阅读说明：本技术 提高比活的α-淀粉酶突变体BasAmy-2及其编码基因和应用 (Alpha-amylase mutant BasAmy-2 capable of improving specific activity and coding gene and application thereof ) 是由 李阳源 王建荣 黄江 聂金梅 陈丽芝 何小梅 杨玲 黄佳乐 于 2017-01-16 设计创作，主要内容包括：本发明涉及基因工程领域,具体涉及提高比活的α-淀粉酶BasAmy突变体及其编码基因和应用。所述突变位点为：氨基酸序列如SEQ ID NO.6所示的α-淀粉酶BasAmy发生以下突变：L29A,L269F,S270P,A275Y,Q278S,S279Q,D356G和L412C。本发明获得的突变体的比活较原酶提高。(The invention relates to the field of genetic engineering, in particular to an alpha-amylase BasAmy mutant capable of improving specific activity and a coding gene and application thereof. The mutation sites are: the alpha-amylase BasAmy with the amino acid sequence shown as SEQ ID NO.6 has the following mutations: L29A, L269F, S270P, a275Y, Q278S, S279Q, D356G and L412C. The specific activity of the mutant obtained by the invention is improved compared with that of proenzyme.)

提高比活的α-淀粉酶突变体BasAmy-2及其编码基因和应用

本申请是以下发明专利申请的分案申请(申请号：2017100308497，申请日：2017-01-16，发明名称：提高比活的α-淀粉酶BasAmy突变体及其编码基因和应用)

技术领域

本发明涉及基因工程领域，具体涉及提高比活的α-淀粉酶突变体BasAmy-2及其编码基因和应用。

背景技术

α-淀粉酶是一种十分重要的酶制剂，能够从淀粉分子内部随机切开α-1,4糖苷键生成糊精和还原糖。α-淀粉酶广泛应用于食品工业、酿造、发酵和纺织品工业。

α-淀粉酶分布十分广泛，遍及微生物至高等植物。相对于其他来源的α-淀粉酶，微生物来源的α-淀粉酶作用温度广，pH值范围广，生产成本低，因此微生物来源的α-淀粉酶被广泛的应用于各个工业领域。作为微生物α-淀粉酶中最重要的一类，芽孢杆菌α-淀粉酶是目前研究和应用最为广泛。

沙漠芽胞杆菌(Bacillus sonorensis)α-淀粉酶简称BasAmy，是一种中温α-淀粉酶，其作用pH广，适用于食品、造纸、饲料等工业领域。相对于其他芽孢杆菌α-淀粉酶，BasAmy比活低，生产成本高，限制了其在食品、造纸、饲料等工业领域的应用。因此，提高BasAmy的比活，降低其生产成本，是BasAmy工业化应用急需解决的问题。

发明内容

本发明对来源于索诺拉沙漠芽胞杆菌的α-淀粉酶BasAmy进行分子改造，从而提高BasAmy的比活，降低生产成本，为α-淀粉酶BasAmy的工业化应用奠定基础。

本发明的目的是提供提高比活的α-淀粉酶BasAmy突变体。

本发明的再一目的是提供提高比活的α-淀粉酶BasAmy突变体的编码基因。

索诺拉沙漠芽胞杆菌的α-淀粉酶BasAmy的核苷酸序列和氨基酸序列分别如SEQID NO.1和SEQ ID NO.6所示。

本发明采用定点饱和突变的方法对SEQ ID NO.6所示的α-淀粉酶BasAmy的第29位、第267位、第270位、第275位和第350位进行分子改造，经过高通量筛选确定了第29位、第267位、第270位、第275位和第350位这5个位点的最优突变氨基酸。其中29位由L突变为A最好，267位则是S突变为Q，270位则是S突变为P，275位则是A突变为Y，350位则是D突变为G。

同时采用易错PCR技术对SEQ ID NO.1所示的α-淀粉酶BasAmy的核苷酸序列进行改造，从而获得一系列的突变位点。经过高通量筛选获得了6个有效突变体分别为A112R，L269F，K274S，Q278S，S279Q和L412C。

在上述有效突变位点的基础上，分别一一进行组合，最终得到了四个提高比活的Bsamy突变体分别命名为BasAmy-1、BasAmy-2、BasAmy-3和BasAmy-4。这四个突变体的相对比活分别是BasAmy的130％，160％，180％和125％。BasAmy-1、BasAmy-2、BasAmy-3和BasAmy-4突变体的核苷酸序列如SEQ ID NO.2到SEQ ID NO.5所示，氨基酸序列如SEQ IDNO.7到SEQ ID NO.10所示。

