具体实施方式
实例1人狗嵌合尿酸酶蛋白UHC在大肠杆菌中重组表达
在本实施例中,所涉及的嵌合蛋白N端前240个氨基酸为狗来源尿酸酶序列,第241-304为人源尿酸酶氨基酸序列。根据上述氨基酸序列,并按照大肠杆菌偏爱的密码子,委托宝生物(大连)有限公司进行全基因合成,其核苷酸序列分别为(Seq ID No:10):CAT ATG GCC CAT TAT CAT AAT GAT TAT AAA AAA AAT GAT GAA GTT GAA TTT GTT CGT ACC GGT TAT GGTAAA GAT ATG GTT AAA GTT CTG CAT ATT CAG CGT GAT GGT AAA TAT CAT TCT ATT AAA GAA GTT GCC ACCTCT GTT CAG CTG ACC CTG TCT TCT AAA AAA GAT TAT GTT TAT GGT GAT AAT TCT GAT ATT ATT CCA ACCGAT ACC ATT AAA AAT ACC GTT CAT GTT CTG GCC AAA TTT AAA GGT ATT AAA TCT ATT GAA ACC TTT GCCATG AAT ATT TGT GAA CAT TTT CTG TCT TCT TTT AAT CAT GTT ATT CGT GCC CAG GTT TAT GTT GAA GAAGTT CCA TGG AAA CGT TTT GAA AAA AAT GGT GTT AAA CAT GTT CAT GCC TTT ATT CAT AAT CCA ACC GGTACC CAT TTT TGT GAA GTT GAA CAG ATG CGT TCT GGT CCA CCA GTT ATT CAT TCT GGT ATT AAA GAT CTGAAA GTT CTG AAA ACC ACC CAG TCT GGT TTT GAA GGT TTT ATT AAA GAT CAG TTT ACC ACC CTG CCA GAAGTT AAA GAT CGT TGT TTT GCC ACC AAA GTT TAT TGT AAA TGG CGT TAT CAT CAG GGT CGT GAT GTT GATTTT GAA GCC ACC TGG GAT ACC GTT CGT GAT ATT GTT CTG GAA AAA TTT GCC GGT CCT TAT GAT AAA GGTGAA TAT TCT CCA TCT GTT CAG AAA ACC CTG TAT GAT ATT CAG GTT CTG TCT CTG TCT CGT GTT CCA GCCATT GAA GAT ATG GAA ATT TCT CTG CCA AAT ATT CAT TAT TTT AAT ATT GAT ATG TCT AAA ATG GGT CTGATT AAT AAA GAA GAA GTT CTG CTG CCA CTG GAT AAT CCT TAT GGT AAA ATT ACC GGT ACC GTT AAA CGTAAA CTG TCT TCT CGT CTG TGA TAA GGA TCC
将全基因合成的重组质粒扩增后,用Nde I和BamH I双酶切,回收目的片段,用T4DNA连接酶与同样经Nde I和BamH I酶切回收的质粒pET-3C(Invitrogen)连接,利用如《CurrentProtocols in Molecular Biology》中所述的标准方法将连接混合物转入大肠杆菌克隆宿主菌DH5α。
在LB/AMP平板上进行转化反应,过夜生长转化体后,挑取转化后单克隆菌落以制备质粒用酶切和PCR验证的方法筛选重组质粒pET-3C-UHC,经DNA测序(TAKARA,大连)后确定含有所需嵌合蛋白UHC cDNA的阳性重组质粒中尿酸酶序列与理论序列完全一致。将上述测序正确的重组质粒转化大肠杆菌表达宿主菌并进行表达。
使用大肠杆菌BL21(DE3)、BL21 Star(DE3)或BL21 Star(DE3)plysS、表达UHC嵌合蛋白。这些菌株仅是许多适用于表达嵌合蛋白中的一些,它们可以分别通过商业渠道自Novagen,Invitrogen及Stratagen获得。利用转化体在含有AMP的LB平板上的生长能力可将其鉴别出来。
使含有UHC重组质粒的大肠杆菌表达重组菌在含有50um/ml的AMP的液体LB培养基中培养过夜,将上述培养液接种大型的培养物,接种比例为1∶25-1∶200。待这些细胞生长至600nm处光密度为一定值后,加入IPTG使其终浓度为0.4mM诱导表达目标蛋白,继续培养细胞3至12个小时。随后通过离心收获细胞,用50mM Tris缓冲液洗涤沉淀,离心放置-20度保存,并进行SDS-PAGE检测(附图2)。
实例2人狗嵌合尿酸酶蛋白UHC突变体DNA构建以及在大肠杆菌中重组表达
以UHC蛋白DNA序列为原始模板,用交错延伸PCR法突变制备含有目标突变的DNA(附图3)。下面以制备S246T-S248G-R249Q和S246T-S248G-R249Q-F266L为例进行说明:
引物编号 |
DNA序列 |
引物1(Seq ID No:11) |
5′CACGACATATGGCCCATTATCATA 3′ |
引物2(Seq ID No:12) |
5′GGATCCTTATCACAGACGAGAA 3′ |
引物3(Seq ID No:13) |
5′ACCCTGGGTCAGGTTCCAGAAATTGAAGATATGGAAATT 3′ |
引物4(Seq ID No:14) |
5′TTCTGGAACCTGACCCAGGGTCAGAACCTGAATATCATA3′ |
引物5(Seq ID No:15) |
5′TTCATTATCTGAATATTGATATGTCTAAAA 3′ |
引物6(Seq ID No:16) |
5′ATCAATATTCAGATAATGAATATTTGGCAG 3′ |
制备UHC246/248/249:第一阶段PCR:模板序列为实例1中全基因合成序列(Seq ID No:8),引物为表2中引物1和引物3,PCR反应体系和方法均采用PCR反应试剂盒(TAKARA,大连),按商家的说明书设置。PCR反应条件为:94℃1min,56℃1min,72℃1min,共30个循环,第一个循环94℃变性10min,最后一个循环72℃延伸10min。按上述PCR条件扩增得到产物UHC246/248/249-a片段;第二阶段PCR:模板序列同第一阶段PCR,引物为引物2和引物4,按上述PCR条件扩增得到UHC246/248/249-b片段;第三阶段PCR:模板为UHC246/248/249-a片段和UHC246/248/249-b片段的1∶1混合溶液,引物为引物1和引物2,按上述PCR条件扩增得到产物UHC246/248/249(SEQID No:6)
