具体实施方式
以下实施例中,莱鲍迪苷D采用液相色谱检测,HPLC在戴安U3000液相色谱仪上进行,色谱分析条件如下:
色谱柱:COSMOSIL-C18250×4.6mm;流动相为0.1%甲酸:乙腈(70:30;V:V);流速1min/mL;柱温55℃;检测波长:210nm。
莱鲍迪苷D标准品购自南京植佰萃生物科技有限公司。
实施例1糖基转移酶UGTZJ1突变体的构建
本实施例提供的糖基转移酶UGTZJ1突变体,其氨基酸序列如SEQ ID NO.2-4任一所示。
编码上述突变体的基因。
上述糖基转移酶UGTZJ1突变体的制备方法,将SEQ ID NO.1所示的糖基转移酶UGTZJ1的氨基酸序列中第48位天冬酰胺突变为苏氨酸或甘氨酸,或将第75位甘氨酸突变为组氨酸。
还可以是携带上述基因的载体或细胞系以及基因工程菌。
该基因工程菌,以大肠杆菌为宿主,同时表达蔗糖合酶基因和上述的糖基转移酶UGTZJ1突变体。
本发明所述的蔗糖合酶基因可以是任意来源的具有蔗糖合酶活性的基因,例如马铃薯来源蔗糖合酶StSUS1基因(核苷酸序列如SEQ ID NO.5)或者拟南芥菜来源的蔗糖合酶基因PSsAt(氨基酸序列如SEQ ID NO.6所示)。
本实施例以大肠杆菌为宿主,同时表达马铃薯来源蔗糖合酶StSUS1基因和上述的糖基转移酶UGTZJ1突变体为例,该基因工程菌的制备方法,包括以下步骤:
(1)将Ziziphus jujuba来源的糖基转移酶UGTZJ1(氨基酸序列如SEQ ID NO.1所示,NCBI登录号XP_015885904.1野生型序列对应的氨基酸),由南京金斯瑞公司合成得到重组表达载体pET28a-UGTZJ1,再将马铃薯来源蔗糖合酶StSUS1基因,经密码子优化后克隆到重组表达载体pET28a-UGTZJ1上得到双酶共表达重组载体pET28a-UGTZJ1-StSUS1;
(2)由结构比对确定突变位点,设计定点突变的突变引物,以携带编码SEQ IDNO.1所示的糖基转移酶UGTZJ1突变体的基因的重组载体pET28a-UGTZJ1-StSUS1为模板,采用基于一步PCR介导的定点突变方法对编码糖基转移酶UGTZJ1突变体的基因进行定点突变;
(3)将突变后的重组载体转化进宿主细胞E.coli BL21(DE3);
(4)分别将测序正确成功突变的菌株接种于10mL的LB培养基中,200r/min摇床,37℃培养8h。取上述菌液按4%(V/V)的接种量,接种至100mL TB,37℃培养当菌体长至OD600为0.2时,添加IPTG至0.01mmo l/L,16℃继续诱导24h后,发酵液8000rpm、4℃离心15min,除去菌体,上清即为粗酶液(含有基因工程菌)。
本实施例利用快速PCR技术,以包含重组表达载体pET28a-UGTZJ1-StSUS1的基因工程菌E.coli BL21(DE3)/pET28a-UGTZJ1-StSUS1的野生型UGTZJ1基因序列(NCBI数据库登陆号:XP_015885904.1)为模板,分别设计并合成引入N48T、N48G和G75H的突变的引物对糖基转移酶野生型UGTZJ1基因进行定点突变(下划线为突变碱基)。分别得到了包含UGTZJ1突变体的基因工程菌E.coli BL21(DE3)/pET28a-UGTZJ1(N48T)-StSUS1、pET28a-UGTZJ1(N48G)-StSUS1、pET28a-UGTZJ1(G75H)-StSUS1。
引入序列N48T突变的定点突变引物为:
正向引物:5'-CTCACACCAGTTACACCTCCCCCAACCCTGAAACCC-3'(SEQ ID NO:7所示)。
反向引物:5'-TCAGGGTTGGGGGAGGTGTAACTGGTGTGAGCAAT-3'(SEQ ID NO:8所示)。
引入序列N48G突变的定点突变引物为:
正向引物:5'-TCACACCAGTTACGGCTCCCCCAACCCTGAAACCC-3'(SEQ ID NO:9所示)
反向引物:5'-TCAGGGTTGGGGGAGCCGTAACTGGTGTGAGCAAT-3'(SEQ ID NO:10所示)
引入序列G75H突变的定点突变引物为;
正向引物:5'-CGTGATATCTCGTCTCACGATTTGATATCTCTTGTA-3'(SEQ ID NO:11所示)
反向引物:5'-AAGAGATATCAAATCGTGAGACGAGATATCACGGTC-3'(SEQ ID NO:12所示)。
实施例2基因工程菌的诱导表达及粗酶液制备
将实施例1中出现的4株分别含有野生型UGTZJ1基因及其突变体基因的不同的基因工程菌分别接种到含有卡那霉素的5mL LB液体培养基(LB(g/L):蛋白胨10,氯化钠10,酵母提取物5)的50mL的摇管中,在摇床上37℃恒温培养8h,转速200rpm。再将培养菌液按4%接种量接入含有100mL诱导培养基TB中(TB(g/L):酵母粉25,胰蛋白胨15,氯化钠10,葡萄糖2,乳糖3)的500mL摇瓶中,于200rpm,37℃培养2h,待OD600达到0.2左右时转16℃诱导24h离心收集菌体。超声破菌,离心取上清液为粗酶液,置于4℃冰箱待用。
