CN113801862A - 一种海洋链霉菌磷脂酶d突变体及其重组表达菌株的制备方法 - Google Patents

一种海洋链霉菌磷脂酶d突变体及其重组表达菌株的制备方法 Download PDF

Info

Publication number
CN113801862A
CN113801862A CN202110962378.XA CN202110962378A CN113801862A CN 113801862 A CN113801862 A CN 113801862A CN 202110962378 A CN202110962378 A CN 202110962378A CN 113801862 A CN113801862 A CN 113801862A
Authority
CN
China
Prior art keywords
ala
leu
gly
ser
asp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202110962378.XA
Other languages
English (en)
Other versions
CN113801862B (zh
Inventor
王永华
胡荣康
王方华
蓝东明
崔瑞国
杨博
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China University of Technology SCUT
Original Assignee
South China University of Technology SCUT
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China University of Technology SCUT filed Critical South China University of Technology SCUT
Priority to CN202110962378.XA priority Critical patent/CN113801862B/zh
Publication of CN113801862A publication Critical patent/CN113801862A/zh
Application granted granted Critical
Publication of CN113801862B publication Critical patent/CN113801862B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6481—Phosphoglycerides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
    • C12Y301/04004—Phospholipase D (3.1.4.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开了一种海洋链霉菌磷脂酶D突变体及其重组表达菌株的制备方法，所述的磷脂酶D突变体是在氨基酸序列为SEQ ID NO:1的亲本序列基础上发生如下突变：P343A；或Y383L；或P343A，Y383L；或P343A，Y383L，S380V，其对应的氨基酸序列为SEQIDNO:2，SEQID NO:3，SEQID NO:4，SEQID NO:5。相比于野生型，本发明突变体1的酶活力是野生型的1.3倍，突变体2的酶活是野生型的1.46倍，突变体3的酶活是野生型的3.9倍，突变体4的酶活是野生型的11.6倍。本发明获得的磷脂酶突变体活力得到显著提高。

Description

一种海洋链霉菌磷脂酶D突变体及其重组表达菌株的制备 方法
技术领域
本发明属于酶的基因工程技术领域，具体地说涉及利用分子生物学定点突变技术获得催化性能显著提高的磷脂酶D突变体及其重组表达制备方法和应用。
背景技术
磷脂是一种含有磷酸的混合脂类，是生物膜的基本成分和组成生命的必需物质。此外，磷脂由于其独特的化学结构和保健功能，已广泛应用于乳化剂、化妆品成分、医药配方和脂质体制剂。在工业中，天然磷脂主要是毛油精炼脱水和脱胶的副产品。近年来，人们对不同分子性质如电荷、极性、大小等的磷脂产生了极大的兴趣，以获得具有优良加工性能和突出生理药理功能的特殊功能磷脂。因此，对天然磷脂进行改性已成为实现磷脂副产物高价值利用的重要途径。
酶法修饰改性是制备功能性磷脂的一个重要方向。作为磷脂酶家族的重要成员，磷脂酶D(PLD)是合成和改造磷脂质的一类重要工具酶。磷脂酶D(PLD)(EC 3.1.4.4)可通过磷脂酰基转移作用(transphosphatidylation)催化磷脂质极性头部基团发生转移反应，从而合成为各种不同的稀有磷脂及系列功能性磷脂衍生物。PLD已被用于合成较少丰富的磷脂，如磷脂酰甘油(phosphatidylglycerol，PG)，磷脂酰乙醇胺(phosphatidylethanolamine，PE)，磷脂酰丝氨酸(phosphatidylserine，PS)和磷脂酰肌醇(phatidylglycerol，PI)。除此之外，通过PLD还合成了大量新型磷脂，其中包括磷脂酰胆碱(phosphatidylcholine，PC)中的胆碱被脂族醇、糖类，核苷、芳香醇或n-杂环醇所取代。尽管PLD可以用于合成大量磷脂，磷脂酶D来源单一、酶活力低下、制备难度大、成本高是当前制约该类酶开发的技术瓶颈，其中酶活力低下成为主要的制约因素，离工业化应用的需求还有很大的差距。利用蛋白质工程对酶分子进行改造成为解决该问题的主要方案，如中国发明专利201610402557.7中利用重组DNA技术对野生型磷脂酶D基因进行定点突变，得到活力较高的磷脂酶D基因。重组表达后，检测高活力磷脂酶D的比酶活较野生型磷脂酶D提高38-140％。酶分子体外定向进化属于蛋白质的非理性设计，属于蛋白质工程的范畴。通常手段是利用分子生物学手段在分子水平创造出分子的多样性，结合灵敏、高通量的筛选技术，
从而能够在短时间内得到理想的突变体。通过合理设计改造酶基因可以提高PLDs的催化性能。
发明内容
为了克服现有技术存在的缺陷，本发明提供一种高活力磷脂酶D。
SEQ ID No.1
DSSATPHLDAVEQTLRQVSPGLEGRVWERTAGNALDAPAGDPAGWLLQTPGCWGDANCAERTGTKRLLARMTENISKATRTVDISTLAPFPNGAFQDAIVAGLKKSVENGNKPKVRVLVGAAPVYHMNVLPSKYRDDLRDKLGKAADGLTLNVASMTTSKTAFSWNHSKLLVVDGQSAITGGINSWKDDYVDTTHPVSDVDLALTGPAAGSAGRYLDQLWTWTCENKSNIASVWFAASPGAGCMPTMEKDANPVPAAATGNVPVIAVGGLGVGIKDSDPSSAFKPELPSAPDTKCVVGLHDNTNADRDYDTVNPEESALRALVGSARSHVEISQQDLNATCPPLPRYDVRLYDALAAKLAAGVKVRIVVSDPENRGAVGSGGYSQIKSLNEISDLLRNRLSLLPGGAQGAKTAMCGNLQLATARSSDSAKWADGKPYAQHHKLVSVDDSAFYIGSKNLYPSWLQDFGYIVESPEAARQLDAELLAPQWKYSQATATFDYARGICQG
本发明的技术方案如下：
一种磷脂酶D突变体，是在氨基酸序列为SEQ ID NO:1的亲本序列基础上发生如下突变：P343A；或Y383L；或P343A，Y383L；或P343A，Y383L，S380V。
所述突变体的基因，由氨基酸序列为SEQ ID No.1(GenBank:RKN69773.1)的克伦基链霉菌(Streptomyces klenkii)作为亲本，在此基础上，通过信号肽在线分析软件Signal P分析其信号肽序列，将去除信号肽后的蛋白序列进行同源建模，获得其三维结构，对其蛋白结构进行分析，通过酶切、连接等构建重组载体，之后由重叠PCR技术对野生型磷脂酶D基因将第343，383位点进行突变，得到突变体基因。
一种磷脂酶D突变体，其氨基酸序列为SEQID NO:2，SEQID NO:3，SEQID NO:4，SEQID NO:5。
