具体实施方式
下面结合实施例对本发明做进一步阐述,但本发明所保护的范围不限于此。
实施例中未详加说明的内容均按本领域现有技术。
相关序列
SEQ ID NO.1 CYP153A A229S-CPRBM3上游引物;
SEQ ID NO.2 CYP153A A229S-CPRBM3下游引物;
SEQ ID NO.3 CYP153A-CPRBM3核苷酸序列;
SEQ ID NO.4 CYP153A A229S-CPRBM3核苷酸序列;
SEQ ID NO.5 CYP153A-CPRBM3氨基酸序列;
SEQ ID NO.6 CYP153A A229S-CPRBM3氨基酸序列;
SEQ ID NO.7突变体A229S核苷酸序列;
SEQ ID NO.8突变体A229S氨基酸序列;
SEQ ID NO.9 CYP153A的上游引物;
SEQ ID NO.10 CYP153A的下游引物;
SEQ ID NO.11 CPRBM3的上游引物;
SEQ ID NO.12 CPRBM3的下游引物。
实施例1
细胞色素氧化酶CYP153A M.aq.突变体的获得
(一)定点饱和突变基因库的获得
基于CYP153A M.aq.三维结构挖掘C10底物专一性的P450酶基因,从PDB下载CYP153A M.aq.与底物癸酸结合三维结构,利用分子生物学工具PYMOL寻找酶与底物结合相关氨基酸,核对酶催化底物的关键氨基酸;其中关键的氨基酸为129Q、130I、131I、143M、145I、146A、229A、303L、306V、307G、311T、354L、357M、418C、420G、455F。以CYP153AM.aq.氨基酸的序列作为探针,从NCBI上进行Blast比对,下载序列相似度在70%-90%之间的序列。对下载下来的同源氨基酸序列与CYP153A M.aq.进行对比,分析CYP153A M.aq.序列中氨基酸的保守性,发现不保守的氨基酸为129Q、131I、143M、229A,因此选用这几个点对为突变位点(见图1)。
(二)突变酶的构建
使用Discovery Studio构建虚拟突变蛋白,随后对突变位点周围
的氨基酸进行局部能量最小化,以使突变位点区域的氨基酸处于能量和集合构象上都比较合理的状态。
使用分子对接程序CDOCKER来寻找虚拟突变蛋白与底物的结合方式。
(三)计算突变酶与底物(癸酸)结合的自由能,确定需构建的突变酶菌体
利用Discovery Studio对突变酶与底物结合的三维结构进行结合自由能的计算,寻找最优突变蛋白(见图2)。
(四)构建突变酶菌体
海杆菌(Marinobacter aquaeolei)的烷烃羟化酶CYP153A与巨大芽孢杆菌(Bacillus megaterium)P450NADH还原酶CPRBM3的融合酶基因,即CYP153A-CPRBM3,CYP153AA229S-CPRBM3融合酶核苷酸序列分别如SEQ ID NO.3、4所示,对应表达的氨基酸序列如SEQ ID NO.5、6所示,突变体A229S核苷酸序列如SEQ ID NO.7所示、突变体A229S氨基酸序列如SEQ ID NO.8所示。
构建重组质粒pET21b-CYP153A-CPRBM3,包括如下步骤:
以经过密码子优化的海杆菌(Marinobacter aquaeolei)的烷烃羟化酶CYP153A与巨大芽孢杆菌(Bacillus megaterium)P450 NADH还原酶CPRBM3的融合酶基因为模板进行PCR扩增,CYP153A的上游引物的核苷酸序列如SEQ ID NO.9所示,下游引物的核苷酸序列如SEQ ID NO.10所示,CPRBM3的上游引物的核苷酸序列如SEQ ID NO.11所示,下游引物的核苷酸序列如SEQ ID NO.12所示,然后将pET21b质粒用Nde I和Xho I进行双酶切,经多片段无缝克隆试剂盒连接,制得重组质粒pET21b-CYP153A-CPRBM3;
PCR扩增体系如下,总体系25μL:
100μM上游引物1.0μL,100μM下游引物1.0μL,模板1.0μL,5U/μL phanta酶12.5μL,ddH2O 9.5μL。
PCR扩增条件如下:
95℃预变性3min;95℃变性15s,60℃退火15s,72℃延伸CYP153A 45S、CPRBM3 55S,循环30次;72℃延伸5min。
细胞色素氧化酶基因突变体的构建是利用不依赖连接酶的快速克隆(quickchange)的方法,在突变点处设计两条引物,利用反向PCR介导,以构建好的pET-21b-CYP153A-CPRBM3的重组质粒为模板进行全序列体外扩增,PCR产物通过DpnⅠ酶进行酶切,去除重组质粒模板,之后,转入大肠杆菌感受态BL21(DE3)中进行环化,得到重组质粒pET21b-CYP153AA229S-CPRBM3。具体为:
以之前实验中构建的pET-21b-CYP153A-CPRBM3为模板,以CYP153A A229S-CPRBM3SEQ ID NO.1为上游引物,以CYP153A A229S-CPRBM3 SEQ ID NO.2为下游引物,PCR扩增pET-21b-CYP153A A229S-CPRBM3,PCR反应体系如下:
上述PCR反应按照如下程序进行:
95℃预变性3min;95℃变性15s,68℃退火15s,72℃延伸4min30s,30个循环;72℃终延伸5min。
PCR结束后通过1%的琼脂糖凝胶电泳分析片段长短如图3,确定成功完成突变。
DpnI酶切原始模板
DpnI酶切体系:
PCR产物 44μL
DpnI 1μL
10X QuickCut Buffer 5μL
反应条件:37℃反应2h。
反应完成后70℃15min失活DpnI。
重组质粒的转化
1.将购买的BL21(DE3)感受态细胞从-80℃拿出,迅速置于冰上融化,加入10μL酶切产物,轻轻混匀,冰浴30min。
2.42℃热激90s,然后迅速置于冰浴中冷却2min。
3.加入900μL LB培养基,37℃,200rpm/min振荡培养60min,使细菌恢复正常生长状态,并表达质粒编码的抗生素抗性基因。
4.2500g离心3min,弃掉900μL上清,用剩余培养基将菌体重悬均匀涂布于带有抗性的LB固体培养基(氨苄青霉素100μg/mL)。
5.待菌液被吸干后,倒置平板于37℃培养12~16h。
挑取10个单菌落,37℃振荡培养6~8h,加入甘油保藏菌种。
实施例2
突变体的筛选
将转有突变质粒的菌株接种于含有氨苄抗生素的平板(含有氨苄青霉素100μg/mL),37℃恒温培养箱培养,挑取10个单菌落于含有1mL液体LB(含氨苄青霉素100μg/mL)的甘油管中培养过夜培养37℃,200rpm,得到我们实验所需的细胞色素氧化酶突变体库,取10个甘油管,每管加入800μL过夜活化的菌液,再加入800μL甘油,取其中两管标记好,送于生工科技有限公司进行测序,剩余8管冷冻保藏,获得大肠杆菌BL21 pET21b-CYP153A A229S-CPRBM3。
实施例3
分离纯化细胞色素氧化酶重组蛋白
种子培养:将筛选得到的正突变体置于50mL的含有100μg/mL氨苄青霉素的LB液体培养基中,在37℃过夜振荡培养;
扩大培养:将菌体接种于1L培养基中,当菌体浓度OD600达到1.0-1.2范围,即对数期,加入终浓度为0.5mM FeCl3、5-ALA,使菌体在20℃适应30min,再加入IPTG,使其终浓度为0.5mM进行诱导过夜(20h)表达目的蛋白。
菌体收集:将发酵所得菌体倒入大离心瓶中,配平,5000rpm离心15min,倒掉上清收集菌体。
菌体的破碎:每升菌离心所得的菌体中加入30mL的重悬缓冲液,充分悬匀,放入冰上进行超声破碎,超声4s,间隔6s,20min,功率400KW。
可溶蛋白的获取:将破碎后的菌体倒入高速离心管中,配平,14000rpm离心1h,取上清。