其中BasAmy-1包含的突变位点为L29A，A112R，S267Q，K274S，Q278S，S279Q，D356G和L412C。

其中BasAmy-2包含的突变位点为L29A，L269F，S270P，A275Y，Q278S，S279Q，D356G和L412C。

其中BasAmy-3包含的突变位点为L29A，A112R，L269F，K274S，A275Y，Q278S，S279Q和L412C。

其中BasAmy-4包含的突变位点为S267Q，L269F，S270P，K274S，A275Y，Q278S，S279Q和L412C。

本发明通过蛋白理性改造和高通量筛选技术对索诺拉沙漠芽胞杆菌的α-淀粉酶BasAmy进行分子改造，得到了四个比活提高的突变体。为索诺拉沙漠芽胞杆菌的α-淀粉酶的工业化应用奠定基础。

附图说明

图1原始α-淀粉酶和突变体BasAmy-1至BasAmy-4的最适反应pH

图2原始α-淀粉酶和突变体BasAmy-1至BasAmy-4的pH稳定性

图3原始α-淀粉酶和突变体BasAmy-1至BasAmy-4的最适反应温度

图4原始α-淀粉酶和突变体BasAmy-1至BasAmy-4的热稳定性

具体实施方式

以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行；所述试剂和生物材料，如无特殊说明，均可从商业途径获得。

实验材料和试剂：

1、菌株与载体

索诺拉沙漠芽胞杆菌(Bacillus sonorensis)的α-淀粉酶购自中国工业微生物菌种保藏管理中心，菌种编号为10848、大肠杆菌菌株Topl0、毕赤酵母X33、载体pPICzαA，载体pGAPzαA，Zeocin购自Invitrogen公司。

2、酶与试剂盒

Q5高保真Taq酶MIX购自NEB公司，质粒提取，胶纯化，限制性内切酶、试剂盒购自上海生工公司。

3、培养基

大肠杆菌培养基为LB(1％蛋白胨，0.5％酵母提取物，1％NaCl，pH7.0)。LBZ为LB培养基加25ug/mLZeocin。

酵母培养基为YPD(1％酵母提取物，2％蛋白胨，2％葡萄糖)。酵母筛选培养基为YPDZ(YPD+100mg/L zeocin)。

酵母诱导培养基BMGY(I％酵母提取物、2％蛋白胨、1.34％YNB、0.00004％Biotin、1％甘油(V/V))和BMMY(除以0.5％甲醇代替甘油，其余成份相与BMGY相同)。

实施例1、索诺拉沙漠芽胞杆菌(Bacillussonorensis)的α-淀粉酶的克隆

将索诺拉沙漠芽胞杆菌接入LB培养基，培养24小时后，提取其基因组DNA。根据已报道索诺拉沙漠芽胞杆菌α-淀粉酶的序列(Genebank:AOFM01000005.1)设计两条引物(R：5'-CTGAATTCATGGTTTACAAATGCAAACGG-3'和F：5'-CTTCTAGACTATCGTTGGACATAAATCGA-3')用于扩增索诺拉沙漠芽胞杆菌α-淀粉酶基因。将扩增的PCR产物纯化回收，分别连接到表达载体pPICzαA和pPGAPzαA，得到表达载体pPICzαA-Basamy和pGAPzαA-Basamy。

实施例2、理性定点突变

以上述pPICzαA-Basamy为模板，以表中的引物进行PCR扩增，具体地扩增反应体系如下：

Q5高保真Taq酶MIX	23uL
		对应突变体引物	1uL
对应突变体引物	1uL
		pPICzαA-Basamy(20ng)	2uL
加水至	50uL

反应程序如下：

琼脂糖电泳检测PCR扩增结果，纯化回收PCR产物。用限制性内切酶DpnI将原始质粒分解，将分解完的产物才用热激法转入大肠杆菌Top10，通过菌液PCR验证重组转化子，提取验证正确的转化子的质粒进行测序，从而确定相应的突变体。将测序正确的突变体，用SacI线性化，转入毕赤酵母X33。

实施例3、高通量筛选高比活突变菌株

将实施例2中的酵母重组转化子用牙签逐个挑至24孔板，每个孔中加入1mL含有BMGY培养基，30℃，220rpm培养24h左右，离心去上清。再分别加入1.6mL BMMY培养基进行诱导培养。培养24h后，离心取上清，将上述上清液分别取出200μL至96孔板，进行α-淀粉酶酶活测定。α-淀粉酶酶活检测参照中华人民共和国国家标准《GB/T 24401-2009》进行测定。经过高通量筛选得到5个有效突变位点分别为L29A，S267Q，S270P，A275Y和D350G。这5个突变体的相对比活如表1所示。