在50mL三角瓶中加入10mL的反应混合物,包含20g/L莱鲍迪苷A、60g/L蔗糖、50mM磷酸钠缓冲调节混合物的pH为7.0,然后分别加入3mg/mL源自4株基因工程菌的粗酶液,反应在30℃、200rpm条件下进行,反应2-24h后取样于95℃加热5min灭活酶,离心取上清,采用高效液相色谱(HPLC)检测莱鲍迪苷浓度,结果见表1。
表1不同菌株对莱鲍迪苷D的合成量
结果以采用基因工程菌E.coli BL21(DE3)/pET28a-UGTZJ1(N48T)-StSUS1诱导表达后获取粗酶液反应的反应液中莱鲍迪苷D的产量最高,为17.04g/L。其次是采用含N48G突变体的菌株和含G75H的菌株的粗酶的反应液,莱鲍迪苷D浓度为14.80和13.54g/L,而采用含野生型UGTZJ1基因的粗酶的反应液中莱鲍迪苷D浓度最低4.06g/L。
实施例3温度对催化反应的影响
采用实施例2中获得产量最高的基因工程菌E.coli BL21(DE3)/pET28a-UGTZJ1(N48T)-StSUS1诱导表达后获取的粗酶液进行反应。在50mL三角瓶中,10mL反应混合物中包含20g/L莱鲍迪苷A,60g/L蔗糖,pH7.0的50mM磷酸钠缓冲,3mg/mL粗酶液,反应分别在20℃、30℃、37℃、42℃、50℃和200rpm条件下进行。加入粗酶液启动反应,反应16h后取样于95℃加热5min灭活酶,离心取上清,测得不同反应温度下反应液中莱鲍迪苷D的含量,结果见表2。
表2不同反应温度下反应液中莱鲍迪苷D的含量
温度(℃) |
莱鲍迪苷D(g/L) |
20 |
15.72 |
30 |
16.38 |
37 |
17.35 |
42 |
5.24 |
50 |
1.08 |
可知该催化反应的最适反应温度为37℃,产量最高可达17.35g/L。
实施例4pH对催化反应的影响
采用基因工程菌E.coli BL21(DE3)/pET28a-UGTZJ1(N48T)-StSUS1诱导表达后获取的粗酶液进行反应。在50mL三角瓶中,10mL反应混合物中包含20g/L莱鲍迪苷A,60g/L蔗糖,3mg/mL粗酶液,50mM磷酸钠缓冲pH分别配置为6.0、6.5、7.0、7.5和8.0,反应在37℃、200rpm条件下进行。加入粗酶液启动反应,反应16h后取样于95℃加热5min灭活酶,离心取上清,测得不同反应pH下反应液中莱鲍迪苷D的含量,结果见表3。
表3不同反应pH下反应液中莱鲍迪苷D的含量
pH |
莱鲍迪苷D(g/L) |
6.0 |
13.26 |
6.5 |
17.07 |
7.0 |
17.32 |
7.5 |
12.12 |
8.0 |
10.51 |
可知该催化反应的最适反应pH为7.0,产量最高可达17.32g/L。
实施例5蔗糖添加量对催化反应的影响
采用基因工程菌E.coli BL21(DE3)/pET28a-UGTZJ1(N48T)-StSUS1诱导表达后获取的粗酶液进行反应。在50mL三角瓶中,10mL反应混合物中包含20g/L莱鲍迪苷A,质量比分别为1:1、1:2、1:3和1:4的蔗糖,pH 7.0的50mM磷酸钠缓冲,3mg/mL粗酶液,反应在37℃、200rpm条件下进行。加入粗酶液启动反应,反应16h后取样于95℃加热5min灭活酶,离心取上清,测得不同底物浓度配比下反应液中莱鲍迪苷D的含量,结果见表4。
表4不同底物浓度配比下反应液中莱鲍迪苷D的含量
莱鲍迪苷A:蔗糖 |
莱鲍迪苷D(g/L) |
1:1 |
16.86 |
1:2 |
18.42 |
1:3 |
17.33 |
1:4 |
17.01 |
可知当该催化反应中莱鲍迪苷A与蔗糖的质量比为1:2时反应液中莱鲍迪苷D产量最高,为18.42/L。
实施例6粗酶液中蛋白浓度对催化反应影响
采用基因工程菌E.coli BL21(DE3)/pET28a-UGTZJ1(N48T)-StSUS1诱导表达后获取的粗酶液进行反应。在50mL三角瓶中,10mL反应混合物中包含50g/L莱鲍迪苷A,100g/L蔗糖,pH 7.0的50mM磷酸钠缓冲,粗酶液蛋白浓度分别为3、6、9、12和15mg/mL,反应在37℃、200rpm条件下进行。加入粗酶液启动反应,反应24h后取样于95℃加热5min灭活酶,离心取上清,测得不同粗酶液浓度下反应液中莱鲍迪苷D的含量,结果见表5。
表5不同粗酶液浓度下反应液中莱鲍迪苷D的含量
粗酶液蛋白浓度(g/L) |
莱鲍迪苷D(g/L) |
3 |
10.40 |
6 |
26.32 |
9 |
35.57 |
12 |
47.56 |
15 |
46.35 |
可知当该催化反应中粗酶液蛋白浓度为12mg/mL时,反应液中莱鲍迪苷D产量最高,为47.56/L。
由实施例3-6可知,最佳反应条件为:
采用基因工程菌E.coli BL21(DE3)/pET28a-UGTZJ1(N48T)-StSUS1诱导表达后获取的粗酶液进行反应。在50mL三角瓶中,10mL反应混合物中包含50g/L莱鲍迪苷A,100g/L蔗糖,pH 7.0的50mM磷酸钠缓冲,粗酶液蛋白浓度12mg/mL,反应在37℃、200rpm条件下进行。加入粗酶液启动反应,反应24h后取样于95℃加热5min灭活酶,离心取上清,反应液中莱鲍迪苷D产量最高,为47.56g/L,转化率达95%。