Ni-NTA亲和层析:将收集的含有可溶性蛋白的上清液体倒入再生好的Ni-NTA柱中;上清液流净后,以wash buffer(20mM PBS,pH8.0,200mM NaCl)冲洗10个柱体积,除去非特异性吸附的蛋白;最后使用elution buffer(20mM PBS,pH8.0,200mM NaCl,250mM咪唑)将目的蛋白洗脱下来,用干净预冷烧杯收集,用超滤管离心浓缩,全程低温操作,将收集完成的粗酶液跑SDS-PAGE蛋白电泳,见图4。
实施例4
大肠杆菌BL21 pET21b-CYP153A A229S-CPRBM3融合酶工程菌发酵
(1)菌种活化:将实施例2中的大肠杆菌BL21 pET21b-CYP153AA229S-CPRBM3以1%的接种量接种至50mL的含有氨苄青霉素的液体LB培养基中,在37℃、200rpm振荡培养12h;
(2)菌体转接:取上述活化菌株3mL接入300mL含有氨苄青霉素的液体培养基中,37℃、200rpm振荡培养至菌液OD600为1.0时,降温至20℃适应1小时后,加入IPTG使培养基中IPTG浓度为0.5mM、加入终浓度0.5mM的FeCl3、0.5mM终浓度5-ALA(5-氨基乙酰丙酸盐酸盐)继续诱导培养18小时,分离细胞,制得诱导细胞;
(3)收集菌体:取上述菌液300mL,5000rpm,4℃离心15min,收集菌体;
(4)用0.85%生理盐水洗涤沉淀三次,并用转化培养基重悬菌体制备菌悬液。转化培养基包含100mM磷酸钾缓冲液(pH7.4),质粒浓度1%的甘油,质量浓度0.4%的葡萄糖,100μg/mL的氨苄抗生素,总体积为30mL,加入0.5g/L的癸酸进行反应,30℃反应2h并在2h取样,反应终止后加入1mL 0.4mol/L HCl终止反应,加入0.1g/L月桂酸作为内标。
发酵液硅烷化处理:取1mL发酵液于1.5mL离心管中,每个样品用1.5mL乙酸乙酯萃取样品溶液中的脂肪酸(每次750μL),旋流混合器混合60s,室温下4000r/m离心10min。取提取液蒸发干燥,干燥后的样品重新溶解在0.5mL乙酸乙酯(色谱纯)、0.5mL正己烷(色谱纯)中,100μL BSTFA-TMCS(99:1,v/v)衍生化试剂加入后室温放置5min,在70℃的烤箱中孵育50min。
气相质谱检测生成产物:气相色谱以氦气为载气,恒流模式,进样体积1μL,分流进样,分流比为1:5,进样温度250℃,50℃保持1min,以15℃/min升至250℃,保持10min。产物色谱图如图5、图6、图7、图8所示。
从气质结果我们能够得知,在底物癸酸为0.5g/L时,2h取样气质结果表示,大肠杆菌BL21 pET21b-CYP153AA229S-CPRBM3对癸酸转化为10-羟基癸酸的转化率为36.22%。
对比例
与大肠杆菌BL21 pET21b-CYP153A A229S-CPRBM3融合酶工程菌的构建方法相同,构建大肠杆菌BL21 pET21b-CYP153A-CPRBM3融合酶工程菌,与实施例4的发酵条件相同,将癸酸转化为10-羟基癸酸的转化率为35.04%。
从对比例的实验结果我们能够发现,经过定点突变的大肠杆菌BL21 pET21b-CYP153A A229S-CPRBM3融合酶工程菌催化癸酸转化为10-羟基癸酸的转化率比大肠杆菌BL21 pET21b-CYP153A -CPRBM3融合酶工程菌提高1.18%。
CYP153A M.aq.对十个碳的脂肪酸(癸酸)的底物转化率相对于其他脂肪酸(碳数为12~14)低,CYP153A M.aq.由于其对癸酸转化率不高,造成利用该酶生产10-羟基癸酸未能广泛应用于工业生产的一大制约因素,因此提高CYP153A M.aq对癸酸的转化率将推动该酶在相关领域的更广泛应用;10-羟基癸酸是化妆品、医药产品和保健品行业的重要原料和中间体,并且价格昂贵,本发明涉及的CYP153A M.aq.突变体A229S提高了癸酸转化为10-羟基癸酸的转化率,对该酶以及酶法生产10-羟基癸酸具有重要的意义。
SEQUENCE LISTING
<110> 齐鲁工业大学
<120> 一种细胞色素氧化酶突变体及其应用
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 27
<212> DNA
<213> 人工序列
<400> 1
cttgccgcac cggacatgcg atcgctc 27
<210> 2
<211> 27
<212> DNA
<213> 人工序列
<400> 2
gagcgatcgc atgtccggtg cggcaag 27
<210> 3
<211> 3195
<212> DNA
<213> 人工序列
<400> 3
atgccgacgt taccacgtac ctttgatgac attcagtctc gcttaatcaa tgctacaagt 60
cgtgtggttc caatgcagcg tcagattcag ggtctgaaat ttctgatgag tgccaaacgc 120
aaaacctttg gtccacgtcg cccaatgccg gaatttgtgg aaacacctat cccggatgtt 180
aatacattag ccttagagga cattgatgtg agtaatccgt ttctgtatcg ccagggccag 240
tggcgcgcat attttaaacg cttacgcgat gaagctccag ttcattatca gaaaaatagc 300
ccatttggtc cgttttggag cgtgacccgc tttgaggaca ttctgtttgt ggataaatca 360
catgatctgt ttagcgccga accacagatc atcttaggtg atcctccgga aggcctgtca 420
gtggaaatgt ttattgcgat ggaccctcct aaacatgatg tgcagcgctc tagtgttcag 480
ggtgtggttg cccctaaaaa tctgaaagaa atggaaggcc tgattcgtag tcgtacgggc 540
gatgtgttag attcattacc gacggataaa ccgtttaatt gggttcctgc ggtgagcaaa 600
gaactgacgg gtagaatgct ggctacctta ctggattttc cgtatgaaga acgtcataaa 660
ctggttgaat ggagcgatcg catggccggt gcggcaagtg ctacgggcgg cgaatttgcg 720
gatgaaaatg ctatgtttga tgatgcggca gatatggcac gctctttttc tcgcctgtgg 780
cgcgataaag aagcccgccg tgcagcaggc gaagaaccgg gctttgattt aatctcactg 840
ctacagtcta ataaagaaac caaggatctg atcaatcgtc ctatggaatt tattggcaat 900
ctgaccctgc tgattgtggg cggtaatgat acgacccgca atagcatgtc aggcggctta 960
gttgccatga atgaatttcc tcgtgaattt gaaaaactga aagccaaacc ggaactgatt 1020
ccgaatatgg tgagcgaaat tattcgttgg cagacaccac tggcctatat gcgccgcatt 1080
gccaaacagg atgttgaact gggcggtcag accatcaaaa aaggtgatcg cgttgttatg 1140
tggtatgcct caggtaatcg cgatgaacgt aaatttgata atccggatca gtttattatc 1200
gatcgtaaag atgcacgcaa tcacatgtct tttggctatg gtgttcatcg ctgtatgggt 1260
aatcgtctgg ccgaattaca gctgcgtatt ctgtgggaag aaatcttaaa acgctttgat 1320