表1原始α-淀粉酶和突变体α-淀粉酶相对比活

编号	相对比活(％)
		原始α-淀粉酶	100
L29A	115
		S267Q	120
S270P	125
		A275Y	119
D350G	128

实施例4、易错PCR非理性改造

以上述pGAPzαA-Basamy为模板，进行易错PCR随机突变扩增，具体地扩增方法是：

第一轮扩增：以载体启动子引物AOX5-F和AOX3-R为引物进行PCR扩增，反应体系如下：

反应程序如下：

回收第一轮PCR产物，去1μL稀释50-100倍用作第二轮PCR的模板；第二，第三轮易错PCR以α-淀粉酶特异性引物R及F替代引物AOX5-F和AOX3-R为反应引物，重复PCR反应。取第二、三轮的产物用XbaI和EcoRI进行双酶切，连接至pGAPzαA载体上的EcoRI和XbaI位点之间。连接产物转化X33，在YPDZ平板培养筛选突变菌株。经过高通量筛选获得了6个有效突变体分别为A112R，L269F，K274S，Q278S，S279Q和L412C。这6个突变体的相对比活如表2所示。

表2原始α-淀粉酶和突变体α-淀粉酶相对比活

编号	相对比活(％)
		原始α-淀粉酶	100
A112R	121
		L269F	130
K274S	126
		Q278S	131
S279Q	123
		L412C	125

实施例5、组合突变

进行组合突变，通过实验最终得到4个组合突变分别命名为BasAmy-1、BasAmy-2、BasAmy-3、BasAmy-4。

其中BasAmy-1包含的突变位点为L29A，A112R，S267Q，K274S，Q278S，S279Q，D356G和L412C。

其中BasAmy-2包含的突变位点为L29A，L269F，S270P，A275Y，Q278S，S279Q，D356G和L412C。

其中BasAmy-3包含的突变位点为L29A，A112R，L269F，K274S，A275Y，Q278S，S279Q和L412C。

其中BasAmy-4包含的突变位点为S267Q，L269F，S270P，K274S，A275Y，Q278S，S279Q和L412C。

实施例6、原始α-淀粉酶及α-淀粉酶突变体的比活分析

分别将原始α-淀粉酶和突变体α-淀粉酶进行纯化，纯化方法为镍柱纯化。将纯化好的α-淀粉酶和突变体α-淀粉酶分别测定相应的酶活并计算出比活。以突变体比活除以原始α-淀粉酶比活，来计算突变体的相对比活。最终BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的相对比活分别为130％，160％，180％和125％。

实施例7、原始α-淀粉酶及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适反应pH及pH稳定性

参照国标方法测定原始α-淀粉酶BasAmy及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适反应pH。BasAmy及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适反应pH如图1所示。由图1可知，突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适pH值几乎和BaAmy一样，均为6.0。

将BasAmy及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4分别在pH4-8条件下室温处理2小时，然后参照国标的方法测定酶活，结果如图2所示。由图2可知突变体BasAmy-1、BasAmy-2、BasAmy-3和BasAmy-4的pH稳定性与BasAmy一致。

实施例8、原始α-淀粉酶及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适反应温度及热稳定性

参照国标方法测定BasAmy及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适反应温度，结果如图3所示。由图3可知，BasAmy及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的最适反应温度均为60℃。

将BasAmy及突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4分别在50℃-90℃条件下水浴处理30分钟，然后参照国标的方法测定酶活，结果如图4所示。由图4可知，突变体BasAmy-1，BasAmy-2，BasAmy-3和BasAmy-4的热稳定性与BasAmy一致。

序列表

<110> 广东溢多利生物科技股份有限公司

<120> 提高比活的α-淀粉酶突变体BasAmy-2及其编码基因和应用

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gtcaccgcgg ttgaagtgaa tcccggcaat cgaaatcagg aaatatctgg agaatatcga 480