一种编码所述磷脂酶D突变体的基因，其核酸序列为SEQID NO:7，SEQID NO:8，SEQID NO:9，SEQID NO:10。
一种磷脂酶D重组表达菌株的制备方法，其特征在于，将权利要求2所述基因克隆到表达载体pET-21a、pET28a或pET32a上，转化大肠杆菌SHuffle T7感受态细胞，获得重组基因工程菌，之后将重组基因工程菌发酵，得到高活力磷脂酶D。
所述的磷脂酶D突变体应用于食品、制药生产中。尤其是，所述的磷脂酶D突变体应用于磷脂酰丝氨酸的生产中。
本发明的实验步骤具体如下：
1、一种构建高活力磷脂酶D突变体编码基因的过程包括如下步骤：
(1)将来自克伦基链霉菌(Streptomyces klenkii)的野生型磷脂酶D(SkPLD)基因与表达载体pET28a连接，构建重组质粒pET28a-PLD，通过重叠PCR定点突变野生型磷脂酶D基因，得到高活力磷脂酶D突变体编码基因；
(2)将含有高活力磷脂酶D突变体编码基因的pET28a-PLD保存。
2、一株含有高活力磷脂酶D基因的大肠杆菌SHuffle T7重组菌株及以此制备高活力磷脂酶D的过程包括如下步骤：
(1)将保存的含有高活力磷脂酶D突变体编码基因的pET28a-PLD转化入大肠杆菌SHuffle T7中，得到重组菌株，之后将重组菌株发酵，得到高活力磷脂酶D。
3、一种磷脂酶D突变体的酶学性质表征的过程包括如下步骤：
以酶联比色法测定突变体对大豆磷脂酰胆碱的酶活及酶动力学。
在本发明中采用如下定义：
1.氨基酸和DNA核酸序列的命名法：
使用氨基酸残基的公认IUPAC命名法，用三字母代码形式。DNA核酸序列采用公认IUPAC命名法。
2.磷脂酶D突变体的标识
采用“原始氨基酸位置替换的氨基酸”来表示磷脂酶D突变体中突变的氨基酸。如P343A，表示位置343的氨基酸由野生型磷脂酶D的Pro替换成Ala。位置的编号对应于SEQ IDNO：1中磷脂酶D的氨基酸序列编号。核苷酸的变化同样采用“原始核苷酸位置替换的核苷酸”来表示，位置编号对应SEQ ID NO：6中野生型磷脂酶D的核苷酸序列编号。本发明中的组合突变，包含如下：SkPLD-P343A-Y383L，SkPLD-P343A-Y383L-S380V。
与现有技术相比，本发明具有如下有益效果：
相比于野生型，本发明突变体1的酶活力是野生型的1.3倍，突变体2的酶活是野生型的1.46倍，突变体3的酶活是野生型的3.9倍，突变体4的酶活是野生型的11.6倍。说明本发明获得的磷脂酶突变体活力得到显著提高。自身酶活力提升进一步提升了该酶的工业利用价值。
附图说明
图1为野生型SkPLD及突变体蛋白纯化SDS-PAGE检测结果图。
图2为磷脂酶D突变体1(SkPLD-P343A)、突变体2(SkPLD-Y383L)、突变体3(SkPLD-P343A-Y383L)、突变体4(SkPLD-P343A-Y383L-S380V)与野生型(SkPLD)相对酶活柱状图。
具体实施方式
下面结合具体实施例对本发明作进一步具体详细描述，但本发明的实施方式不限于此，对于未特别注明的工艺参数，可参照常规技术进行。
实施例1：磷脂酶SkPLD突变体表达载体及表达菌株的构建
(1)参照克伦基链霉菌(Streptomyces klenkii)的野生型磷脂酶D(SkPLD)完整氨基酸序列(GenBank:RKN69773.1)，通过信号肽在线分析软件Signal P分析其信号肽序列，将1-33位氨基酸从完整序列中进行删除，获得磷脂酶SkPLD成熟肽编码序列(SEQ IDNO.1)；
(2)根据(1)所得氨基酸序列，按照大肠杆菌密码子偏好性设计磷脂酶SkPLD基因编码序列，其碱基序列如SEQ ID NO.1所示(野生型)。在序列上游引入ECoR I，下游引入NotI酶切位点，所得磷脂酶SkPLD基因序列由生工生物工程(上海)股份有限公司合成；
(3)将(2)所合成的磷脂酶SkPLD基因用限制性内切酶ECoR I和Not I分别对纯化的基因片段和质粒pET28a进行双酶切消化，连接，转化至大肠杆菌E.coli DH5α感受态细胞。涂布于LB(含50μg/mL卡那霉素)平板。挑取阳性克隆通过ECoR I和Not I双酶切鉴定及基因测序，获得野生型SkPLD的pET28a-SkPLD重组质粒；
(4)采用两步重叠延伸PCR方法构建本发明所述突变体SEQID NO:2，SEQID NO:3，SEQID NO:4，SEQID NO:5。首先进行引物全长的拼接，然后以含有目的基因的质粒模板进行扩增。反应条件如下：
反应条件1：
Figure BDA0003222556650000041
其中突变体1构建所用上游引物和下游引物序列为：
上游引物(P343A-F)：cgcggcagcgccgggcaggtc
下游引物(P343A-R)：gacctgcccggcgctgccgcg
突变体2构建所用上游引物和下游引物序列为：
上游引物(Y383L-F)：gggatttgatctgagataaaccgccagagccaaccg
下游引物(Y383L-R)：cggttggctctggcggtttatctcagatcaaatccc
突变体3构建所用上游引物和下游引物序列为：
上游引物(P343A-F)：cgcggcagcgccgggcaggtc
下游引物(P343A-R)：gacctgcccggcgctgccgcg
上游引物(Y383L-F)：gggatttgatctgagataaaccgccagagccaaccg
下游引物(Y383L-R)：cggttggctctggcggtttatctcagatcaaatccc
突变体4构建所用上游引物和下游引物序列为：
上游引物(P343A-F)：cgcggcagcgccgggcaggtc
下游引物(P343A-R)：gacctgcccggcgctgccgcg
上游引物(Y383L-F)：gggatttgatctgagataaaccgccagagccaaccg
下游引物(Y383L-R)：cggttggctctggcggtttatctcagatcaaatccc
上游引物(S380V-F)：gagagtaaccgccaacgccaaccgcgccac
下游引物(S380V-R)：gtggcgcggttggcgttggcggttactctc
PCR扩增条件:98℃，3min；98℃，10s；58℃，15s；72℃，10s；20个循环；72℃，2min。扩增产物经DNA纯化试剂盒纯化得到全长引物。
反应条件2：
Figure BDA0003222556650000051
PCR扩增条件:98℃，3min；98℃，10s；58℃，15s；72℃，408s；31个循环；72℃，2min。PCR产物利用DNA纯化试剂盒进行产物纯化得到磷脂酶突变基因。
用Dpn I酶切消化模板质粒，Dpn I酶切消化体系如下：
Figure BDA0003222556650000052
将Dpn I酶切消化体系置于37℃条件下，2h。将消化产物转化至E.coli DH5α感受态细胞。涂布于LB(含50μg/mL卡那霉素)平板。挑取阳性克隆通过ECoR I和Not I双酶切鉴定及基因测序，获得pET28a-SkPLD-突变体质粒。
(5)分别将(3)、(4)所得的重组质粒转化到大肠杆菌SHuffle T7感受态细胞，挑选阳性克隆并测序验证，即获得重组pET28a-SkPLD野生型及突变体的SHuffle T7大肠杆菌表达菌株。
实施例2：野生型SkPLD及其突变体重组表达菌株发酵及重组蛋白纯化
(1)将重组大肠杆菌SkPLD野生型及突变体表达菌株接种于含卡那霉素(50μg/mL)的种子培养基(NaCl 10g/L，蛋白胨10g/L，酵母提取物5g/L，pH7.2～7.4)中，于37℃，200r/min摇瓶培养至对数生长期，作为种子液；
(2)将(1)中所述种子液按5％的接种量接种到自诱导液体发酵培养基(酶水解酪蛋白10g/L，酵母浸膏5g/L，葡萄糖0.5g/L，乳糖2g/L，甘油5g/L，磷酸氢二钠3.6g/L，磷酸二氢钾3.4g/L，氯化铵2.7g/L，硫酸钠0.7g/L，硫酸镁1g/L，pH 7.2～7.4)中，于37℃，200r/min摇瓶培养至OD600＝0.6～0.8，再于20℃，200r/min条件下诱导培养24h；
(3)将(2)中所得发酵液离心(8000r/min,5min)，收集菌体沉淀，用Tris-HCl缓冲液(pH 8.0)重悬并超声破碎细胞，将细胞破碎液离心(10000r/min,20min)，取上清，即为制备得到的磷脂酶SkPLD粗酶液；