aatatcgaag ttgtggaaga accagaacgt gtgcagagca attttgttcg cggctatagc 1380
cgcttaatgg ttaaactgac acctaatagt atgggcggca ttccttcacc aagccgagag 1440
cagtcagcta aaaaagagcg caaaaccgta gaaaacgctc ataatacgcc gcttcttgtg 1500
ctatacggtt caaatatggg aacagccgaa ggaacggcgc gtgatttagc ggatattgcg 1560
atgagcaaag gattcgcacc gcaagtcgca acgcttgatt cccacgcagg aaaccttccg 1620
cgtgaaggag ctgttttaat tgtaacggct tcttataacg gtcatcctcc tgataacgca 1680
aaggaatttg ttgactggtt agaccaagcg tctgctgatg aagtaaaagg cgtgcgctac 1740
tccgtatttg gatgcggtga taaaaactgg gcgacaacgt atcaaaaagt gcctgctttt 1800
attgatgaaa ctcttgccgc taaaggggca gaaaacatag ctgaacgcgg tgaagcagat 1860
gcaagcgacg actttgaagg cacatacgaa gaatggcgtg aacacatgtg gagtgactta 1920
gcagcctact ttaacttaga cattgaaaac agcgaagaaa atgcgtctac gctttcactt 1980
caatttgtcg acagcgctgc ggacatgccg cttgcgaaaa tgcaccgtgc gttttcagca 2040
aacgtcgtag caagcaaaga gcttcaaaag ccaggcagtg cacgaagcac gcgtcatctt 2100
gaaattgaac ttccaaaaga agcttcttat caagaaggag atcatttagg tgttattcct 2160
cgcaactatg aaggaatagt aaatcgtgta gcaacaagat ttggtctaga tgcatcacag 2220
caaatccgtt tggaagctga agaagaaaaa ttagctcatt tgccactcgg taaaacagta 2280
tcagtagaag agcttctgca atacgtggag cttcaagatc ctgttacgcg cacgcagctt 2340
cgcgcaatgg ctgctaaaac agtctgcccg ccgcataaag tagagcttga agtcttgctt 2400
gaaaagcagg cgtacaaaga acaagtgctg gcaaaacgtt taacaatgct tgaactgctt 2460
gaaaaatatc cggcgtgtga aatggaattc agcgaattta tcgcacttct tccaagcatg 2520
cgtccgcgct attactcaat ttcttcatca cctcgtgtcg atgaaaaaca agcaagcatc 2580
acggtcagcg ttgtttcagg agaagcgtgg agcggatacg gagaatacaa aggaattgca 2640
tcgaactatc ttgccaatct gcaagaagga gatacgatta cgtgctttgt ttccacaccg 2700
cagtcaggat ttacgctgcc aaaaggccct gaaacaccac ttatcatggt aggaccggga 2760
acaggcgtcg cgccgtttag aggctttgtg caggctcgca agcagttaaa agaacaagga 2820
cagtcgcttg gagaagcgca tttatacttt ggctgccgtt cacctcatga agattatctg 2880
tatcaaaaag agcttgaaaa cgcccaaaat gaaggcatca ttacgcttca taccgctttt 2940
tctcgcgtac caaatcagcc gaaaacatac gttcaacacg tgatggaaca agacggcaag 3000
aaattgattg aacttcttga ccaaggagcg cacttctata tttgcggaga cggaagccaa 3060
atggcacctg acgttgaagc aacgcttatg aaaagctatg ctgaagttca ccaagtgagt 3120
gaagcagacg ctcgcttatg gctgcagcag ctagaagaaa agggccgata cgcaaaagac 3180
gtgtgggctg ggtaa 3195
<210> 4
<211> 3195
<212> DNA
<213> 人工序列
<400> 4
atgccgacgt taccacgtac ctttgatgac attcagtctc gcttaatcaa tgctacaagt 60
cgtgtggttc caatgcagcg tcagattcag ggtctgaaat ttctgatgag tgccaaacgc 120
aaaacctttg gtccacgtcg cccaatgccg gaatttgtgg aaacacctat cccggatgtt 180
aatacattag ccttagagga cattgatgtg agtaatccgt ttctgtatcg ccagggccag 240
tggcgcgcat attttaaacg cttacgcgat gaagctccag ttcattatca gaaaaatagc 300
ccatttggtc cgttttggag cgtgacccgc tttgaggaca ttctgtttgt ggataaatca 360
catgatctgt ttagcgccga accacagatc atcttaggtg atcctccgga aggcctgtca 420
gtggaaatgt ttattgcgat ggaccctcct aaacatgatg tgcagcgctc tagtgttcag 480
ggtgtggttg cccctaaaaa tctgaaagaa atggaaggcc tgattcgtag tcgtacgggc 540
gatgtgttag attcattacc gacggataaa ccgtttaatt gggttcctgc ggtgagcaaa 600
gaactgacgg gtagaatgct ggctacctta ctggattttc cgtatgaaga acgtcataaa 660
ctggttgaat ggagcgatcg catgtccggt gcggcaagtg ctacgggcgg cgaatttgcg 720
gatgaaaatg ctatgtttga tgatgcggca gatatggcac gctctttttc tcgcctgtgg 780
cgcgataaag aagcccgccg tgcagcaggc gaagaaccgg gctttgattt aatctcactg 840
ctacagtcta ataaagaaac caaggatctg atcaatcgtc ctatggaatt tattggcaat 900
ctgaccctgc tgattgtggg cggtaatgat acgacccgca atagcatgtc aggcggctta 960
