓度的菌液；将50μl菌液、900μl柠檬酸钠-磷酸二氢钠缓冲液（pH5.0、100mM）和50μl ABTS水溶液混合，得到反应体系，ABTS在反应体系中的初始浓度为1mM；在40℃水浴中反应3min，然后检测OD420nm吸光值的变化。
三、结果分析
步骤一和步骤二的结果见图5。结果显示：重组菌甲的活体细胞具有漆酶活性，可以氧化底物ABTS，全细胞酶活随着细胞数量的增加而增加，可达到每5.2×108个菌细胞的活性为0.12mU；重组菌乙的活体细胞不具有漆酶活性，不能氧化底物ABTS。
结果表明，利用本发明提供的重组质粒pAutoDisplay将CotA展示在细菌表面得到的重组菌具有良好的催化活性。由于漆酶的分离纯化步骤繁多，耗时耗力，导致酶的应用成本较高，另外，纯化的漆酶往往容易受环境因素影响失去活性，也不利于漆酶的应用。所以，利用本发明可以实现全细胞漆酶，具有制备方便快捷、使用成本低、稳定性高的优势，对于将漆酶广泛应用于工业生产中具有重要的意义。
序列表
<110>中国科学院生物物理研究所
<120>用于在细菌表面呈递目的蛋白的DNA分子及其应用
<130>CGGNAY133349
<160>6
<210>1
<211>1708
<212>DNA
<213>人工序列
<220>
<223>
<400>1
catatgtacc tggaccgctt ccgtcagtgc cctagcagcc tgcaaatccc gcgtagtgcc60
tggcgcctgc acgcattagc cgcagcatta gccctggccg gtatggcccg tttagcacct120
gccgcagccc aagcccctca accgccggtt gcaggtgcac cgcatgccca agacgcaggc180
catcatcacc atcatcatgg atccaagctt gaattcggta cctcgtcgac gggctcgagt240
gggagctctg ccggtattag tctgagcgtt gcaagcggcg cagcatggca tggtgcaacc300
caggttctgc agagtgccac cctgggcaaa ggtggcacct gggtggtgaa tgcagacagt360
cgtgtgcagg acatgagcat gcgcggcggc cgtgtggaat ttcaagcacc ggccccggag420
gcaagctaca agaccctgac cctgcagaca ctggacggta acggcgtgtt cgtgctgaac480
accaacgtgg cagccggtca aaacgaccag ctgcgtgtga ccggtcgtgc agatggccag540
caccgtgttc tggtgcgcaa cgccggtggt gaagcagata gccgtggtgc acgcctgggt600
ctggttcaca cccagggcca aggcaacgcc acctttcgtc tggccaacgt tggcaaggca660
gtggatctgg gcacctggcg ctatagtctg gccgaagacc cgaagaccca cgtgtggagt720
ctgcagcgtg ccggtcaggc cttaagtggc gcagccaatg ccgccgtgaa tgccgcagat780
ctgagcagta tcgccctggc agaaagtaat gccctggaca agcgcctggg tgagctgcgt840
ttacgtgcag acgcaggtgg tccgtgggca cgcaccttca gcgaacgcca gcagatcagc900
aaccgtcatg cccgtgcata cgaccagacc gtgagcggcc tggagattgg tctggatcgt960
ggttggagtg ccagcggtgg tcgctggtat gcaggtggtt tactgggcta cacctatgcc1020
gaccgtacct atccgggcga tggtggtggt aaggttaagg gcttacacgt gggcggctat1080
gcagcatacg tgggcgacgg cggctactac ctggataccg ttctgcgtct gggccgctac1140
gaccaacagt acaacatcgc cggtacagac ggtggccgtg ttacagccga ctaccgtacc1200
agcggtgcag catggagttt agagggcggt cgccgcttcg aactgccgaa cgactggttt1260
gccgaaccgc aagccgaggt tatgctgtgg cgcaccagcg gcaaacgtta ccgcgcaagc1320
aatggcctgc gcgttaaggt ggatgccaat accgccacac tgggtcgctt aggcttacgc1380
ttcggccgcc gtattgcact ggccggtggc aatatcgtgc agccgtatgc ccgtctgggc1440
tggacccagg agtttaagag caccggcgac gtgcgcacca atggtattgg ccatgcaggc1500
gcaggtcgtc acggtcgtgt tgaactgggc gcaggtgttg acgcagcctt aggtaaaggc1560
cacaacctgt acgcaagcta cgagtatgcc gccggtgacc gtatcaacat cccgtggagc1620