(4)将(3)中所得磷脂酶粗酶液，用0.45μm的滤膜抽滤。滤液利用镍柱亲和层析柱纯化，流速4mL/min，最后用含10-500mM咪唑的Tris-HCl缓冲液(pH 8.0)梯度洗脱，目标蛋白在250mM咪唑浓度处被洗脱下来。将洗脱下来的目的蛋白过G-25脱盐柱后，上样Q柱层析，用含有700mM NaCl的Tris-HCl缓冲液(pH 7.3)洗脱得到目的蛋白(见附图1)。
实施例3:磷脂酶酶学性质分析
(1)磷脂酶活力的测定方法：野生型磷脂酶SkPLD及突变体磷脂酶活力的测定均采用标准的酶联比色法进行测定，以大豆磷脂酰胆碱作为反应底物。采用酶联比色法进行活性检测：磷脂酶D催化水解L-α-卵磷脂生成胆碱，胆碱在胆碱氧化酶的作用下生成过氧化氢，过氧化氢在过氧化酶的作用下与4-氨基氨替比林和苯酚生成醌亚胺显色物质，在500nm波长下有吸光值。对于底物制备，1mmol大豆磷脂酰胆碱溶解在5mL氯仿中。氮气吹干后，加入SDS和Triton X-100，得到包含10mm大豆磷脂酰胆碱、12.5mM SDS和40mm Triton X-100的溶液。使用800W/s的探针超声器对溶液进行涡旋和超声10分钟。反应混合物(100μL)由0.4mM大豆PC、50mM Tris-HCl(pH 8.0)、20mm CaCl2和10μL酶样组成。在40℃振荡孵育10min后，加入25μL含50mM EDTA和50mM Tris-HCl(pH 8.0)的溶液并在100℃加热变性5分钟。冷却反应混合物到室温后,加入25μL的50mM Tris-HCl(pH值8.0)包含42mM苯酚,4-氨基氨替比林50mM,0.5U辣根过氧化物酶和0.25U胆碱氧化酶混合溶液。37℃孵育60min后，在500nm处测量反应混合物的吸光度。
结果如图2所示，相比于野生型，本发明突变体1的酶活力是野生型的1.3倍，突变体2的酶活是野生型的1.46倍，突变体3的酶活是野生型的3.9倍，突变体4的酶活是野生型的11.6倍。说明本发明获得的磷脂酶突变体活力得到显著提高。自身酶活力提升进一步提升了该酶的工业利用价值。
综上所述，与野生型相比，本发明获得的磷脂酶突变体显著提高了其自身酶活力，更适合应用于食品、医药等工业领域。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 华南理工大学
<120> 一种海洋链霉菌磷脂酶D突变体及其重组表达菌株的制备方法
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 506
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Asp Ser Ser Ala Thr Pro His Leu Asp Ala Val Glu Gln Thr Leu Arg
1 5 10 15
Gln Val Ser Pro Gly Leu Glu Gly Arg Val Trp Glu Arg Thr Ala Gly
20 25 30
Asn Ala Leu Asp Ala Pro Ala Gly Asp Pro Ala Gly Trp Leu Leu Gln
35 40 45
Thr Pro Gly Cys Trp Gly Asp Ala Asn Cys Ala Glu Arg Thr Gly Thr
50 55 60
Lys Arg Leu Leu Ala Arg Met Thr Glu Asn Ile Ser Lys Ala Thr Arg
65 70 75 80
Thr Val Asp Ile Ser Thr Leu Ala Pro Phe Pro Asn Gly Ala Phe Gln
85 90 95
Asp Ala Ile Val Ala Gly Leu Lys Lys Ser Val Glu Asn Gly Asn Lys
100 105 110
Pro Lys Val Arg Val Leu Val Gly Ala Ala Pro Val Tyr His Met Asn
115 120 125
Val Leu Pro Ser Lys Tyr Arg Asp Asp Leu Arg Asp Lys Leu Gly Lys
130 135 140
Ala Ala Asp Gly Leu Thr Leu Asn Val Ala Ser Met Thr Thr Ser Lys
145 150 155 160
Thr Ala Phe Ser Trp Asn His Ser Lys Leu Leu Val Val Asp Gly Gln
165 170 175
Ser Ala Ile Thr Gly Gly Ile Asn Ser Trp Lys Asp Asp Tyr Val Asp
180 185 190
Thr Thr His Pro Val Ser Asp Val Asp Leu Ala Leu Thr Gly Pro Ala
195 200 205
Ala Gly Ser Ala Gly Arg Tyr Leu Asp Gln Leu Trp Thr Trp Thr Cys
210 215 220
Glu Asn Lys Ser Asn Ile Ala Ser Val Trp Phe Ala Ala Ser Pro Gly
225 230 235 240
Ala Gly Cys Met Pro Thr Met Glu Lys Asp Ala Asn Pro Val Pro Ala
245 250 255
Ala Ala Thr Gly Asn Val Pro Val Ile Ala Val Gly Gly Leu Gly Val
260 265 270
Gly Ile Lys Asp Ser Asp Pro Ser Ser Ala Phe Lys Pro Glu Leu Pro
275 280 285
Ser Ala Pro Asp Thr Lys Cys Val Val Gly Leu His Asp Asn Thr Asn
290 295 300
Ala Asp Arg Asp Tyr Asp Thr Val Asn Pro Glu Glu Ser Ala Leu Arg
305 310 315 320
Ala Leu Val Gly Ser Ala Arg Ser His Val Glu Ile Ser Gln Gln Asp
325 330 335
Leu Asn Ala Thr Cys Pro Pro Leu Pro Arg Tyr Asp Val Arg Leu Tyr
340 345 350
Asp Ala Leu Ala Ala Lys Leu Ala Ala Gly Val Lys Val Arg Ile Val
355 360 365
Val Ser Asp Pro Glu Asn Arg Gly Ala Val Gly Ser Gly Gly Tyr Ser
370 375 380
Gln Ile Lys Ser Leu Asn Glu Ile Ser Asp Leu Leu Arg Asn Arg Leu
385 390 395 400
Ser Leu Leu Pro Gly Gly Ala Gln Gly Ala Lys Thr Ala Met Cys Gly
405 410 415
Asn Leu Gln Leu Ala Thr Ala Arg Ser Ser Asp Ser Ala Lys Trp Ala
420 425 430
Asp Gly Lys Pro Tyr Ala Gln His His Lys Leu Val Ser Val Asp Asp
435 440 445
Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu Tyr Pro Ser Trp Leu Gln
450 455 460
Asp Phe Gly Tyr Ile Val Glu Ser Pro Glu Ala Ala Arg Gln Leu Asp
465 470 475 480
Ala Glu Leu Leu Ala Pro Gln Trp Lys Tyr Ser Gln Ala Thr Ala Thr
485 490 495
Phe Asp Tyr Ala Arg Gly Ile Cys Gln Gly
500 505
<210> 2
<211> 506