atcaaagcgt ggacaggatt caatttccct ggacgcggca gcacatacag tgattttaaa 540

tggcattggt atcattttga tgggacggat tgggacgaat cccgaaagct gaatcgcatc 600

tacaagttcc gcggagatgg gaaggcatgg gattgggagg tttccagcga aaacggcaac 660

tacgattatt taatgtatgc ggatgtcgat tatgaccacc ccgatgttgt ggcagaaatg 720

aaacggtggg gaacctggta tgcaaaagag cttcaattgg atgggttccg gcttgatgcc 780

gtcaagcata ttaagttctc tttttttcct gattggttga agtatgtgcg ttctcagact 840

ggcaaggaaa tgtttacggt tgcggaatac tggcaaaata accttggaga aatcgaaaac 900

tacttgcaaa aaaccgattt tcaacattct gtattcgatg tgccgcttca ttttaacctt 960

caggccgcat cttcacacgg aggcagctat gatatgagga atttgctgaa cggaacggtt 1020

gtttccaaac atcctttgaa agcggttaca tttgtcgaca accatggtac acagccgggg 1080

caatcattgg agtcgaccgt ccaaacatgg ttcaagccgc ttgcctacgc ttttattttg 1140

acaagagagg ccgggtaccc gcaggttttt tatggagata tgtatgggac aaaaggtcct 1200

acatcgcggg aaattccttc tcttaaaagt aaatgtgagc cgattttgaa agcgcgcaag 1260

tattatgctt atggaacaca gcatgattat ttcgatcatc cagatgccat cggctggacg 1320

agggaaggcg atcaatccgt cgctgcatca ggcttggccg ctttaatcac agacggaccg 1380

ggcggatcaa agcggatgta tgtgggcagg cagcatgccg gtgagacatg gcatgacatc 1440

actgggaacc gttcagattc cgtcgtgatc aattcggacg gctggggaga gttttatgta 1500

aacggcggtt cggtttcgat ttatgtccaa cgatag 1536

<210> 4

<211> 1536

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 4

atggtttaca aatgcaaacg gatattatgt tgtgtgctgc tgtttttcat agtgctgccg 60

gcttctaaaa catatgcggc aagcgctaac ggcacgctga tgcagtattt tgaatggaat 120

ctgcctaatg acggccagca ttggaagcgc ttacaaaatg atgcgggata tttatccgac 180

attgggataa cggctgtttg gattccgccc gcctacaagg gaacgagcca ggctgacgtt 240

ggatacggcc catacgattt gtacgattta ggggagttcc tgcaaaaagg gacggtgcgg 300

acgaaatacg ggatgaaaac agagcttcag tcaagagtcg gttcgcttca ttcccagaac 360

atccaagtgt atggcgatgt tgtccttaat cataaggctg gggcggatct gacggaggat 420

gtcaccgcgg ttgaagtgaa tcccggcaat cgaaatcagg aaatatctgg agaatatcga 480

atcaaagcgt ggacaggatt caatttccct ggacgcggca gcacatacag tgattttaaa 540

tggcattggt atcattttga tgggacggat tgggacgaat cccgaaagct gaatcgcatc 600

tacaagttcc gcggagatgg gaaggcatgg gattgggagg tttccagcga aaacggcaac 660

tacgattatt taatgtatgc ggatgtcgat tatgaccacc ccgatgttgt ggcagaaatg 720

aaacggtggg gaacctggta tgcaaaagag cttcaattgg atgggttccg gcttgatgcc 780

gtcaagcata ttaagttctc ttttttttct gattggttgt cgtatgtgcg ttctcagact 840

ggcaaggaaa tgtttacggt tgcggaatac tggcaaaata accttggaga aatcgaaaac 900

tacttgcaaa aaaccgattt tcaacattct gtattcgatg tgccgcttca ttttaacctt 960

caggccgcat cttcacacgg aggcagctat gatatgagga atttgctgaa cggaacggtt 1020