ttccacgccg gttaccgtta cagcttttaa gatccggctg ctaacaaagc ccgaaaggaa1680
gctgagttgg ctgctgccac cgctgagc1708
<210>2
<211>42
<212>PRT
<213>人工序列
<220>
<223>
<400>2
Met Tyr Leu Asp Arg Phe Arg Gln Cys Pro Ser Ser Leu Gln Ile Pro
151015
Arg Ser Ala Trp Arg Leu His Ala Leu Ala Ala Ala Leu Ala Leu Ala
202530
Gly Met Ala Arg Leu Ala Pro Ala Ala Ala
3540
<210>3
<211>187
<212>PRT
<213>人工序列
<220>
<223>
<400>3
Ala Gly Ile Ser Leu Ser Val Ala Ser Gly Ala Ala Trp His Gly Ala
151015
Thr Gln Val Leu Gln Ser Ala Thr Leu Gly Lys Gly Gly Thr Trp Val
202530
Val Asn Ala Asp Ser Arg Val Gln Asp Met Ser Met Arg Gly Gly Arg
354045
Val Glu Phe Gln Ala Pro Ala Pro Glu Ala Ser Tyr Lys Thr Leu Thr
505560
Leu Gln Thr Leu Asp Gly Asn Gly Val Phe Val Leu Asn Thr Asn Val
65707580
Ala Ala Gly Gln Asn Asp Gln Leu Arg Val Thr Gly Arg Ala Asp Gly
859095
Gln His Arg Val Leu Val Arg Asn Ala Gly Gly Glu Ala Asp Ser Arg
100105110
Gly Ala Arg Leu Gly Leu Val His Thr Gln Gly Gln Gly Asn Ala Thr
115120125
Phe Arg Leu Ala Asn Val Gly Lys Ala Val Asp Leu Gly Thr Trp Arg
130135140
Tyr Ser Leu Ala Glu Asp Pro Lys Thr His Val Trp Ser Leu Gln Arg
145150155160
Ala Gly Gln Ala Leu Ser Gly Ala Ala Asn Ala Ala Val Asn Ala Ala
165170175
Asp Leu Ser Ser Ile Ala Leu Ala Glu Ser Asn
180185
<210>4
<211>279
<212>PRT
<213>人工序列
<220>
<223>
<400>4
Ala Leu Asp Lys Arg Leu Gly Glu Leu Arg Leu Arg Ala Asp Ala Gly
151015
Gly Pro Trp Ala Arg Thr Phe Ser Glu Arg Gln Gln Ile Ser Asn Arg
202530
His Ala Arg Ala Tyr Asp Gln Thr Val Ser Gly Leu Glu Ile Gly Leu
354045
Asp Arg Gly Trp Ser Ala Ser Gly Gly Arg Trp Tyr Ala Gly Gly Leu
505560
Leu Gly Tyr Thr Tyr Ala Asp Arg Thr Tyr Pro Gly Asp Gly Gly Gly
65707580
Lys Val Lys Gly Leu His Val Gly Gly Tyr Ala Ala Tyr Val Gly Asp
859095
Gly Gly Tyr Tyr Leu Asp Thr Val Leu Arg Leu Gly Arg Tyr Asp Gln
100105110
Gln Tyr Asn Ile Ala Gly Thr Asp Gly Gly Arg Val Thr Ala Asp Tyr
115120125
Arg Thr Ser Gly Ala Ala Trp Ser Leu Glu Gly Gly Arg Arg Phe Glu
130135140
Leu Pro Asn Asp Trp Phe Ala Glu Pro Gln Ala Glu Val Met Leu Trp
145150155160
Arg Thr Ser Gly Lys Arg Tyr Arg Ala Ser Asn Gly Leu Arg Val Lys
165170175
Val Asp Ala Asn Thr Ala Thr Leu Gly Arg Leu Gly Leu Arg Phe Gly
180185190
Arg Arg Ile Ala Leu Ala Gly Gly Asn Ile Val Gln Pro Tyr Ala Arg
195200205
Leu Gly Trp Thr Gln Glu Phe Lys Ser Thr Gly Asp Val Arg Thr Asn
210215220
Gly Ile Gly His Ala Gly Ala Gly Arg His Gly Arg Val Glu Leu Gly
225230235240
Ala Gly Val Asp Ala Ala Leu Gly Lys Gly His Asn Leu Tyr Ala Ser
245250255