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Asp Ser Ser Ala Thr Pro His Leu Asp Ala Val Glu Gln Thr Leu Arg
1 5 10 15
Gln Val Ser Pro Gly Leu Glu Gly Arg Val Trp Glu Arg Thr Ala Gly
20 25 30
Asn Ala Leu Asp Ala Pro Ala Gly Asp Pro Ala Gly Trp Leu Leu Gln
35 40 45
Thr Pro Gly Cys Trp Gly Asp Ala Asn Cys Ala Glu Arg Thr Gly Thr
50 55 60
Lys Arg Leu Leu Ala Arg Met Thr Glu Asn Ile Ser Lys Ala Thr Arg
65 70 75 80
Thr Val Asp Ile Ser Thr Leu Ala Pro Phe Pro Asn Gly Ala Phe Gln
85 90 95
Asp Ala Ile Val Ala Gly Leu Lys Lys Ser Val Glu Asn Gly Asn Lys
100 105 110
Pro Lys Val Arg Val Leu Val Gly Ala Ala Pro Val Tyr His Met Asn
115 120 125
Val Leu Pro Ser Lys Tyr Arg Asp Asp Leu Arg Asp Lys Leu Gly Lys
130 135 140
Ala Ala Asp Gly Leu Thr Leu Asn Val Ala Ser Met Thr Thr Ser Lys
145 150 155 160
Thr Ala Phe Ser Trp Asn His Ser Lys Leu Leu Val Val Asp Gly Gln
165 170 175
Ser Ala Ile Thr Gly Gly Ile Asn Ser Trp Lys Asp Asp Tyr Val Asp
180 185 190
Thr Thr His Pro Val Ser Asp Val Asp Leu Ala Leu Thr Gly Pro Ala
195 200 205
Ala Gly Ser Ala Gly Arg Tyr Leu Asp Gln Leu Trp Thr Trp Thr Cys
210 215 220
Glu Asn Lys Ser Asn Ile Ala Ser Val Trp Phe Ala Ala Ser Pro Gly
225 230 235 240
Ala Gly Cys Met Pro Thr Met Glu Lys Asp Ala Asn Pro Val Pro Ala
245 250 255
Ala Ala Thr Gly Asn Val Pro Val Ile Ala Val Gly Gly Leu Gly Val
260 265 270
Gly Ile Lys Asp Ser Asp Pro Ser Ser Ala Phe Lys Pro Glu Leu Pro
275 280 285
Ser Ala Pro Asp Thr Lys Cys Val Val Gly Leu His Asp Asn Thr Asn
290 295 300
Ala Asp Arg Asp Tyr Asp Thr Val Asn Pro Glu Glu Ser Ala Leu Arg
305 310 315 320
Ala Leu Val Gly Ser Ala Arg Ser His Val Glu Ile Ser Gln Gln Asp
325 330 335
Leu Asn Ala Thr Cys Pro Ala Leu Pro Arg Tyr Asp Val Arg Leu Tyr
340 345 350
Asp Ala Leu Ala Ala Lys Leu Ala Ala Gly Val Lys Val Arg Ile Val
355 360 365
Val Ser Asp Pro Glu Asn Arg Gly Ala Val Gly Ser Gly Gly Tyr Ser
370 375 380
Gln Ile Lys Ser Leu Asn Glu Ile Ser Asp Leu Leu Arg Asn Arg Leu
385 390 395 400
Ser Leu Leu Pro Gly Gly Ala Gln Gly Ala Lys Thr Ala Met Cys Gly
405 410 415
Asn Leu Gln Leu Ala Thr Ala Arg Ser Ser Asp Ser Ala Lys Trp Ala
420 425 430
Asp Gly Lys Pro Tyr Ala Gln His His Lys Leu Val Ser Val Asp Asp
435 440 445
Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu Tyr Pro Ser Trp Leu Gln
450 455 460
Asp Phe Gly Tyr Ile Val Glu Ser Pro Glu Ala Ala Arg Gln Leu Asp
465 470 475 480
Ala Glu Leu Leu Ala Pro Gln Trp Lys Tyr Ser Gln Ala Thr Ala Thr
485 490 495
Phe Asp Tyr Ala Arg Gly Ile Cys Gln Gly
500 505
<210> 3
<211> 506
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Asp Ser Ser Ala Thr Pro His Leu Asp Ala Val Glu Gln Thr Leu Arg
1 5 10 15
Gln Val Ser Pro Gly Leu Glu Gly Arg Val Trp Glu Arg Thr Ala Gly
20 25 30
Asn Ala Leu Asp Ala Pro Ala Gly Asp Pro Ala Gly Trp Leu Leu Gln
35 40 45
Thr Pro Gly Cys Trp Gly Asp Ala Asn Cys Ala Glu Arg Thr Gly Thr
50 55 60
Lys Arg Leu Leu Ala Arg Met Thr Glu Asn Ile Ser Lys Ala Thr Arg
65 70 75 80
Thr Val Asp Ile Ser Thr Leu Ala Pro Phe Pro Asn Gly Ala Phe Gln
85 90 95
Asp Ala Ile Val Ala Gly Leu Lys Lys Ser Val Glu Asn Gly Asn Lys
100 105 110
Pro Lys Val Arg Val Leu Val Gly Ala Ala Pro Val Tyr His Met Asn
115 120 125
Val Leu Pro Ser Lys Tyr Arg Asp Asp Leu Arg Asp Lys Leu Gly Lys
130 135 140
Ala Ala Asp Gly Leu Thr Leu Asn Val Ala Ser Met Thr Thr Ser Lys
145 150 155 160
Thr Ala Phe Ser Trp Asn His Ser Lys Leu Leu Val Val Asp Gly Gln
165 170 175
Ser Ala Ile Thr Gly Gly Ile Asn Ser Trp Lys Asp Asp Tyr Val Asp
180 185 190
Thr Thr His Pro Val Ser Asp Val Asp Leu Ala Leu Thr Gly Pro Ala
195 200 205
Ala Gly Ser Ala Gly Arg Tyr Leu Asp Gln Leu Trp Thr Trp Thr Cys
210 215 220
Glu Asn Lys Ser Asn Ile Ala Ser Val Trp Phe Ala Ala Ser Pro Gly