本发明不局限于上述特定的实施方案范围内,上述实施方案仅仅是为了能够对本发明的使用过程进行详细地说明,而且有相等功能的生产方法和技术细节也属于本发明内容的一部分。事实上,本领域技术人员根据前文的描述,就能够根据各自需要找到不同的调整方案,这些调整都应在本文所附的权利要求书的范围内。
序列表
<110> 广东广业清怡食品科技有限公司
南京朗奈生物技术有限公司
翁源广业清怡食品科技有限公司
<120> 一种糖基转移酶UGTZJ1突变体及其应用
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Val Tyr Ser Tyr Arg Asn Arg Thr Ser Ser Ala Val Ile Trp Asn Thr
195 200 205
Met Asp Cys Leu Glu Gln Ser Arg Leu Leu Gln Val Gln Gln Glu His
210 215 220
Lys Val Pro Ile Phe Pro Ile Ala Pro Met His Thr Ile Ala Pro His
225 230 235 240
Thr Ser Ser Ser Leu Leu Lys Glu Asp Arg Ser Cys Met Glu Trp Leu
245 250 255
Asp Lys Gln Ser His Asn Ser Val Ile Tyr Val Ser Leu Gly Ser Ile
260 265 270
Ala Phe Leu Ser Glu Lys Glu Val Gly Glu Met Ala Trp Gly Leu Ala
275 280 285
Asn Ser Glu Gln Pro Phe Leu Trp Val Val Arg Pro Gly Ser Ile Ser
290 295 300
Gly Ser Asp Trp Ile Glu Leu Leu Pro Gln Gly Phe Arg Asn Gly Val
305 310 315 320
Gly Glu Arg Gly Phe Ile Val Lys Trp Ala Pro Gln Lys Glu Val Leu
325 330 335
Ala His Glu Ala Val Gly Gly Phe Trp Ser His Cys Gly Trp Asn Ser
340 345 350
Thr Leu Glu Ser Ile Cys Glu Gly Val Pro Met Ile Cys Arg Pro Cys
355 360 365
Phe Gly Asp Gln Arg Val Asn Ala Arg Tyr Val Ser His Glu Trp Arg
370 375 380
Ile Gly Ile Glu Leu Glu Lys Leu Glu Arg Glu Glu Ile Glu Arg Ala
385 390 395 400
Val Arg Lys Leu Ile Leu Gly Asp Glu Gly Lys Glu Met Arg Glu Arg
405 410 415
Ala Lys Asp Leu Lys Glu Lys Val Thr Val Tyr Thr Arg Glu Gly Gly
420 425 430
Ser Ser Tyr Asn Phe Leu Asn Glu Leu Val Asn Leu Ile Met Ser Phe
435 440 445
<210> 5
<211> 2418
<212> DNA
<213> 马铃薯来源蔗糖合酶(Sucrose synthase)
<400> 5
atggccgaac gtgtcctgac ccgtgtccat agtctgcgtg aacgtgttga tgctaccctg 60
gctgcccacc gtaatgaaat cctgctgttt ctgagtcgta ttgaaagcca cggcaaaggt 120
atcctgaaac cgcacgaact gctggcagaa tttgatgcta ttcgccagga tgacaaaaac 180
aaactgaacg aacatgcatt cgaagaactg ctgaaaagca cccaagaagc tatcgtcctg 240
ccgccgtggg tggcactggc aattcgtctg cgcccgggcg tttgggaata catccgtgtt 300
aacgtcaatg cgctggttgt ggaagaactg agtgtgccgg aatatctgca gtttaaagaa 360
gaactggtcg atggcgcgtc caacggtaat ttcgtgctgg aactggactt tgaaccgttc 420
accgcctcat ttccgaaacc gaccctgacg aaatcgattg gcaacggtgt tgaatttctg 480
aatcgtcatc tgagcgccaa aatgttccac gataaagaat ctatgacccc gctgctggaa 540
tttctgcgcg cacatcacta taaaggtaaa accatgatgc tgaacgatcg tattcagaac 600