gttgccatga atgaatttcc tcgtgaattt gaaaaactga aagccaaacc ggaactgatt 1020
ccgaatatgg tgagcgaaat tattcgttgg cagacaccac tggcctatat gcgccgcatt 1080
gccaaacagg atgttgaact gggcggtcag accatcaaaa aaggtgatcg cgttgttatg 1140
tggtatgcct caggtaatcg cgatgaacgt aaatttgata atccggatca gtttattatc 1200
gatcgtaaag atgcacgcaa tcacatgtct tttggctatg gtgttcatcg ctgtatgggt 1260
aatcgtctgg ccgaattaca gctgcgtatt ctgtgggaag aaatcttaaa acgctttgat 1320
aatatcgaag ttgtggaaga accagaacgt gtgcagagca attttgttcg cggctatagc 1380
cgcttaatgg ttaaactgac acctaatagt atgggcggca ttccttcacc aagccgagag 1440
cagtcagcta aaaaagagcg caaaaccgta gaaaacgctc ataatacgcc gcttcttgtg 1500
ctatacggtt caaatatggg aacagccgaa ggaacggcgc gtgatttagc ggatattgcg 1560
atgagcaaag gattcgcacc gcaagtcgca acgcttgatt cccacgcagg aaaccttccg 1620
cgtgaaggag ctgttttaat tgtaacggct tcttataacg gtcatcctcc tgataacgca 1680
aaggaatttg ttgactggtt agaccaagcg tctgctgatg aagtaaaagg cgtgcgctac 1740
tccgtatttg gatgcggtga taaaaactgg gcgacaacgt atcaaaaagt gcctgctttt 1800
attgatgaaa ctcttgccgc taaaggggca gaaaacatag ctgaacgcgg tgaagcagat 1860
gcaagcgacg actttgaagg cacatacgaa gaatggcgtg aacacatgtg gagtgactta 1920
gcagcctact ttaacttaga cattgaaaac agcgaagaaa atgcgtctac gctttcactt 1980
caatttgtcg acagcgctgc ggacatgccg cttgcgaaaa tgcaccgtgc gttttcagca 2040
aacgtcgtag caagcaaaga gcttcaaaag ccaggcagtg cacgaagcac gcgtcatctt 2100
gaaattgaac ttccaaaaga agcttcttat caagaaggag atcatttagg tgttattcct 2160
cgcaactatg aaggaatagt aaatcgtgta gcaacaagat ttggtctaga tgcatcacag 2220
caaatccgtt tggaagctga agaagaaaaa ttagctcatt tgccactcgg taaaacagta 2280
tcagtagaag agcttctgca atacgtggag cttcaagatc ctgttacgcg cacgcagctt 2340
cgcgcaatgg ctgctaaaac agtctgcccg ccgcataaag tagagcttga agtcttgctt 2400
gaaaagcagg cgtacaaaga acaagtgctg gcaaaacgtt taacaatgct tgaactgctt 2460
gaaaaatatc cggcgtgtga aatggaattc agcgaattta tcgcacttct tccaagcatg 2520
cgtccgcgct attactcaat ttcttcatca cctcgtgtcg atgaaaaaca agcaagcatc 2580
acggtcagcg ttgtttcagg agaagcgtgg agcggatacg gagaatacaa aggaattgca 2640
tcgaactatc ttgccaatct gcaagaagga gatacgatta cgtgctttgt ttccacaccg 2700
cagtcaggat ttacgctgcc aaaaggccct gaaacaccac ttatcatggt aggaccggga 2760
acaggcgtcg cgccgtttag aggctttgtg caggctcgca agcagttaaa agaacaagga 2820
cagtcgcttg gagaagcgca tttatacttt ggctgccgtt cacctcatga agattatctg 2880
tatcaaaaag agcttgaaaa cgcccaaaat gaaggcatca ttacgcttca taccgctttt 2940
tctcgcgtac caaatcagcc gaaaacatac gttcaacacg tgatggaaca agacggcaag 3000
aaattgattg aacttcttga ccaaggagcg cacttctata tttgcggaga cggaagccaa 3060
atggcacctg acgttgaagc aacgcttatg aaaagctatg ctgaagttca ccaagtgagt 3120
gaagcagacg ctcgcttatg gctgcagcag ctagaagaaa agggccgata cgcaaaagac 3180
gtgtgggctg ggtaa 3195
<210> 5
<211> 1064
<212> PRT
<213> 人工序列
<400> 5
Met Pro Thr Leu Pro Arg Thr Phe Asp Asp Ile Gln Ser Arg Leu Ile
1 5 10 15
Asn Ala Thr Ser Arg Val Val Pro Met Gln Arg Gln Ile Gln Gly Leu
20 25 30
Lys Phe Leu Met Ser Ala Lys Arg Lys Thr Phe Gly Pro Arg Arg Pro
35 40 45
Met Pro Glu Phe Val Glu Thr Pro Ile Pro Asp Val Asn Thr Leu Ala
50 55 60
Leu Glu Asp Ile Asp Val Ser Asn Pro Phe Leu Tyr Arg Gln Gly Gln
65 70 75 80
Trp Arg Ala Tyr Phe Lys Arg Leu Arg Asp Glu Ala Pro Val His Tyr
85 90 95
Gln Lys Asn Ser Pro Phe Gly Pro Phe Trp Ser Val Thr Arg Phe Glu
100 105 110
Asp Ile Leu Phe Val Asp Lys Ser His Asp Leu Phe Ser Ala Glu Pro
115 120 125
Gln Ile Ile Leu Gly Asp Pro Pro Glu Gly Leu Ser Val Glu Met Phe
130 135 140
Ile Ala Met Asp Pro Pro Lys His Asp Val Gln Arg Ser Ser Val Gln