225 230 235 240
Ala Gly Cys Met Pro Thr Met Glu Lys Asp Ala Asn Pro Val Pro Ala
245 250 255
Ala Ala Thr Gly Asn Val Pro Val Ile Ala Val Gly Gly Leu Gly Val
260 265 270
Gly Ile Lys Asp Ser Asp Pro Ser Ser Ala Phe Lys Pro Glu Leu Pro
275 280 285
Ser Ala Pro Asp Thr Lys Cys Val Val Gly Leu His Asp Asn Thr Asn
290 295 300
Ala Asp Arg Asp Tyr Asp Thr Val Asn Pro Glu Glu Ser Ala Leu Arg
305 310 315 320
Ala Leu Val Gly Ser Ala Arg Ser His Val Glu Ile Ser Gln Gln Asp
325 330 335
Leu Asn Ala Thr Cys Pro Pro Leu Pro Arg Tyr Asp Val Arg Leu Tyr
340 345 350
Asp Ala Leu Ala Ala Lys Leu Ala Ala Gly Val Lys Val Arg Ile Val
355 360 365
Val Ser Asp Pro Glu Asn Arg Gly Ala Val Gly Ser Gly Gly Leu Ser
370 375 380
Gln Ile Lys Ser Leu Asn Glu Ile Ser Asp Leu Leu Arg Asn Arg Leu
385 390 395 400
Ser Leu Leu Pro Gly Gly Ala Gln Gly Ala Lys Thr Ala Met Cys Gly
405 410 415
Asn Leu Gln Leu Ala Thr Ala Arg Ser Ser Asp Ser Ala Lys Trp Ala
420 425 430
Asp Gly Lys Pro Tyr Ala Gln His His Lys Leu Val Ser Val Asp Asp
435 440 445
Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu Tyr Pro Ser Trp Leu Gln
450 455 460
Asp Phe Gly Tyr Ile Val Glu Ser Pro Glu Ala Ala Arg Gln Leu Asp
465 470 475 480
Ala Glu Leu Leu Ala Pro Gln Trp Lys Tyr Ser Gln Ala Thr Ala Thr
485 490 495
Phe Asp Tyr Ala Arg Gly Ile Cys Gln Gly
500 505
<210> 4
<211> 506
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Asp Ser Ser Ala Thr Pro His Leu Asp Ala Val Glu Gln Thr Leu Arg
1 5 10 15
Gln Val Ser Pro Gly Leu Glu Gly Arg Val Trp Glu Arg Thr Ala Gly
20 25 30
Asn Ala Leu Asp Ala Pro Ala Gly Asp Pro Ala Gly Trp Leu Leu Gln
35 40 45
Thr Pro Gly Cys Trp Gly Asp Ala Asn Cys Ala Glu Arg Thr Gly Thr
50 55 60
Lys Arg Leu Leu Ala Arg Met Thr Glu Asn Ile Ser Lys Ala Thr Arg
65 70 75 80
Thr Val Asp Ile Ser Thr Leu Ala Pro Phe Pro Asn Gly Ala Phe Gln
85 90 95
Asp Ala Ile Val Ala Gly Leu Lys Lys Ser Val Glu Asn Gly Asn Lys
100 105 110
Pro Lys Val Arg Val Leu Val Gly Ala Ala Pro Val Tyr His Met Asn
115 120 125
Val Leu Pro Ser Lys Tyr Arg Asp Asp Leu Arg Asp Lys Leu Gly Lys
130 135 140
Ala Ala Asp Gly Leu Thr Leu Asn Val Ala Ser Met Thr Thr Ser Lys
145 150 155 160
Thr Ala Phe Ser Trp Asn His Ser Lys Leu Leu Val Val Asp Gly Gln
165 170 175
Ser Ala Ile Thr Gly Gly Ile Asn Ser Trp Lys Asp Asp Tyr Val Asp
180 185 190
Thr Thr His Pro Val Ser Asp Val Asp Leu Ala Leu Thr Gly Pro Ala
195 200 205
Ala Gly Ser Ala Gly Arg Tyr Leu Asp Gln Leu Trp Thr Trp Thr Cys
210 215 220
Glu Asn Lys Ser Asn Ile Ala Ser Val Trp Phe Ala Ala Ser Pro Gly
225 230 235 240
Ala Gly Cys Met Pro Thr Met Glu Lys Asp Ala Asn Pro Val Pro Ala
245 250 255
Ala Ala Thr Gly Asn Val Pro Val Ile Ala Val Gly Gly Leu Gly Val
260 265 270
Gly Ile Lys Asp Ser Asp Pro Ser Ser Ala Phe Lys Pro Glu Leu Pro
275 280 285
Ser Ala Pro Asp Thr Lys Cys Val Val Gly Leu His Asp Asn Thr Asn
290 295 300
Ala Asp Arg Asp Tyr Asp Thr Val Asn Pro Glu Glu Ser Ala Leu Arg
305 310 315 320
Ala Leu Val Gly Ser Ala Arg Ser His Val Glu Ile Ser Gln Gln Asp
325 330 335
Leu Asn Ala Thr Cys Pro Ala Leu Pro Arg Tyr Asp Val Arg Leu Tyr
340 345 350
Asp Ala Leu Ala Ala Lys Leu Ala Ala Gly Val Lys Val Arg Ile Val
355 360 365
Val Ser Asp Pro Glu Asn Arg Gly Ala Val Gly Ser Gly Gly Leu Ser
370 375 380
Gln Ile Lys Ser Leu Asn Glu Ile Ser Asp Leu Leu Arg Asn Arg Leu
385 390 395 400
Ser Leu Leu Pro Gly Gly Ala Gln Gly Ala Lys Thr Ala Met Cys Gly
405 410 415
Asn Leu Gln Leu Ala Thr Ala Arg Ser Ser Asp Ser Ala Lys Trp Ala
420 425 430
Asp Gly Lys Pro Tyr Ala Gln His His Lys Leu Val Ser Val Asp Asp
435 440 445
Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu Tyr Pro Ser Trp Leu Gln