agcaatacgc tgcaaaatgt gctgcgcaaa gcggaagaat acctgatcat gctgccgccg 660
gaaaccccgt acttcgaatt tgaacataaa ttccaggaaa ttggcctgga aaaaggctgg 720
ggtgatacgg cagaacgtgt gctggaaatg gtttgcatgc tgctggatct gctggaagct 780
ccggacagct gtaccctgga aaaatttctg ggtcgcattc cgatggtttt caacgtcgtg 840
atcctgtctc cgcacggcta ttttgcgcag gaaaatgtcc tgggttaccc ggataccggc 900
ggtcaggttg tctatattct ggaccaagtg ccggccctgg aacgtgaaat gctgaaacgc 960
atcaaagaac agggcctgga tattatcccg cgtattctga tcgtcacccg tctgctgccg 1020
gacgcagtgg gcaccacgtg cggtcaacgt attgaaaaag tgtatggcgc tgaacattca 1080
cacatcctgc gtgttccgtt tcgcaccgaa aaaggtattg tccgtaaatg gatctcgcgc 1140
tttgaagtgt ggccgtacat ggaaacgttc attgaagatg ttgcaaaaga aatctcagcg 1200
gaactgcagg ccaaaccgga cctgattatc ggcaactata gcgaaggtaa tctggcggcc 1260
tctctgctgg cccataaact gggcgtgacc caatgtacga ttgcacacgc tctggaaaaa 1320
accaaatatc cggattcgga catctactgg aaaaaattcg atgaaaaata ccatttcagc 1380
tctcagttca ccgcagatct gattgctatg aaccacacgg actttattat caccagtacg 1440
ttccaggaaa tcgcgggctc caaagatacc gtgggtcaat acgaaagtca tatggccttt 1500
acgatgccgg gcctgtatcg cgtggttcac ggtatcaacg ttttcgatcc gaaattcaac 1560
attgtctccc cgggtgcaga catcaatctg tatttttcat actcggaaac cgaaaaacgt 1620
ctgacggctt tccatccgga aatcgatgaa ctgctgtata gcgatgtgga aaacgacgaa 1680
cacctgtgcg ttctgaaaga tcgcaccaaa ccgattctgt ttacgatggc gcgtctggac 1740
cgcgttaaaa atctgaccgg cctggtcgaa tggtacgcca aaaacccgcg tctgcgcggt 1800
ctggtgaatc tggtcgtggt tggcggtgat cgtcgcaaag aatctaaaga cctggaagaa 1860
caggcggaaa tgaagaaaat gtacgaactg atcgaaaccc ataacctgaa tggccagttc 1920
cgttggatca gttcccaaat gaaccgtgtt cgcaatggcg aactgtatcg ctacatcgca 1980
gatacgaaag gtgcttttgt ccagccggcg ttttacgaag ccttcggcct gaccgtcgtg 2040
gaagcgatga cgtgcggtct gccgaccttc gcaacgaatc atggcggccc ggcagaaatt 2100
atcgttcacg gcaaaagtgg ttttcatatt gatccgtatc acggcgaaca ggcagctgat 2160
ctgctggccg actttttcga aaaatgtaaa aaagacccgt cacattggga aaccatttcg 2220
atgggcggtc tgaaacgcat cgaagaaaaa tatacctggc aaatttacag cgaatctctg 2280
ctgacgctgg cggccgtgta cggtttctgg aaacacgttt ctaaactgga tcgtctggaa 2340
attcgtcgct atctggaaat gttttatgcg ctgaaatacc gcaaaatggc ggaagccgtg 2400
ccgctggcag ctgaataa 2418
<210> 6
<211> 809
<212> PRT
<213> 拟南芥菜来源的蔗糖合酶(Sucrose synthase)
<400> 6