gtttccaaac atcctttgaa agcggttaca tttgtcgaca accatgacac acagccgggg 1080

caatcattgg agtcgaccgt ccaaacatgg ttcaagccgc ttgcctacgc ttttattttg 1140

acaagagagg ccgggtaccc gcaggttttt tatggagata tgtatgggac aaaaggtcct 1200

acatcgcggg aaattccttc tcttaaaagt aaatgtgagc cgattttgaa agcgcgcaag 1260

tattatgctt atggaacaca gcatgattat ttcgatcatc cagatgccat cggctggacg 1320

agggaaggcg atcaatccgt cgctgcatca ggcttggccg ctttaatcac agacggaccg 1380

ggcggatcaa agcggatgta tgtgggcagg cagcatgccg gtgagacatg gcatgacatc 1440

actgggaacc gttcagattc cgtcgtgatc aattcggacg gctggggaga gttttatgta 1500

aacggcggtt cggtttcgat ttatgtccaa cgatag 1536

<210> 5

<211> 1536

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 5

atggtttaca aatgcaaacg gatattatgt tgtgtgctgc tgtttttcat agtgctgccg 60

gcttctaaaa catatgcggc aagcctgaac ggcacgctga tgcagtattt tgaatggaat 120

ctgcctaatg acggccagca ttggaagcgc ttacaaaatg atgcgggata tttatccgac 180

attgggataa cggctgtttg gattccgccc gcctacaagg gaacgagcca ggctgacgtt 240

ggatacggcc catacgattt gtacgattta ggggagttcc tgcaaaaagg gacggtgcgg 300

acgaaatacg ggatgaaaac agagcttcag tcagctgtcg gttcgcttca ttcccagaac 360

atccaagtgt atggcgatgt tgtccttaat cataaggctg gggcggatct gacggaggat 420

gtcaccgcgg ttgaagtgaa tcccggcaat cgaaatcagg aaatatctgg agaatatcga 480

atcaaagcgt ggacaggatt caatttccct ggacgcggca gcacatacag tgattttaaa 540

tggcattggt atcattttga tgggacggat tgggacgaat cccgaaagct gaatcgcatc 600

tacaagttcc gcggagatgg gaaggcatgg gattgggagg tttccagcga aaacggcaac 660

tacgattatt taatgtatgc ggatgtcgat tatgaccacc ccgatgttgt ggcagaaatg 720

aaacggtggg gaacctggta tgcaaaagag cttcaattgg atgggttccg gcttgatgcc 780

gtcaagcata ttaagttcca gttttttcct gattggttgt cgtatgtgcg ttctcagact 840

ggcaaggaaa tgtttacggt tgcggaatac tggcaaaata accttggaga aatcgaaaac 900

tacttgcaaa aaaccgattt tcaacattct gtattcgatg tgccgcttca ttttaacctt 960

caggccgcat cttcacacgg aggcagctat gatatgagga atttgctgaa cggaacggtt 1020

gtttccaaac atcctttgaa agcggttaca tttgtcgaca accatgacac acagccgggg 1080

caatcattgg agtcgaccgt ccaaacatgg ttcaagccgc ttgcctacgc ttttattttg 1140

acaagagagg ccgggtaccc gcaggttttt tatggagata tgtatgggac aaaaggtcct 1200

acatcgcggg aaattccttc tcttaaaagt aaatgtgagc cgattttgaa agcgcgcaag 1260

tattatgctt atggaacaca gcatgattat ttcgatcatc cagatgccat cggctggacg 1320

agggaaggcg atcaatccgt cgctgcatca ggcttggccg ctttaatcac agacggaccg 1380

ggcggatcaa agcggatgta tgtgggcagg cagcatgccg gtgagacatg gcatgacatc 1440

actgggaacc gttcagattc cgtcgtgatc aattcggacg gctggggaga gttttatgta 1500

aacggcggtt cggtttcgat ttatgtccaa cgatag 1536

<210> 6

<211> 511

<212> PRT

<213> 索诺拉沙漠芽胞杆菌(Bacillus sonorensis)