450 455 460
Asp Phe Gly Tyr Ile Val Glu Ser Pro Glu Ala Ala Arg Gln Leu Asp
465 470 475 480
Ala Glu Leu Leu Ala Pro Gln Trp Lys Tyr Ser Gln Ala Thr Ala Thr
485 490 495
Phe Asp Tyr Ala Arg Gly Ile Cys Gln Gly
500 505
<210> 5
<211> 506
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Asp Ser Ser Ala Thr Pro His Leu Asp Ala Val Glu Gln Thr Leu Arg
1 5 10 15
Gln Val Ser Pro Gly Leu Glu Gly Arg Val Trp Glu Arg Thr Ala Gly
20 25 30
Asn Ala Leu Asp Ala Pro Ala Gly Asp Pro Ala Gly Trp Leu Leu Gln
35 40 45
Thr Pro Gly Cys Trp Gly Asp Ala Asn Cys Ala Glu Arg Thr Gly Thr
50 55 60
Lys Arg Leu Leu Ala Arg Met Thr Glu Asn Ile Ser Lys Ala Thr Arg
65 70 75 80
Thr Val Asp Ile Ser Thr Leu Ala Pro Phe Pro Asn Gly Ala Phe Gln
85 90 95
Asp Ala Ile Val Ala Gly Leu Lys Lys Ser Val Glu Asn Gly Asn Lys
100 105 110
Pro Lys Val Arg Val Leu Val Gly Ala Ala Pro Val Tyr His Met Asn
115 120 125
Val Leu Pro Ser Lys Tyr Arg Asp Asp Leu Arg Asp Lys Leu Gly Lys
130 135 140
Ala Ala Asp Gly Leu Thr Leu Asn Val Ala Ser Met Thr Thr Ser Lys
145 150 155 160
Thr Ala Phe Ser Trp Asn His Ser Lys Leu Leu Val Val Asp Gly Gln
165 170 175
Ser Ala Ile Thr Gly Gly Ile Asn Ser Trp Lys Asp Asp Tyr Val Asp
180 185 190
Thr Thr His Pro Val Ser Asp Val Asp Leu Ala Leu Thr Gly Pro Ala
195 200 205
Ala Gly Ser Ala Gly Arg Tyr Leu Asp Gln Leu Trp Thr Trp Thr Cys
210 215 220
Glu Asn Lys Ser Asn Ile Ala Ser Val Trp Phe Ala Ala Ser Pro Gly
225 230 235 240
Ala Gly Cys Met Pro Thr Met Glu Lys Asp Ala Asn Pro Val Pro Ala
245 250 255
Ala Ala Thr Gly Asn Val Pro Val Ile Ala Val Gly Gly Leu Gly Val
260 265 270
Gly Ile Lys Asp Ser Asp Pro Ser Ser Ala Phe Lys Pro Glu Leu Pro
275 280 285
Ser Ala Pro Asp Thr Lys Cys Val Val Gly Leu His Asp Asn Thr Asn
290 295 300
Ala Asp Arg Asp Tyr Asp Thr Val Asn Pro Glu Glu Ser Ala Leu Arg
305 310 315 320
Ala Leu Val Gly Ser Ala Arg Ser His Val Glu Ile Ser Gln Gln Asp
325 330 335
Leu Asn Ala Thr Cys Pro Ala Leu Pro Arg Tyr Asp Val Arg Leu Tyr
340 345 350
Asp Ala Leu Ala Ala Lys Leu Ala Ala Gly Val Lys Val Arg Ile Val
355 360 365
Val Ser Asp Pro Glu Asn Arg Gly Ala Val Gly Val Gly Gly Leu Ser
370 375 380
Gln Ile Lys Ser Leu Asn Glu Ile Ser Asp Leu Leu Arg Asn Arg Leu
385 390 395 400
Ser Leu Leu Pro Gly Gly Ala Gln Gly Ala Lys Thr Ala Met Cys Gly
405 410 415
Asn Leu Gln Leu Ala Thr Ala Arg Ser Ser Asp Ser Ala Lys Trp Ala
420 425 430
Asp Gly Lys Pro Tyr Ala Gln His His Lys Leu Val Ser Val Asp Asp
435 440 445
Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu Tyr Pro Ser Trp Leu Gln
450 455 460
Asp Phe Gly Tyr Ile Val Glu Ser Pro Glu Ala Ala Arg Gln Leu Asp
465 470 475 480
Ala Glu Leu Leu Ala Pro Gln Trp Lys Tyr Ser Gln Ala Thr Ala Thr
485 490 495
Phe Asp Tyr Ala Arg Gly Ile Cys Gln Gly
500 505
<210> 6
<211> 1518
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gatagctctg cgaccccgca cctggatgcg gttgaacaga ccctgcgtca ggttagccct 60
ggtctggaag gccgcgtttg ggaacgtacc gcgggtaacg ctctggatgc gccggcgggt 120
gatccggcgg gttggctgtt gcagacccca ggttgctggg gtgatgcgaa ctgcgcggaa 180
cgtaccggca ccaaacgtct gctggcacgt atgaccgaaa acatctctaa agcgacccgc 240
accgttgaca tctctaccct ggcgccgttc ccgaacggtg cgttccagga tgcgatcgtt 300
gcgggtctga aaaagtctgt tgaaaacggt aacaaaccga aagttcgcgt tctggtgggt 360
gcagcgccgg tttaccacat gaacgttctg ccgtccaaat accgtgatga tctgcgtgat 420
aaactgggca aagccgcgga tggcctgacc ctgaacgttg caagcatgac caccagcaaa 480
accgcattca gctggaacca ctctaaactg ctggttgtgg atggtcagtc tgcgatcacc 540
ggtggcatca acagctggaa agatgattac gttgacacca cccacccggt gagcgacgtt 600
gatctggcgc tgaccggtcc ggcggcgggt agcgcgggtc gttacctgga ccagctgtgg 660
acctggacct gcgaaaacaa atccaacatc gcaagcgttt ggtttgcggc ctctccgggc 720