Tyr Glu Tyr Ala Ala Gly Asp Arg Ile Asn Ile Pro Trp Ser Phe His
260265270
Ala Gly Tyr Arg Tyr Ser Phe
275
<210>5
<211>717
<212>DNA
<213>人工序列
<220>
<223>
<400>5
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag717
<210>6
<211>1539
<212>DNA
<213>人工序列
<220>
<223>
<400>6
atgacacttg aaaaatttgt ggatgctctc ccaatcccag atacactaaa gccagtacag60
caatcaaaag aaaaaacata ctacgaagtc accatggagg aatgcactca tcagctccat120
cgcgatctcc ctccaacccg cctgtggggc tacaacggct tatttccggg accgaccatt180
gaggttaaaa gaaatgaaaa cgtatatgta aaatggatga ataaccttcc ttccacgcat240
ttccttccga ttgatcacac cattcatcac agtgacagcc agcatgaaga gcccgaggta300
aagactgttg ttcatttaca cggcggcgtc acgccagatg atagtgacgg gtatccggag360
gcttggtttt ccaaagactt tgaacaaaca ggaccttatt tcaaaagaga ggtttatcat420
tatccaaacc agcagcgcgg ggctatattg tggtatcacg atcacgccat ggcgctcacc480
aggctaaatg tctatgccgg acttgtcggt gcatatatca ttcatgaccc aaaggaaaaa540
cgcttaaaac tgccttcaga cgaatacgat gtgccgcttc ttatcacaga ccgcacgatc600
aatgaggatg gttctttgtt ttatccgagc gcaccggaaa acccttctcc gtcactgcct660
aatccttcaa tcgttccggc tttttgcgga gaaaccatac tcgtcaacgg gaaggtatgg720
ccatacttgg aagtcgagcc aaggaaatac cgattccgtg tcatcaacgc ctccaataca780
agaacctata acctgtcact cgataatggc ggagatttta ttcagattgg ttcagatgga840
gggctcctgc cgcgatctgt taaactgaat tctttcagcc ttgcgcctgc tgaacgttac900
gatatcatca ttgacttcac agcgtatgaa ggagaatcga tcattttggc aaacagcgcg960
ggctgcggcg gtgacgtcaa tcctgaaaca gatgcgaata tcatgcaatt cagagtcaca1020
aaaccattgg cacaaaaaga cgaaagcaga aagccgaagt acctcgcctc atacccttcg1080
gtacagcatg aaagaataca aaacatcaga acgttaaaac tggcaggcac ccaggacgaa1140
tacggcagac ccgtccttct gcttaataac aaacgctggc acgatcccgt cacagaaaca1200
ccaaaagtcg gcacaactga aatatggtcc attatcaacc cgacacgcgg aacacatccg1260
atccacctgc atctagtctc cttccgtgta ttagaccggc ggccgtttga tatcgcccgt1320
tatcaagaaa gcggggaatt gtcctatacc ggtccggctg tcccgccgcc gccaagtgaa1380
aagggctgga aagacaccat tcaagcgcat gcaggtgaag tcctgagaat cgcggcgaca1440
ttcggtccgt acagcggacg atacgtatgg cattgccata ttctagagca tgaagactat1500
gacatgatga gaccgatgga tataactgat ccccataaa1539
Claims (4)
1.一种DNA分子，如序列表的序列1所示。
2.含有权利要求1所述DNA分子的重组质粒、转基因细胞系或重组菌。
3.权利要求1所述的DNA分子或权利要求2所述重组质粒的应用；所述应用为在细菌表面呈递目的蛋白；所述细菌为大肠杆菌。
4.一种在细菌表面呈递目的蛋白的方法，包括如下步骤：
(1)在权利要求2所述重组质粒中插入目的蛋白的编码基因，插入位点为N端信号肽的编码序列和Passenger结构域的编码序列之间；所述N端信号肽的编码序列如序列表的序列1自5’末端第4-129位核苷酸所示；所述Passenger结构域的编码序列如序列表的序列1自5’末端第250-810位核苷酸所示；
(2)将步骤(1)得到的质粒导入宿主细菌；所述宿主细菌为大肠杆菌；
(3)培养步骤(2)得到的细菌，从而在细菌表面呈递所述目的蛋白。
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