<400> 6

Met Val Thr Leu Cys Leu Ala Ile Leu Cys Cys Val Leu Leu Pro Pro

1 5 10 15

Ile Val Leu Pro Ala Ser Leu Thr Thr Ala Ala Ser Leu Ala Gly Thr

20 25 30

Leu Met Gly Thr Pro Gly Thr Ala Leu Pro Ala Ala Gly Gly His Thr

35 40 45

Leu Ala Leu Gly Ala Ala Ala Gly Thr Leu Ser Ala Ile Gly Ile Thr

50 55 60

Ala Val Thr Ile Pro Pro Ala Thr Leu Gly Thr Ser Gly Ala Ala Val

65 70 75 80

Gly Thr Gly Pro Thr Ala Leu Thr Ala Leu Gly Gly Pro Leu Gly Leu

85 90 95

Gly Thr Val Ala Thr Leu Thr Gly Met Leu Thr Gly Leu Gly Ser Ala

100 105 110

Val Gly Ser Leu His Ser Gly Ala Ile Gly Val Thr Gly Ala Val Val

115 120 125

Leu Ala His Leu Ala Gly Ala Ala Leu Thr Gly Ala Val Thr Ala Val

130 135 140

Gly Val Ala Pro Gly Ala Ala Ala Gly Gly Ile Ser Gly Gly Thr Ala

145 150 155 160

Ile Leu Ala Thr Thr Gly Pro Ala Pro Pro Gly Ala Gly Ser Thr Thr

165 170 175

Ser Ala Pro Leu Thr His Thr Thr His Pro Ala Gly Thr Ala Thr Ala

180 185 190

Gly Ser Ala Leu Leu Ala Ala Ile Thr Leu Pro Ala Gly Ala Gly Leu

195 200 205

Ala Thr Ala Thr Gly Val Ser Ser Gly Ala Gly Ala Thr Ala Thr Leu

210 215 220

Met Thr Ala Ala Val Ala Thr Ala His Pro Ala Val Val Ala Gly Met

225 230 235 240

Leu Ala Thr Gly Thr Thr Thr Ala Leu Gly Leu Gly Leu Ala Gly Pro

245 250 255

Ala Leu Ala Ala Val Leu His Ile Leu Pro Ser Pro Leu Ser Ala Thr

260 265 270

Leu Leu Ala Val Ala Gly Ser Thr Gly Leu Gly Met Pro Thr Val Ala

275 280 285

Gly Thr Thr Gly Ala Ala Leu Gly Gly Ile Gly Ala Thr Leu Gly Leu

290 295 300

Thr Ala Pro Gly His Ser Val Pro Ala Val Pro Leu His Pro Ala Leu

305 310 315 320

Gly Ala Ala Ser Ser His Gly Gly Ser Thr Ala Met Ala Ala Leu Leu

325 330 335

Ala Gly Thr Val Val Ser Leu His Pro Leu Leu Ala Val Thr Pro Val

340 345 350

Ala Ala His Ala Thr Gly Pro Gly Gly Ser Leu Gly Ser Thr Val Gly

355 360 365

Thr Thr Pro Leu Pro Leu Ala Thr Ala Pro Ile Leu Thr Ala Gly Ala

370 375 380

Gly Thr Pro Gly Val Pro Thr Gly Ala Met Thr Gly Thr Leu Gly Pro

385 390 395 400

Thr Ser Ala Gly Ile Pro Ser Leu Leu Ser Leu Leu Gly Pro Ile Leu

405 410 415

Leu Ala Ala Leu Thr Thr Ala Thr Gly Thr Gly His Ala Thr Pro Ala

420 425 430

His Pro Ala Ala Ile Gly Thr Thr Ala Gly Gly Ala Gly Ser Val Ala

435 440 445

Ala Ser Gly Leu Ala Ala Leu Ile Thr Ala Gly Pro Gly Gly Ser Leu

450 455 460

Ala Met Thr Val Gly Ala Gly His Ala Gly Gly Thr Thr His Ala Ile

465 470 475 480

Thr Gly Ala Ala Ser Ala Ser Val Val Ile Ala Ser Ala Gly Thr Gly

485 490 495

Gly Pro Thr Val Ala Gly Gly Ser Val Ser Ile Thr Val Gly Ala

500 505 510

<210> 7

<211> 511

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 7

Met Val Tyr Lys Cys Lys Arg Ile Leu Cys Cys Val Leu Leu Phe Phe

1 5 10 15

Ile Val Leu Pro Ala Ser Lys Thr Tyr Ala Ala Ser Ala Asn Gly Thr

20 25 30

Leu Met Gln Tyr Phe Glu Trp Asn Leu Pro Asn Asp Gly Gln His Trp

35 40 45

Lys Arg Leu Gln Asn Asp Ala Gly Tyr Leu Ser Asp Ile Gly Ile Thr

50 55 60

Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val

65 70 75 80

Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Leu Gln Lys

85 90 95

Gly Thr Val Arg Thr Lys Tyr Gly Met Lys Thr Glu Leu Gln Ser Arg

100 105 110

Val Gly Ser Leu His Ser Gln Asn Ile Gln Val Tyr Gly Asp Val Val

115 120 125

Leu Asn His Lys Ala Gly Ala Asp Leu Thr Glu Asp Val Thr Ala Val

130 135 140

Glu Val Asn Pro Gly Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Arg

145 150 155 160

Ile Lys Ala Trp Thr Gly Phe Asn Phe Pro Gly Arg Gly Ser Thr Tyr

165 170 175

Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp

180 185 190

Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Arg Gly Asp Gly Lys

195 200 205

Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu

210 215 220

Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Met

225 230 235 240

Lys Arg Trp Gly Thr Trp Tyr Ala Lys Glu Leu Gln Leu Asp Gly Phe

245 250 255

Arg Leu Asp Ala Val Lys His Ile Lys Phe Gln Phe Leu Ser Asp Trp

260 265 270

Leu Ser Ala Val Arg Ser Gln Thr Gly Lys Glu Met Phe Thr Val Ala

275 280 285

Glu Tyr Trp Gln Asn Asn Leu Gly Glu Ile Glu Asn Tyr Leu Gln Lys

290 295 300

Thr Asp Phe Gln His Ser Val Phe Asp Val Pro Leu His Phe Asn Leu

305 310 315 320

Gln Ala Ala Ser Ser His Gly Gly Ser Tyr Asp Met Arg Asn Leu Leu

325 330 335

Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ala Val Thr Phe Val