145 150 155 160
Gly Val Val Ala Pro Lys Asn Leu Lys Glu Met Glu Gly Leu Ile Arg
165 170 175
Ser Arg Thr Gly Asp Val Leu Asp Ser Leu Pro Thr Asp Lys Pro Phe
180 185 190
Asn Trp Val Pro Ala Val Ser Lys Glu Leu Thr Gly Arg Met Leu Ala
195 200 205
Thr Leu Leu Asp Phe Pro Tyr Glu Glu Arg His Lys Leu Val Glu Trp
210 215 220
Ser Asp Arg Met Ala Gly Ala Ala Ser Ala Thr Gly Gly Glu Phe Ala
225 230 235 240
Asp Glu Asn Ala Met Phe Asp Asp Ala Ala Asp Met Ala Arg Ser Phe
245 250 255
Ser Arg Leu Trp Arg Asp Lys Glu Ala Arg Arg Ala Ala Gly Glu Glu
260 265 270
Pro Gly Phe Asp Leu Ile Ser Leu Leu Gln Ser Asn Lys Glu Thr Lys
275 280 285
Asp Leu Ile Asn Arg Pro Met Glu Phe Ile Gly Asn Leu Thr Leu Leu
290 295 300
Ile Val Gly Gly Asn Asp Thr Thr Arg Asn Ser Met Ser Gly Gly Leu
305 310 315 320
Val Ala Met Asn Glu Phe Pro Arg Glu Phe Glu Lys Leu Lys Ala Lys
325 330 335
Pro Glu Leu Ile Pro Asn Met Val Ser Glu Ile Ile Arg Trp Gln Thr
340 345 350
Pro Leu Ala Tyr Met Arg Arg Ile Ala Lys Gln Asp Val Glu Leu Gly
355 360 365
Gly Gln Thr Ile Lys Lys Gly Asp Arg Val Val Met Trp Tyr Ala Ser
370 375 380
Gly Asn Arg Asp Glu Arg Lys Phe Asp Asn Pro Asp Gln Phe Ile Ile
385 390 395 400
Asp Arg Lys Asp Ala Arg Asn His Met Ser Phe Gly Tyr Gly Val His
405 410 415
Arg Cys Met Gly Asn Arg Leu Ala Glu Leu Gln Leu Arg Ile Leu Trp
420 425 430
Glu Glu Ile Leu Lys Arg Phe Asp Asn Ile Glu Val Val Glu Glu Pro
435 440 445
Glu Arg Val Gln Ser Asn Phe Val Arg Gly Tyr Ser Arg Leu Met Val
450 455 460
Lys Leu Thr Pro Asn Ser Met Gly Gly Ile Pro Ser Pro Ser Arg Glu
465 470 475 480
Gln Ser Ala Lys Lys Glu Arg Lys Thr Val Glu Asn Ala His Asn Thr
485 490 495
Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr
500 505 510
Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln
515 520 525
Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala
530 535 540
Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala
545 550 555 560
Lys Glu Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys
565 570 575
Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr
580 585 590
Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys
595 600 605
Gly Ala Glu Asn Ile Ala Glu Arg Gly Glu Ala Asp Ala Ser Asp Asp
610 615 620
Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Leu
625 630 635 640
Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Glu Asn Ala Ser
645 650 655
Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala
660 665 670
Lys Met His Arg Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu
675 680 685
Gln Lys Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu Leu
690 695 700
Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro
705 710 715 720
Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Ala Thr Arg Phe Gly Leu
725 730 735
Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala
740 745 750
His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr
755 760 765
Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala
770 775 780
Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Val Leu Leu
785 790 795 800
Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met
805 810 815
Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu
820 825 830
Phe Ile Ala Leu Leu Pro Ser Met Arg Pro Arg Tyr Tyr Ser Ile Ser
835 840 845
Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val
850 855 860
Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala
865 870 875 880
Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe
885 890 895
Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Gly Pro Glu Thr
900 905 910
Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly
915 920 925
Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly
930 935 940
Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu
945 950 955 960
Tyr Gln Lys Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu
965 970 975
His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln
980 985 990
His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln
995 1000 1005
Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro
1010 1015 1020
Asp Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Glu Val His Gln
1025 1030 1035
Val Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu
1040 1045 1050
Lys Gly Arg Tyr Ala Lys Asp Val Trp Ala Gly
1055 1060
<210> 6
<211> 1064
<212> PRT
<213> 人工序列
<400> 6
Met Pro Thr Leu Pro Arg Thr Phe Asp Asp Ile Gln Ser Arg Leu Ile
1 5 10 15
Asn Ala Thr Ser Arg Val Val Pro Met Gln Arg Gln Ile Gln Gly Leu
20 25 30
Lys Phe Leu Met Ser Ala Lys Arg Lys Thr Phe Gly Pro Arg Arg Pro
35 40 45
Met Pro Glu Phe Val Glu Thr Pro Ile Pro Asp Val Asn Thr Leu Ala
50 55 60
Leu Glu Asp Ile Asp Val Ser Asn Pro Phe Leu Tyr Arg Gln Gly Gln
65 70 75 80
Trp Arg Ala Tyr Phe Lys Arg Leu Arg Asp Glu Ala Pro Val His Tyr
85 90 95
Gln Lys Asn Ser Pro Phe Gly Pro Phe Trp Ser Val Thr Arg Phe Glu
100 105 110
Asp Ile Leu Phe Val Asp Lys Ser His Asp Leu Phe Ser Ala Glu Pro
115 120 125
Gln Ile Ile Leu Gly Asp Pro Pro Glu Gly Leu Ser Val Glu Met Phe
130 135 140
Ile Ala Met Asp Pro Pro Lys His Asp Val Gln Arg Ser Ser Val Gln
145 150 155 160
Gly Val Val Ala Pro Lys Asn Leu Lys Glu Met Glu Gly Leu Ile Arg
165 170 175
Ser Arg Thr Gly Asp Val Leu Asp Ser Leu Pro Thr Asp Lys Pro Phe
180 185 190
Asn Trp Val Pro Ala Val Ser Lys Glu Leu Thr Gly Arg Met Leu Ala
195 200 205
Thr Leu Leu Asp Phe Pro Tyr Glu Glu Arg His Lys Leu Val Glu Trp
210 215 220
Ser Asp Arg Met Ser Gly Ala Ala Ser Ala Thr Gly Gly Glu Phe Ala
225 230 235 240
Asp Glu Asn Ala Met Phe Asp Asp Ala Ala Asp Met Ala Arg Ser Phe
245 250 255
Ser Arg Leu Trp Arg Asp Lys Glu Ala Arg Arg Ala Ala Gly Glu Glu
260 265 270
Pro Gly Phe Asp Leu Ile Ser Leu Leu Gln Ser Asn Lys Glu Thr Lys
275 280 285
Asp Leu Ile Asn Arg Pro Met Glu Phe Ile Gly Asn Leu Thr Leu Leu
290 295 300
Ile Val Gly Gly Asn Asp Thr Thr Arg Asn Ser Met Ser Gly Gly Leu
305 310 315 320
Val Ala Met Asn Glu Phe Pro Arg Glu Phe Glu Lys Leu Lys Ala Lys
325 330 335
Pro Glu Leu Ile Pro Asn Met Val Ser Glu Ile Ile Arg Trp Gln Thr
340 345 350
Pro Leu Ala Tyr Met Arg Arg Ile Ala Lys Gln Asp Val Glu Leu Gly
355 360 365
Gly Gln Thr Ile Lys Lys Gly Asp Arg Val Val Met Trp Tyr Ala Ser
370 375 380
Gly Asn Arg Asp Glu Arg Lys Phe Asp Asn Pro Asp Gln Phe Ile Ile
385 390 395 400
Asp Arg Lys Asp Ala Arg Asn His Met Ser Phe Gly Tyr Gly Val His
405 410 415
Arg Cys Met Gly Asn Arg Leu Ala Glu Leu Gln Leu Arg Ile Leu Trp
420 425 430
Glu Glu Ile Leu Lys Arg Phe Asp Asn Ile Glu Val Val Glu Glu Pro
435 440 445
Glu Arg Val Gln Ser Asn Phe Val Arg Gly Tyr Ser Arg Leu Met Val
450 455 460
Lys Leu Thr Pro Asn Ser Met Gly Gly Ile Pro Ser Pro Ser Arg Glu
465 470 475 480
Gln Ser Ala Lys Lys Glu Arg Lys Thr Val Glu Asn Ala His Asn Thr
485 490 495
Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly Thr
500 505 510
Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro Gln
515 520 525
Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly Ala