Met Ala Asn Pro Lys Leu Thr Arg Val Leu Ser Thr Arg Asp Arg Val
1 5 10 15
Gln Asp Thr Leu Ser Ala His Arg Asn Glu Leu Val Ala Leu Leu Ser
20 25 30
Arg Tyr Val Asp Gln Gly Lys Gly Ile Leu Gln Pro His Asn Leu Ile
35 40 45
Asp Glu Leu Glu Ser Val Ile Gly Asp Asp Glu Thr Lys Lys Ser Leu
50 55 60
Ser Asp Gly Pro Phe Gly Glu Ile Leu Lys Ser Ala Met Glu Ala Ile
65 70 75 80
Val Val Pro Pro Phe Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val
85 90 95
Trp Glu Tyr Val Arg Val Asn Val Phe Glu Leu Ser Val Glu Gln Leu
100 105 110
Thr Val Ser Glu Tyr Leu Arg Phe Lys Glu Glu Leu Val Asp Gly Pro
115 120 125
Asn Ser Asp Pro Phe Cys Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala
130 135 140
Asn Val Pro Arg Pro Ser Arg Ser Ser Ser Ile Gly Asn Gly Val Gln
145 150 155 160
Phe Leu Asn Arg His Leu Ser Ser Val Met Phe Arg Asn Lys Asp Cys
165 170 175
Leu Glu Pro Leu Leu Asp Phe Leu Arg Val His Lys Tyr Lys Gly His
180 185 190
Pro Leu Met Leu Asn Asp Arg Ile Gln Ser Ile Ser Arg Leu Gln Ile
195 200 205
Gln Leu Ser Lys Ala Glu Asp His Ile Ser Lys Leu Ser Gln Glu Thr
210 215 220
Pro Phe Ser Glu Phe Glu Tyr Ala Leu Gln Gly Met Gly Phe Glu Lys
225 230 235 240
Gly Trp Gly Asp Thr Ala Gly Arg Val Leu Glu Met Met His Leu Leu
245 250 255
Ser Asp Ile Leu Gln Ala Pro Asp Pro Ser Ser Leu Glu Lys Phe Leu
260 265 270
Gly Met Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly
275 280 285
Tyr Phe Gly Gln Ala Asn Val Leu Gly Leu Pro Asp Thr Gly Gly Gln
290 295 300
Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Thr Glu Met Leu
305 310 315 320
Leu Arg Ile Lys Arg Gln Gly Leu Asp Ile Ser Pro Ser Ile Leu Ile
325 330 335
Val Thr Arg Leu Ile Pro Asp Ala Lys Gly Thr Thr Cys Asn Gln Arg
340 345 350
Leu Glu Arg Val Ser Gly Thr Glu His Thr His Ile Leu Arg Val Pro
355 360 365
Phe Arg Ser Glu Lys Gly Ile Leu Arg Lys Trp Ile Ser Arg Phe Asp
370 375 380
Val Trp Pro Tyr Leu Glu Asn Tyr Ala Gln Asp Ala Ala Ser Glu Ile
385 390 395 400
Val Gly Glu Leu Gln Gly Val Pro Asp Phe Ile Ile Gly Asn Tyr Ser
405 410 415
Asp Gly Asn Leu Val Ala Ser Leu Met Ala His Arg Met Gly Val Thr
420 425 430
Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser
435 440 445
Asp Ile Tyr Trp Lys Asp Phe Asp Asn Lys Tyr His Phe Ser Cys Gln
450 455 460
Phe Thr Ala Asp Leu Ile Ala Met Asn Asn Ala Asp Phe Ile Ile Thr