340 345 350

Asp Asn His Gly Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln

355 360 365

Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala

370 375 380

Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Pro

385 390 395 400

Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Cys Glu Pro Ile Leu

405 410 415

Lys Ala Arg Lys Tyr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp

420 425 430

His Pro Asp Ala Ile Gly Trp Thr Arg Glu Gly Asp Gln Ser Val Ala

435 440 445

Ala Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys

450 455 460

Arg Met Tyr Val Gly Arg Gln His Ala Gly Glu Thr Trp His Asp Ile

465 470 475 480

Thr Gly Asn Arg Ser Asp Ser Val Val Ile Asn Ser Asp Gly Trp Gly

485 490 495

Glu Phe Tyr Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg

500 505 510

<210> 8

<211> 511

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 8

Met Val Tyr Lys Cys Lys Arg Ile Leu Cys Cys Val Leu Leu Phe Phe

1 5 10 15

Ile Val Leu Pro Ala Ser Lys Thr Tyr Ala Ala Ser Ala Asn Gly Thr

20 25 30

Leu Met Gln Tyr Phe Glu Trp Asn Leu Pro Asn Asp Gly Gln His Trp

35 40 45

Lys Arg Leu Gln Asn Asp Ala Gly Tyr Leu Ser Asp Ile Gly Ile Thr

50 55 60

Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val

65 70 75 80

Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Leu Gln Lys

85 90 95

Gly Thr Val Arg Thr Lys Tyr Gly Met Lys Thr Glu Leu Gln Ser Ala

100 105 110

Val Gly Ser Leu His Ser Gln Asn Ile Gln Val Tyr Gly Asp Val Val

115 120 125

Leu Asn His Lys Ala Gly Ala Asp Leu Thr Glu Asp Val Thr Ala Val

130 135 140

Glu Val Asn Pro Gly Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Arg

145 150 155 160

Ile Lys Ala Trp Thr Gly Phe Asn Phe Pro Gly Arg Gly Ser Thr Tyr

165 170 175

Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp

180 185 190

Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Arg Gly Asp Gly Lys

195 200 205

Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu

210 215 220

Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Met

225 230 235 240

Lys Arg Trp Gly Thr Trp Tyr Ala Lys Glu Leu Gln Leu Asp Gly Phe

245 250 255

Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Phe Pro Asp Trp

260 265 270

Leu Lys Tyr Val Arg Ser Gln Thr Gly Lys Glu Met Phe Thr Val Ala

275 280 285

Glu Tyr Trp Gln Asn Asn Leu Gly Glu Ile Glu Asn Tyr Leu Gln Lys

290 295 300

Thr Asp Phe Gln His Ser Val Phe Asp Val Pro Leu His Phe Asn Leu

305 310 315 320

Gln Ala Ala Ser Ser His Gly Gly Ser Tyr Asp Met Arg Asn Leu Leu

325 330 335

Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ala Val Thr Phe Val

340 345 350

Asp Asn His Gly Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln

355 360 365

Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala

370 375 380

Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Pro

385 390 395 400

Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Cys Glu Pro Ile Leu

405 410 415

Lys Ala Arg Lys Tyr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp

420 425 430

His Pro Asp Ala Ile Gly Trp Thr Arg Glu Gly Asp Gln Ser Val Ala

435 440 445

Ala Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys

450 455 460

Arg Met Tyr Val Gly Arg Gln His Ala Gly Glu Thr Trp His Asp Ile

465 470 475 480

Thr Gly Asn Arg Ser Asp Ser Val Val Ile Asn Ser Asp Gly Trp Gly

485 490 495

Glu Phe Tyr Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg

500 505 510

<210> 9

<211> 511

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 9

Met Val Tyr Lys Cys Lys Arg Ile Leu Cys Cys Val Leu Leu Phe Phe

1 5 10 15

Ile Val Leu Pro Ala Ser Lys Thr Tyr Ala Ala Ser Ala Asn Gly Thr

20 25 30

Leu Met Gln Tyr Phe Glu Trp Asn Leu Pro Asn Asp Gly Gln His Trp

35 40 45

Lys Arg Leu Gln Asn Asp Ala Gly Tyr Leu Ser Asp Ile Gly Ile Thr

50 55 60

Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val

65 70 75 80

Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Leu Gln Lys

85 90 95

Gly Thr Val Arg Thr Lys Tyr Gly Met Lys Thr Glu Leu Gln Ser Arg

100 105 110

Val Gly Ser Leu His Ser Gln Asn Ile Gln Val Tyr Gly Asp Val Val

115 120 125

Leu Asn His Lys Ala Gly Ala Asp Leu Thr Glu Asp Val Thr Ala Val

130 135 140

Glu Val Asn Pro Gly Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Arg

145 150 155 160

Ile Lys Ala Trp Thr Gly Phe Asn Phe Pro Gly Arg Gly Ser Thr Tyr