530 535 540
Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn Ala
545 550 555 560
Lys Glu Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val Lys
565 570 575
Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala Thr
580 585 590
Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala Lys
595 600 605
Gly Ala Glu Asn Ile Ala Glu Arg Gly Glu Ala Asp Ala Ser Asp Asp
610 615 620
Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp Leu
625 630 635 640
Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Glu Asn Ala Ser
645 650 655
Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu Ala
660 665 670
Lys Met His Arg Ala Phe Ser Ala Asn Val Val Ala Ser Lys Glu Leu
675 680 685
Gln Lys Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu Leu
690 695 700
Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile Pro
705 710 715 720
Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Ala Thr Arg Phe Gly Leu
725 730 735
Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu Ala
740 745 750
His Leu Pro Leu Gly Lys Thr Val Ser Val Glu Glu Leu Leu Gln Tyr
755 760 765
Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met Ala
770 775 780
Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Val Leu Leu
785 790 795 800
Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr Met
805 810 815
Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Glu Phe Ser Glu
820 825 830
Phe Ile Ala Leu Leu Pro Ser Met Arg Pro Arg Tyr Tyr Ser Ile Ser
835 840 845
Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser Val
850 855 860
Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile Ala
865 870 875 880
Ser Asn Tyr Leu Ala Asn Leu Gln Glu Gly Asp Thr Ile Thr Cys Phe
885 890 895
Val Ser Thr Pro Gln Ser Gly Phe Thr Leu Pro Lys Gly Pro Glu Thr
900 905 910
Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly
915 920 925
Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu Gly
930 935 940
Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr Leu
945 950 955 960
Tyr Gln Lys Glu Leu Glu Asn Ala Gln Asn Glu Gly Ile Ile Thr Leu
965 970 975
His Thr Ala Phe Ser Arg Val Pro Asn Gln Pro Lys Thr Tyr Val Gln
980 985 990
His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp Gln
995 1000 1005
Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro
1010 1015 1020
Asp Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Glu Val His Gln
1025 1030 1035
Val Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu
1040 1045 1050
Lys Gly Arg Tyr Ala Lys Asp Val Trp Ala Gly
1055 1060
<210> 7
<211> 1410
<212> DNA
<213> 人工序列
<400> 7
atgccgacgt taccacgtac ctttgatgac attcagtctc gcttaatcaa tgctacaagt 60
cgtgtggttc caatgcagcg tcagattcag ggtctgaaat ttctgatgag tgccaaacgc 120
aaaacctttg gtccacgtcg cccaatgccg gaatttgtgg aaacacctat cccggatgtt 180
aatacattag ccttagagga cattgatgtg agtaatccgt ttctgtatcg ccagggccag 240
tggcgcgcat attttaaacg cttacgcgat gaagctccag ttcattatca gaaaaatagc 300
ccatttggtc cgttttggag cgtgacccgc tttgaggaca ttctgtttgt ggataaatca 360
catgatctgt ttagcgccga accacagatc atcttaggtg atcctccgga aggcctgtca 420
gtggaaatgt ttattgcgat ggaccctcct aaacatgatg tgcagcgctc tagtgttcag 480
ggtgtggttg cccctaaaaa tctgaaagaa atggaaggcc tgattcgtag tcgtacgggc 540
gatgtgttag attcattacc gacggataaa ccgtttaatt gggttcctgc ggtgagcaaa 600
gaactgacgg gtagaatgct ggctacctta ctggattttc cgtatgaaga acgtcataaa 660
ctggttgaat ggagcgatcg catgtccggt gcggcaagtg ctacgggcgg cgaatttgcg 720
gatgaaaatg ctatgtttga tgatgcggca gatatggcac gctctttttc tcgcctgtgg 780