465 470 475 480
Ser Thr Tyr Gln Glu Ile Ala Gly Thr Lys Asn Thr Val Gly Gln Tyr
485 490 495
Glu Ser His Gly Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His
500 505 510
Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala
515 520 525
Asp Met Thr Ile Tyr Phe Pro Tyr Ser Glu Glu Thr Arg Arg Leu Thr
530 535 540
Ala Leu His Gly Ser Ile Glu Glu Met Leu Tyr Ser Pro Asp Gln Thr
545 550 555 560
Asp Glu His Val Gly Thr Leu Ser Asp Arg Ser Lys Pro Ile Leu Phe
565 570 575
Ser Met Ala Arg Leu Asp Lys Val Lys Asn Ile Ser Gly Leu Val Glu
580 585 590
Met Tyr Ser Lys Asn Thr Lys Leu Arg Glu Leu Val Asn Leu Val Val
595 600 605
Ile Ala Gly Asn Ile Asp Val Asn Lys Ser Lys Asp Arg Glu Glu Ile
610 615 620
Val Glu Ile Glu Lys Met His Asn Leu Met Lys Asn Tyr Lys Leu Asp
625 630 635 640
Gly Gln Phe Arg Trp Ile Thr Ala Gln Thr Asn Arg Ala Arg Asn Gly
645 650 655
Glu Leu Tyr Arg Tyr Ile Ala Asp Thr Arg Gly Ala Phe Ala Gln Pro
660 665 670
Ala Phe Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys
675 680 685
Gly Leu Pro Thr Phe Ala Thr Cys His Gly Gly Pro Ala Glu Ile Ile
690 695 700
Glu His Gly Leu Ser Gly Phe His Ile Asp Pro Tyr His Pro Glu Gln
705 710 715 720
Ala Gly Asn Ile Met Ala Asp Phe Phe Glu Arg Cys Lys Glu Asp Pro
725 730 735
Asn His Trp Lys Lys Val Ser Asp Ala Gly Leu Gln Arg Ile Tyr Glu
740 745 750
Arg Tyr Thr Trp Lys Ile Tyr Ser Glu Arg Leu Met Thr Leu Ala Gly
755 760 765
Val Tyr Gly Phe Trp Lys Tyr Val Ser Lys Leu Glu Arg Arg Glu Thr
770 775 780
Arg Arg Tyr Leu Glu Met Phe Tyr Ile Leu Lys Phe Arg Asp Leu Val
785 790 795 800
Lys Thr Val Pro Ser Thr Ala Asp Asp
805
<210> 7
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ctcacaccag ttacacctcc cccaaccctg aaaccc 36
<210> 8
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tcagggttgg gggaggtgta actggtgtga gcaat 35
<210> 9
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tcacaccagt tacggctccc ccaaccctga aaccc 35
<210> 10
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tcagggttgg gggagccgta actggtgtga gcaat 35
<210> 11
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cgtgatatct cgtctcacga tttgatatct cttgta 36
<210> 12
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aagagatatc aaatcgtgag acgagatatc acggtc 36