gctggctgta tgccgacgat ggaaaaagat gctaacccgg ttccggcggc tgcgaccggt 780
aacgttccgg tgatcgcggt gggcggtctg ggtgttggca tcaaagattc cgatccgagc 840
agcgcgttca aaccggaact gccgagcgcc ccggatacca aatgcgttgt tggtctgcac 900
gataacacca acgcggaccg tgattacgat accgttaacc cggaagaaag cgcgctgcgt 960
gctctggtgg gcagcgcgcg ttcccacgtt gaaatctctc agcaggatct gaacgcgacc 1020
tgcccgccgc tgccgcgtta tgacgtgcgc ctgtacgatg cactggcggc gaaactggcg 1080
gctggcgtga aagtgcgtat cgttgtgagc gacccggaaa accgtggcgc ggttggctct 1140
ggcggttact ctcagatcaa atccctgaac gaaatctccg acctgctgcg taaccgtctg 1200
agcctgctgc cgggcggtgc tcagggtgct aaaaccgcta tgtgcggtaa cctgcaactg 1260
gcgaccgcgc gcagcagcga ctctgctaaa tgggctgatg gtaaaccgta cgcgcagcac 1320
cacaaactgg ttagcgttga tgattctgca ttctacatcg gtagcaaaaa cctgtacccg 1380
agctggctgc aagatttcgg ttacatcgtt gaaagcccgg aagcggcgcg tcagctggat 1440
gcggaactgc tggcgccgca gtggaaatac agccaggcga ccgcgacctt cgattacgcg 1500
cgtggcatct gccagggt 1518
<210> 7
<211> 1518
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gatagctctg cgaccccgca cctggatgcg gttgaacaga ccctgcgtca ggttagccct 60
ggtctggaag gccgcgtttg ggaacgtacc gcgggtaacg ctctggatgc gccggcgggt 120
gatccggcgg gttggctgtt gcagacccca ggttgctggg gtgatgcgaa ctgcgcggaa 180
cgtaccggca ccaaacgtct gctggcacgt atgaccgaaa acatctctaa agcgacccgc 240
accgttgaca tctctaccct ggcgccgttc ccgaacggtg cgttccagga tgcgatcgtt 300
gcgggtctga aaaagtctgt tgaaaacggt aacaaaccga aagttcgcgt tctggtgggt 360
gcagcgccgg tttaccacat gaacgttctg ccgtccaaat accgtgatga tctgcgtgat 420
aaactgggca aagccgcgga tggcctgacc ctgaacgttg caagcatgac caccagcaaa 480
accgcattca gctggaacca ctctaaactg ctggttgtgg atggtcagtc tgcgatcacc 540
ggtggcatca acagctggaa agatgattac gttgacacca cccacccggt gagcgacgtt 600
gatctggcgc tgaccggtcc ggcggcgggt agcgcgggtc gttacctgga ccagctgtgg 660
acctggacct gcgaaaacaa atccaacatc gcaagcgttt ggtttgcggc ctctccgggc 720
gctggctgta tgccgacgat ggaaaaagat gctaacccgg ttccggcggc tgcgaccggt 780
aacgttccgg tgatcgcggt gggcggtctg ggtgttggca tcaaagattc cgatccgagc 840
agcgcgttca aaccggaact gccgagcgcc ccggatacca aatgcgttgt tggtctgcac 900
gataacacca acgcggaccg tgattacgat accgttaacc cggaagaaag cgcgctgcgt 960
gctctggtgg gcagcgcgcg ttcccacgtt gaaatctctc agcaggatct gaacgcgacc 1020
tgcccggcgc tgccgcgtta tgacgtgcgc ctgtacgatg cactggcggc gaaactggcg 1080
gctggcgtga aagtgcgtat cgttgtgagc gacccggaaa accgtggcgc ggttggctct 1140
ggcggttact ctcagatcaa atccctgaac gaaatctccg acctgctgcg taaccgtctg 1200
agcctgctgc cgggcggtgc tcagggtgct aaaaccgcta tgtgcggtaa cctgcaactg 1260
gcgaccgcgc gcagcagcga ctctgctaaa tgggctgatg gtaaaccgta cgcgcagcac 1320
cacaaactgg ttagcgttga tgattctgca ttctacatcg gtagcaaaaa cctgtacccg 1380
agctggctgc aagatttcgg ttacatcgtt gaaagcccgg aagcggcgcg tcagctggat 1440
gcggaactgc tggcgccgca gtggaaatac agccaggcga ccgcgacctt cgattacgcg 1500
cgtggcatct gccagggt 1518
<210> 8
<211> 1518
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gatagctctg cgaccccgca cctggatgcg gttgaacaga ccctgcgtca ggttagccct 60
ggtctggaag gccgcgtttg ggaacgtacc gcgggtaacg ctctggatgc gccggcgggt 120
gatccggcgg gttggctgtt gcagacccca ggttgctggg gtgatgcgaa ctgcgcggaa 180
cgtaccggca ccaaacgtct gctggcacgt atgaccgaaa acatctctaa agcgacccgc 240
accgttgaca tctctaccct ggcgccgttc ccgaacggtg cgttccagga tgcgatcgtt 300
gcgggtctga aaaagtctgt tgaaaacggt aacaaaccga aagttcgcgt tctggtgggt 360
gcagcgccgg tttaccacat gaacgttctg ccgtccaaat accgtgatga tctgcgtgat 420
aaactgggca aagccgcgga tggcctgacc ctgaacgttg caagcatgac caccagcaaa 480
accgcattca gctggaacca ctctaaactg ctggttgtgg atggtcagtc tgcgatcacc 540
ggtggcatca acagctggaa agatgattac gttgacacca cccacccggt gagcgacgtt 600
gatctggcgc tgaccggtcc ggcggcgggt agcgcgggtc gttacctgga ccagctgtgg 660