制备UHC246/248/249/266:第一阶段PCR:模板序列为UHC246/248/249序列(Seq ID No:8),引物为表2中引物1和引物5,按上述PCR条件扩增得到产物UHC246/248/249/266-a片段;第二阶段PCR:模板序列同第一阶段PCR,引物为引物2和引物4,按上述PCR条件扩增得到UHC246/248/249/266-b片段;第三阶段PCR:模板为UHC246/248/249/266-a片段和UHC246/248/249/266-b片段的1∶1混合溶液,引物为引物1和引物2,按上述PCR条件扩增得到产物UHC246/248/249/266(SEQ ID No:8)。
引物1和引物2的5’末端分别含有Nde I和BamH I酶切序列,将含各突变序列的DNA序列用Nde I和BamH I进行双酶切,连接、转化、筛选、表达方法同实例1。
实例3UHC嵌合蛋白及其突变体蛋白大肠杆菌表达产物纯化
取50g菌体沉淀加入500ml破菌液,破菌液pH为8.2,含有25mM Tris-HCl,0.1mg/ml溶菌酶中,37℃搅拌2小时,将上述菌液超声破菌(500W 4S,间隔6S,30次);离心弃去上清,沉淀用2L pH 10.20.1M Na2CO3溶解,室温搅拌过夜;离心弃去沉淀,在上清中补入15%饱和硫酸铵,4度沉淀2小时;离心弃去上清,沉淀用2L pH 10.20.1M Na2CO3溶解,室温搅拌过夜;离心弃去沉淀,上清进行QAE琼脂糖阴离子交换层析柱(GE)纯化,目标蛋白全部挂柱后,用线性梯度为0至0.5M的NaCl(pH 10.20.1M Na2CO3)进行洗脱,目标蛋白在0.3M NaCl时被洗脱;将上述洗脱组分进行Sephacryl S 200分子筛纯化,收集主峰,即为目标蛋白峰;将上述组分进行黄嘌呤亲和层析(Sigma)纯化,将上述组分用pH 10.20.1M Na2CO3稀释至0.5mg/ml后上样,用含有60um黄嘌呤的pH 10.20.1M Na2CO3洗脱液洗脱目标蛋白。此时进行SDS-PAGE和HPLC检测纯度,均可达到95%以上(附图4)。
实例4UHC嵌合蛋白及其突变体蛋白活性检测
在25℃、pH8.5时,每分钟转化1μmol尿酸为尿囊素的酶量定义为一个国际单位(IU)。尿酸在293nm处有特征吸收峰,当其被尿酸酶降解后,产物在此波长范围内无吸收峰,定时检测293nm处吸光度的变化来确定尿酸的减少量,然后利用尿酸的摩尔消光系数(1.23×104M-1·CM-1)算出尿酸浓度,根据尿酸浓度的变化可计算尿酸酶活性。将紫外分光光度计调至293nm,待机器稳定后,用0.1M四硼酸钠溶液作为空白调零,取3ml 0.1mM尿酸溶液反应溶解加入石英比色杯中,补入10ul-50ul UHC嵌合蛋白及其突变体蛋白活性,每30秒进行一次读数,测量3min内OD293变化值。根据公式C=A/Kb(C为溶液尿酸浓度,A为293nm吸光值,K为摩尔消光系数-1.23×104M-1·CM-1,b为比色杯的内径),计算不同时间点OD293对应的尿酸浓度;根据ΔM=ΔCV(ΔM为尿酸减少的摩尔数,ΔC为尿酸浓度变化,C为反应液体积)计算减少的尿酸物质的量;根据U=ΔM/TV1(U为每毫升血浆含有的尿酸酶活性单位,T为反应分钟,V1为加入反应体系的UHC嵌合蛋白及其突变体蛋白体积)计算尿酸酶活性。
蛋白 |
酶比活(IU/mg) |
UHC |
6.3 |
UHC246/248/249 |
7.1 |
UHC246/248/249/266 |
7.3 |
UHC245/252/253 |
6.9 |
UHC245/252/253/266 |
6.8 |
实例5UHC嵌合蛋白及其突变体蛋白体外稳定性检测
抗疏水介导的聚集能力:将1mg/ml的UHC蛋白、UHC246/248/249蛋白、UHC246/248/249/266蛋白、UHC245/252/253蛋白、UHC245/252/253/266蛋白4度放置12小时,分别测量280nm处和350nm处吸光值,根据公式AI=100×OD350/(OD280-OD350)计算不同蛋白的聚集度(表3),并计算比活保留率
蛋白 |
聚集度(aggregation index) |
比活保留率 |
UHC |
20.5 |
79.5% |
UHC246/248/249 |
11.3 |
91.2% |
UHC246/248/249/266 |
10.2 |
90.5% |
UHC245/252/253 |
15.7 |
85.3% |
UHC245/252/253/266 |
16.2 |
83.9% |
热稳定性:将1mg/ml的UHC蛋白、UHC246/248/249蛋白、UHC246/248/249/266蛋白、UHC245/252/253蛋白、UHC245/252/253/266蛋白30度放置5天,取出测量酶活,比较酶活保留率。
蛋白 |
酶比活保留率 |
UHC |
60.3% |
UHC246/248/249 |
65.5% |
UHC246/248/249/266 |
67.8% |
UHC245/252/253 |
71.2% |
UHC245/252/253/266 |
69.8% |
实例6UHC嵌合蛋白及其突变体蛋白体内稳定性检测
取2.0-2.5kg新西兰长耳兔20只,随机分为5组,每组分别静脉注射不同类型UHC嵌合蛋白及其突变体蛋白,注射剂量为1.0mg/kg,分别在1h、3h、6h、12h、24小时取血,用3.2%柠檬酸三钠抗凝,混匀后12000转离心10分钟,取出上层血浆测量酶活,进行药代动力学分析,计算不同组的AUC,结果见下表
蛋白 |
AUC(酶活×小时) |
UHC |
3.26 |
UHC246/248/249 |
4.11 |
UHC246/248/249/266 |
4.53 |
UHC245/252/253 |
4.78 |
UHC245/252/253/266 |
3.99 |
实例7体内药效学实验
取100日龄“罗曼蛋鸡”70只,随机抽取15只作为空白对照组(常规饲料,自由饮水),其余55只喂食造模饲料(造模饲料底料为玉米粉、补充鱼粉、骨粉,蛋白质含量大于20%、含钙量大于4%),每日控制进水100ml内,造模2-3周将血液尿酸浓度大于480umol/L的蛋鸡作为高尿酸血症模型筛选出来(45只),随机分为3组,每组15只:尿酸酶UHC组,皮下注射UHC蛋白,剂量为1mg/kg,每天注射一次;阳性对照药物苯溴马隆组,灌喂苯溴马隆,剂量为5mg/kg,每天灌喂一次;模型对照组,继续喂食造模饲料。给药三周后,从鸡翅静脉取血用尿酸测定试剂盒测量血液尿酸浓度,结果如下:
组别 |
给药0天血液尿酸浓度(um/L) |
给药10天血液尿酸浓度(um/L) |
给药21天血液尿酸浓度(um/L) |
正常组 |
234±15 |
241±29 |
220±34 |
UHC组 |
529±123 |
411±78 |
266±69 |
苯溴马隆组 |
527±109 |
455±67 |
416±98 |
模型对照组 |
534±136 |
551±121 |
520±131 |