165 170 175

Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp

180 185 190

Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Arg Gly Asp Gly Lys

195 200 205

Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu

210 215 220

Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Met

225 230 235 240

Lys Arg Trp Gly Thr Trp Tyr Ala Lys Glu Leu Gln Leu Asp Gly Phe

245 250 255

Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Phe Ser Asp Trp

260 265 270

Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys Glu Met Phe Thr Val Ala

275 280 285

Glu Tyr Trp Gln Asn Asn Leu Gly Glu Ile Glu Asn Tyr Leu Gln Lys

290 295 300

Thr Asp Phe Gln His Ser Val Phe Asp Val Pro Leu His Phe Asn Leu

305 310 315 320

Gln Ala Ala Ser Ser His Gly Gly Ser Tyr Asp Met Arg Asn Leu Leu

325 330 335

Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ala Val Thr Phe Val

340 345 350

Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln

355 360 365

Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala

370 375 380

Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Pro

385 390 395 400

Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Cys Glu Pro Ile Leu

405 410 415

Lys Ala Arg Lys Tyr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp

420 425 430

His Pro Asp Ala Ile Gly Trp Thr Arg Glu Gly Asp Gln Ser Val Ala

435 440 445

Ala Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys

450 455 460

Arg Met Tyr Val Gly Arg Gln His Ala Gly Glu Thr Trp His Asp Ile

465 470 475 480

Thr Gly Asn Arg Ser Asp Ser Val Val Ile Asn Ser Asp Gly Trp Gly

485 490 495

Glu Phe Tyr Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg

500 505 510

<210> 10

<211> 511

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 10

Met Val Tyr Lys Cys Lys Arg Ile Leu Cys Cys Val Leu Leu Phe Phe

1 5 10 15

Ile Val Leu Pro Ala Ser Lys Thr Tyr Ala Ala Ser Leu Asn Gly Thr

20 25 30

Leu Met Gln Tyr Phe Glu Trp Asn Leu Pro Asn Asp Gly Gln His Trp

35 40 45

Lys Arg Leu Gln Asn Asp Ala Gly Tyr Leu Ser Asp Ile Gly Ile Thr

50 55 60

Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val

65 70 75 80

Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Leu Gln Lys

85 90 95

Gly Thr Val Arg Thr Lys Tyr Gly Met Lys Thr Glu Leu Gln Ser Ala

100 105 110

Val Gly Ser Leu His Ser Gln Asn Ile Gln Val Tyr Gly Asp Val Val

115 120 125

Leu Asn His Lys Ala Gly Ala Asp Leu Thr Glu Asp Val Thr Ala Val

130 135 140

Glu Val Asn Pro Gly Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Arg

145 150 155 160

Ile Lys Ala Trp Thr Gly Phe Asn Phe Pro Gly Arg Gly Ser Thr Tyr

165 170 175

Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp

180 185 190

Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Arg Gly Asp Gly Lys

195 200 205

Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu

210 215 220

Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Met

225 230 235 240

Lys Arg Trp Gly Thr Trp Tyr Ala Lys Glu Leu Gln Leu Asp Gly Phe

245 250 255

Arg Leu Asp Ala Val Lys His Ile Lys Phe Gln Phe Phe Pro Asp Trp

260 265 270

Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys Glu Met Phe Thr Val Ala

275 280 285

Glu Tyr Trp Gln Asn Asn Leu Gly Glu Ile Glu Asn Tyr Leu Gln Lys

290 295 300

Thr Asp Phe Gln His Ser Val Phe Asp Val Pro Leu His Phe Asn Leu

305 310 315 320

Gln Ala Ala Ser Ser His Gly Gly Ser Tyr Asp Met Arg Asn Leu Leu

325 330 335

Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ala Val Thr Phe Val

340 345 350

Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln

355 360 365

Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala

370 375 380

Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Pro

385 390 395 400

Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Cys Glu Pro Ile Leu

405 410 415

Lys Ala Arg Lys Tyr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp

420 425 430

His Pro Asp Ala Ile Gly Trp Thr Arg Glu Gly Asp Gln Ser Val Ala

435 440 445

Ala Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys

450 455 460

Arg Met Tyr Val Gly Arg Gln His Ala Gly Glu Thr Trp His Asp Ile

465 470 475 480

Thr Gly Asn Arg Ser Asp Ser Val Val Ile Asn Ser Asp Gly Trp Gly

485 490 495

Glu Phe Tyr Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg

500 505 510