cgcgataaag aagcccgccg tgcagcaggc gaagaaccgg gctttgattt aatctcactg 840
ctacagtcta ataaagaaac caaggatctg atcaatcgtc ctatggaatt tattggcaat 900
ctgaccctgc tgattgtggg cggtaatgat acgacccgca atagcatgtc aggcggctta 960
gttgccatga atgaatttcc tcgtgaattt gaaaaactga aagccaaacc ggaactgatt 1020
ccgaatatgg tgagcgaaat tattcgttgg cagacaccac tggcctatat gcgccgcatt 1080
gccaaacagg atgttgaact gggcggtcag accatcaaaa aaggtgatcg cgttgttatg 1140
tggtatgcct caggtaatcg cgatgaacgt aaatttgata atccggatca gtttattatc 1200
gatcgtaaag atgcacgcaa tcacatgtct tttggctatg gtgttcatcg ctgtatgggt 1260
aatcgtctgg ccgaattaca gctgcgtatt ctgtgggaag aaatcttaaa acgctttgat 1320
aatatcgaag ttgtggaaga accagaacgt gtgcagagca attttgttcg cggctatagc 1380
cgcttaatgg ttaaactgac acctaatagt 1410
<210> 8
<211> 470
<212> PRT
<213> 人工序列
<400> 8
Met Pro Thr Leu Pro Arg Thr Phe Asp Asp Ile Gln Ser Arg Leu Ile
1 5 10 15
Asn Ala Thr Ser Arg Val Val Pro Met Gln Arg Gln Ile Gln Gly Leu
20 25 30
Lys Phe Leu Met Ser Ala Lys Arg Lys Thr Phe Gly Pro Arg Arg Pro
35 40 45
Met Pro Glu Phe Val Glu Thr Pro Ile Pro Asp Val Asn Thr Leu Ala
50 55 60
Leu Glu Asp Ile Asp Val Ser Asn Pro Phe Leu Tyr Arg Gln Gly Gln
65 70 75 80
Trp Arg Ala Tyr Phe Lys Arg Leu Arg Asp Glu Ala Pro Val His Tyr
85 90 95
Gln Lys Asn Ser Pro Phe Gly Pro Phe Trp Ser Val Thr Arg Phe Glu
100 105 110
Asp Ile Leu Phe Val Asp Lys Ser His Asp Leu Phe Ser Ala Glu Pro
115 120 125
Gln Ile Ile Leu Gly Asp Pro Pro Glu Gly Leu Ser Val Glu Met Phe
130 135 140
Ile Ala Met Asp Pro Pro Lys His Asp Val Gln Arg Ser Ser Val Gln
145 150 155 160
Gly Val Val Ala Pro Lys Asn Leu Lys Glu Met Glu Gly Leu Ile Arg
165 170 175
Ser Arg Thr Gly Asp Val Leu Asp Ser Leu Pro Thr Asp Lys Pro Phe
180 185 190
Asn Trp Val Pro Ala Val Ser Lys Glu Leu Thr Gly Arg Met Leu Ala
195 200 205
Thr Leu Leu Asp Phe Pro Tyr Glu Glu Arg His Lys Leu Val Glu Trp
210 215 220
Ser Asp Arg Met Ser Gly Ala Ala Ser Ala Thr Gly Gly Glu Phe Ala
225 230 235 240
Asp Glu Asn Ala Met Phe Asp Asp Ala Ala Asp Met Ala Arg Ser Phe
245 250 255
Ser Arg Leu Trp Arg Asp Lys Glu Ala Arg Arg Ala Ala Gly Glu Glu
260 265 270
Pro Gly Phe Asp Leu Ile Ser Leu Leu Gln Ser Asn Lys Glu Thr Lys
275 280 285
Asp Leu Ile Asn Arg Pro Met Glu Phe Ile Gly Asn Leu Thr Leu Leu
290 295 300
Ile Val Gly Gly Asn Asp Thr Thr Arg Asn Ser Met Ser Gly Gly Leu
305 310 315 320
Val Ala Met Asn Glu Phe Pro Arg Glu Phe Glu Lys Leu Lys Ala Lys
325 330 335
Pro Glu Leu Ile Pro Asn Met Val Ser Glu Ile Ile Arg Trp Gln Thr
340 345 350
Pro Leu Ala Tyr Met Arg Arg Ile Ala Lys Gln Asp Val Glu Leu Gly
355 360 365
Gly Gln Thr Ile Lys Lys Gly Asp Arg Val Val Met Trp Tyr Ala Ser
370 375 380
Gly Asn Arg Asp Glu Arg Lys Phe Asp Asn Pro Asp Gln Phe Ile Ile
385 390 395 400
Asp Arg Lys Asp Ala Arg Asn His Met Ser Phe Gly Tyr Gly Val His
405 410 415
Arg Cys Met Gly Asn Arg Leu Ala Glu Leu Gln Leu Arg Ile Leu Trp
420 425 430
Glu Glu Ile Leu Lys Arg Phe Asp Asn Ile Glu Val Val Glu Glu Pro
435 440 445
Glu Arg Val Gln Ser Asn Phe Val Arg Gly Tyr Ser Arg Leu Met Val
450 455 460
Lys Leu Thr Pro Asn Ser
465 470
<210> 9
<211> 42
<212> DNA
<213> 人工序列
<400> 9
taagaaggag atatacatat gatgccgacg ttaccacgta cc 42
<210> 10
<211> 39
<212> DNA
<213> 人工序列
<400> 10
tgccgcccat actattaggt gtcagtttaa ccattaagc 39
<210> 11
<211> 28
<212> DNA
<213> 人工序列
<400> 11
acctaatagt atgggcggca ttccttca 28
<210> 12
<211> 41
<212> DNA
<213> 人工序列
<400> 12
gtggtggtgg tggtgctcga gttacccagc ccacacgtct t 41