结果显示,UHC蛋白注射高尿酸血症动物后,可显著降低血液尿酸水平。
序列表
SEQUENCE LISTING
<110>重庆富进生物医药有限公司
<120>人源化重组尿酸酶及其突变体
<160>16
<210>SEQ ID NO:1
<211>304
<212>PRT
<213>Homo sapiens
<400>1
Met Ala His Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Val Glu Phe
1 5 10 15
Val Arg Thr Gly Tyr Gly Lys Glu Met Val Lys Val Leu His Ile Gln
20 25 30
Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln
35 40 45
Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp
50 55 60
Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys
65 70 75 80
Phe Lys Glu Ile Lys Ser Ile Glu Ala Phe Gly Val Asn Ile Cys Glu
85 90 95
His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Met
100 105 110
Glu Glu Ile Pro Trp Lys His Leu Glu Lys Asn Glu Val Lys His Val
115 120 125
His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu
130 135 140
Gln Leu Arg Ser Gly Pro Gln Val Ile His Ser Gly Ile Lys Asp Leu
145 150 155 160
Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp
165 170 175
Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln
180 185 190
Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Lys
195 200 205
Ala Thr Trp Asp Thr Ile Arg Asp Leu Val Met Glu Lys Ser Ala Gly
210 215 220
Pro Tyr Asp Lys Gly Glu Tyr Leu Thr Ser Val Gln Lys Thr Leu Cys
225 230 235 240
Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Ala Ile Glu Asp Met
245 250 255
Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys
260 265 270
Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro
275 280 285
Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu
290 295 300
<210>SEQ ID NO:2
<211>304
<212>PRT
<213>Sus scrofa
<400>2
Met Ala His Tyr Arg Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe
1 5 10 15
Val Arg Thr Gly Tyr Gly Lys Asp Met Ile Lys Val Leu His Ile Gln
20 25 30
Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln
35 40 45
Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp
50 55 60
Val Ile Pro Thr Asp Thr Ile Lys Asn Thr Val Asn Val Leu Ala Lys
65 70 75 80
Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Val Thr Ile Cys Glu
85 90 95
His Phe Leu Ser Ser Phe Lys His Val Ile Arg Ala Gln Val Tyr Val
100 105 110
Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val
115 120 125
His Ala Phe Ile Tyr Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu
130 135 140
Gln Ile Arg Asn Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu
145 150 155 160
Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp
165 170 175
Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln
180 185 190
Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu
195 200 205
Ala Thr Trp Asp Thr Val Arg Ser Ile Val Leu Gln Lys Phe Ala Gly
210 215 220
Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr
225 230 235 240
Asp Ile Gln Val Leu Thr Leu Gly Gln Val Pro Glu Ile Glu Asp Met
245 250 255
Glu Ile Ser Leu Pro Asn Ile His Tyr Leu Asn Ile Asp Met Ser Lys
260 265 270
Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro
275 280 285
Tyr Gly Arg Ile Thr Gly Thr Val Lys Arg Lys Leu Thr Ser Arg Leu
290 295 300
<210>SEQ ID NO:3
<211>304
<212>PRT
<213>Canis familiaris
<400>3
Met Ala His Tyr His Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe
1 5 10 15
Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln
20 25 30
Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln
35 40 45
Leu Thr Leu Ser Ser Lys Lys Asp Tyr Val Tyr Gly Asp Asn Ser Asp
50 55 60
Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys
65 70 75 80
Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Met Asn Ile Cys Glu
85 90 95
His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val
100 105 110
Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val
115 120 125
His Ala Phe Ile His Asn Pro Thr Gly Thr His Phe Cys Glu Val Glu
130 135 140
Gln Met Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu
145 150 155 160
Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp
165 170 175
Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Lys
180 185 190
Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu
195 200 205
Ala Thr Trp Asp Thr Val Arg Asp Ile Val Leu Glu Lys Phe Ala Gly
210 215 220
Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr
225 230 235 240
Asp Ile Gln Val His Ser Leu Ser Arg Val Pro Glu Met Glu Asp Met
245 250 255
Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys
260 265 270
Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro
275 280 285
Tyr Gly Arg Ile Thr Gly Thr Ala Lys Arg Lys Leu Ala Ser Lys Leu
290 295 300
<210>SEQ ID NO:4
<211>304
<212>PRT
<213>Bos taurus
<400>4
Met Ala His Tyr His Asn Asp Tyr Gln Lys Asn Asp Glu Val Glu Phe
1 5 10 15
Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln
20 25 30
Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln
35 40 45
Leu Thr Leu Asn Ser Arg Arg Glu Tyr Leu His Gly Asp Asn Ser Asp
50 55 60
Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val Gln Val Leu Ala Lys
65 70 75 80
Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Met Asn Ile Cys Glu
85 90 95
His Phe Leu Ser Ser Phe Asn His Val Ile Arg Val Gln Val Tyr Val
100 105 110
Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val
115 120 125
His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu
130 135 140
Gln Leu Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu
145 150 155 160
Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Leu Lys Asp
165 170 175
Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln
180 185 190
Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu
195 200 205
Ala Thr Trp Glu Ala Val Arg Gly Ile Val Leu Lys Lys Phe Ala Gly
210 215 220
Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr
225 230 235 240
Asp Ile Gln Val Leu Ser Leu Ser Gln Leu Pro Glu Ile Glu Asp Met
245 250 255
Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys
260 265 270
Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro
275 280 285
Tyr Gly Arg Ile Thr Gly Thr Val Lys Arg Lys Leu Thr Ser Arg Leu
290 295 300
<210>SEQ ID NO:5
<211>304
<212>PRT
<213>Artificial Sequence
<400>5
Met Ala His Tyr His Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe
1 5 10 15
Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln
20 25 30
Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln
35 40 45
Leu Thr Leu Ser Ser Lys Lys Asp Tyr Val Tyr Gly Asp Asn Ser Asp
50 55 60
Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys
65 70 75 80
Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Met Asn Ile Cys Glu
85 90 95
His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val
100 105 110
Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val
115 120 125
His Ala Phe Ile His Asn Pro Thr Gly Thr His Phe Cys Glu Val Glu
130 135 140
Gln Met Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu
145 150 155 160
Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp
165 170 175
Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Lys
180 185 190
Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu
195 200 205
Ala Thr Trp Asp Thr Val Arg Asp Ile Val Leu Glu Lys Phe Ala Gly
210 215 220
Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr
225 230 235 240
Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Ala Ile Glu Asp Met
245 250 255
Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys
260 265 270
Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro
275 280 285
Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu
290 295 300
<210>SEQ ID NO:6
<2ll>304
<212>PRT
<213>Artificial Sequence
<400>6
Met Ala His Tyr His Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe
1 5 10 15
Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln
20 25 30
Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln
35 40 45
Leu Thr Leu Ser Ser Lys Lys Asp Tyr Val Tyr Gly Asp Asn Ser Asp
50 55 60
Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys
65 70 75 80
Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Met Asn Ile Cys Glu
85 90 95
His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val
100 105 110
Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val
115 120 125
His Ala Phe Ile His Asn Pro Thr Gly Thr His Phe Cys Glu Val Glu
130 135 140
Gln Met Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu
145 150 155 160
Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp
165 170 175
Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Lys
180 185 190
Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu
195 200 205
Ala Thr Trp Asp Thr Val Arg Asp Ile Val Leu Glu Lys Phe Ala Gly
210 215 220
Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr
225 230 235 240
Asp Ile Gln Val Leu Thr Leu Gly Gln Val Pro Ala Ile Glu Asp Met
245 250 255
Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys
260 265 270
Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro
275 280 285
Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu
290 295 300
<210>SEQ ID NO:7
<211>304
<212>PRT
<213>Artificial Sequence
<400>7
Met Ala His Tyr His Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe
1 5 10 15
Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln
20 25 30
Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln
35 40 45
Leu Thr Leu Ser Ser Lys Lys Asp Tyr Val Tyr Gly Asp Asn Ser Asp
50 55 60
Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys
65 70 75 80
Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Met Asn Ile Cys Glu
85 90 95
His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val
100 105 110
Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val
115 120 125
His Ala Phe Ile His Asn Pro Thr Gly Thr His Phe Cys Glu Val Glu
130 135 140
Gln Met Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu
145 150 155 160
Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp
165 170 175
Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Lys
180 185 190
Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu
195 200 205
Ala Thr Trp Asp Thr Val Arg Asp Ile Val Leu Glu Lys Phe Ala Gly
210 215 220
Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr
225 230 235 240
Asp Ile Gln Val His Ser Leu Ser Arg Val Pro Glu Met Glu Asp Met
245 250 255
Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys
260 265 270
Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro
275 280 285
Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu
290 295 300
<210>SEQ ID NO:8
<211>304
<212>PRT
<213>Artificial Sequence
<400>8
Met Ala His Tyr His Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe
1 5 10 15
Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln
20 25 30
Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln
35 40 45
Leu Thr Leu Ser Ser Lys Lys Asp Tyr Val Tyr Gly Asp Asn Ser Asp
50 55 60
Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys
65 70 75 80
Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Met Asn Ile Cys Glu
85 90 95
His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val
100 105 110
Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val
115 120 125
His Ala Phe Ile His Asn Pro Thr Gly Thr His Phe Cys Glu Val Glu
130 135 140
Gln Met Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu
145 150 155 160
Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp
165 170 175
Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Lys
180 185 190
Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu
195 200 205
Ala Thr Trp Asp Thr Val Arg Asp Ile Val Leu Glu Lys Phe Ala Gly
210 215 220
Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr
225 230 235 240
Asp Ile Gln Val Leu Thr Leu Gly Gln Val Pro Ala Ile Glu Asp Met
245 250 255
Glu Ile Ser Leu Pro Asn Ile His Tyr Leu Asn Ile Asp Met Ser Lys
260 265 270
Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro
275 280 285
Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu
290 295 300
<210>SEQ ID NO:9
<211>304
<212>PRT
<213>Artificial Sequence
<400>9
Met Ala His Tyr His Asn Asp Tyr Lys Lys Asn Asp Glu Val Glu Phe
1 5 10 15
Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln
20 25 30
Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln
35 40 45
Leu Thr Leu Ser Ser Lys Lys Asp Tyr Val Tyr Gly Asp Asn Ser Asp
50 55 60
Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys
65 70 75 80
Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Met Asn Ile Cys Glu
85 90 95
His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val
100 105 110
Glu Glu Val Pro Trp Lys Arg Phe Glu Lys Asn Gly Val Lys His Val
115 120 125
His Ala Phe Ile His Asn Pro Thr Gly Thr His Phe Cys Glu Val Glu
130 135 140
Gln Met Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu
145 150 155 160
Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp
165 170 175
Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Lys
180 185 190
Val Tyr Cys Lys Trp Arg Tyr His Gln Gly Arg Asp Val Asp Phe Glu
195 200 205
Ala Thr Trp Asp Thr Val Arg Asp Ile Val Leu Glu Lys Phe Ala Gly
210 215 220
Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr
225 230 235 240
Asp Ile Gln Val His Ser Leu Ser Arg Val Pro Glu Met Glu Asp Met
245 250 255
Glu Ile Ser Leu Pro Asn Ile His Tyr Leu Asn Ile Asp Met Ser Lys
260 265 270
Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro
275 280 285
Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu
290 295 300
<210>SEQ ID NO:10
<211>927
<212>DNA
<213>Artificial Sequence
<223>Synthetic Primer
<400>10
catatggccc attatcataa tgattataaa aaaaatgatg aagttgaatt tgttcgtacc 60
ggttatggta aagatatggt taaagttctg catattcagc gtgatggtaa atatcattct 120
attaaagaag ttgccacctc tgttcagctg accctgtctt ctaaaaaaga ttatgtttat 180
ggtgataatt ctgatattat tccaaccgat accattaaaa ataccgttca tgttctggcc 240
aaatttaaag gtattaaatc tattgaaacc tttgccatga atatttgtga acattttctg 300
tcttctttta atcatgttat tcgtgcccag gtttatgttg aagaagttcc atggaaacgt 360
tttgaaaaaa atggtgttaa acatgttcat gcctttattc ataatccaac cggtacccat 420
ttttgtgaag ttgaacagat gcgttctggt ccaccagtta ttcattctgg tattaaagat 480
ctgaaagttc tgaaaaccac ccagtctggt tttgaaggtt ttattaaaga tcagtttacc 540
accctgccag aagttaaaga tcgttgtttt gccaccaaag tttattgtaa atggcgttat 600
catcagggtc gtgatgttga ttttgaagcc acctgggata ccgttcgtga tattgttctg 660
gaaaaatttg ccggtcctta tgataaaggt gaatattctc catctgttca gaaaaccctg 720
tatgatattc aggttctgtc tctgtctcgt gttccagcca ttgaagatat ggaaatttct 780
ctgccaaata ttcattattt taatattgat atgtctaaaa tgggtctgat taataaagaa 840
gaagttctgc tgccactgga taatccttat ggtaaaatta ccggtaccgt taaacgtaaa 900
ctgtcttctc gtctgtgata aggatcc 927
<210>SEQ ID NO:11
<211>24
<212>DNA
<213>Artificial Sequence
<223>Synthetic Primer
<400>11
cacgacatat ggcccattat cata 24
<210>SEQ ID NO:12
<211>22
<212>DNA
<213>Artificial Sequence
<223>Synthetic Primer
<400>12
ggatccttat cacagacgag aa 22
<210>SEQ ID NO:13
<211>39
<212>DNA
<213>Artificial Sequence
<223>Synthetic Primer
<400>13
accctgggtc aggttccaga aattgaagat atggaaatt 39
<210>SEQ ID NO:14
<211>39
<212>DNA
<213>Artificial Sequence
<223>Synthetic Primer
<400>14
ttctggaacc tgacccaggg tcagaacctg aatatcata 39
<210>SEQ ID NO:15
<211>30
<212>DNA
<213>Artificial Sequence
<223>Synthetic Primer
<400>15
ttcattatct gaatattgat atgtctaaaa 30
<210>SEQ ID NO:16
<211>30
<212>DNA
<213>Artificial Sequence
<223>Synthetic Primer
<400>16
atcaatattc agataatgaa tatttggcag 30