acctggacct gcgaaaacaa atccaacatc gcaagcgttt ggtttgcggc ctctccgggc 720
gctggctgta tgccgacgat ggaaaaagat gctaacccgg ttccggcggc tgcgaccggt 780
aacgttccgg tgatcgcggt gggcggtctg ggtgttggca tcaaagattc cgatccgagc 840
agcgcgttca aaccggaact gccgagcgcc ccggatacca aatgcgttgt tggtctgcac 900
gataacacca acgcggaccg tgattacgat accgttaacc cggaagaaag cgcgctgcgt 960
gctctggtgg gcagcgcgcg ttcccacgtt gaaatctctc agcaggatct gaacgcgacc 1020
tgcccgccgc tgccgcgtta tgacgtgcgc ctgtacgatg cactggcggc gaaactggcg 1080
gctggcgtga aagtgcgtat cgttgtgagc gacccggaaa accgtggcgc ggttggctct 1140
ggcggtttct ctcagatcaa atccctgaac gaaatctccg acctgctgcg taaccgtctg 1200
agcctgctgc cgggcggtgc tcagggtgct aaaaccgcta tgtgcggtaa cctgcaactg 1260
gcgaccgcgc gcagcagcga ctctgctaaa tgggctgatg gtaaaccgta cgcgcagcac 1320
cacaaactgg ttagcgttga tgattctgca ttctacatcg gtagcaaaaa cctgtacccg 1380
agctggctgc aagatttcgg ttacatcgtt gaaagcccgg aagcggcgcg tcagctggat 1440
gcggaactgc tggcgccgca gtggaaatac agccaggcga ccgcgacctt cgattacgcg 1500
cgtggcatct gccagggt 1518
<210> 9
<211> 1518
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gatagctctg cgaccccgca cctggatgcg gttgaacaga ccctgcgtca ggttagccct 60
ggtctggaag gccgcgtttg ggaacgtacc gcgggtaacg ctctggatgc gccggcgggt 120
gatccggcgg gttggctgtt gcagacccca ggttgctggg gtgatgcgaa ctgcgcggaa 180
cgtaccggca ccaaacgtct gctggcacgt atgaccgaaa acatctctaa agcgacccgc 240
accgttgaca tctctaccct ggcgccgttc ccgaacggtg cgttccagga tgcgatcgtt 300
gcgggtctga aaaagtctgt tgaaaacggt aacaaaccga aagttcgcgt tctggtgggt 360
gcagcgccgg tttaccacat gaacgttctg ccgtccaaat accgtgatga tctgcgtgat 420
aaactgggca aagccgcgga tggcctgacc ctgaacgttg caagcatgac caccagcaaa 480
accgcattca gctggaacca ctctaaactg ctggttgtgg atggtcagtc tgcgatcacc 540
ggtggcatca acagctggaa agatgattac gttgacacca cccacccggt gagcgacgtt 600
gatctggcgc tgaccggtcc ggcggcgggt agcgcgggtc gttacctgga ccagctgtgg 660
acctggacct gcgaaaacaa atccaacatc gcaagcgttt ggtttgcggc ctctccgggc 720
gctggctgta tgccgacgat ggaaaaagat gctaacccgg ttccggcggc tgcgaccggt 780
aacgttccgg tgatcgcggt gggcggtctg ggtgttggca tcaaagattc cgatccgagc 840
agcgcgttca aaccggaact gccgagcgcc ccggatacca aatgcgttgt tggtctgcac 900
gataacacca acgcggaccg tgattacgat accgttaacc cggaagaaag cgcgctgcgt 960
gctctggtgg gcagcgcgcg ttcccacgtt gaaatctctc agcaggatct gaacgcgacc 1020
tgcccggcgc tgccgcgtta tgacgtgcgc ctgtacgatg cactggcggc gaaactggcg 1080
gctggcgtga aagtgcgtat cgttgtgagc gacccggaaa accgtggcgc ggttggctct 1140
ggcggtttct ctcagatcaa atccctgaac gaaatctccg acctgctgcg taaccgtctg 1200
agcctgctgc cgggcggtgc tcagggtgct aaaaccgcta tgtgcggtaa cctgcaactg 1260
gcgaccgcgc gcagcagcga ctctgctaaa tgggctgatg gtaaaccgta cgcgcagcac 1320
cacaaactgg ttagcgttga tgattctgca ttctacatcg gtagcaaaaa cctgtacccg 1380
agctggctgc aagatttcgg ttacatcgtt gaaagcccgg aagcggcgcg tcagctggat 1440
gcggaactgc tggcgccgca gtggaaatac agccaggcga ccgcgacctt cgattacgcg 1500
cgtggcatct gccagggt 1518
<210> 10
<211> 1518
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gatagctctg cgaccccgca cctggatgcg gttgaacaga ccctgcgtca ggttagccct 60
ggtctggaag gccgcgtttg ggaacgtacc gcgggtaacg ctctggatgc gccggcgggt 120
gatccggcgg gttggctgtt gcagacccca ggttgctggg gtgatgcgaa ctgcgcggaa 180
cgtaccggca ccaaacgtct gctggcacgt atgaccgaaa acatctctaa agcgacccgc 240
accgttgaca tctctaccct ggcgccgttc ccgaacggtg cgttccagga tgcgatcgtt 300
gcgggtctga aaaagtctgt tgaaaacggt aacaaaccga aagttcgcgt tctggtgggt 360
gcagcgccgg tttaccacat gaacgttctg ccgtccaaat accgtgatga tctgcgtgat 420
aaactgggca aagccgcgga tggcctgacc ctgaacgttg caagcatgac caccagcaaa 480
accgcattca gctggaacca ctctaaactg ctggttgtgg atggtcagtc tgcgatcacc 540
ggtggcatca acagctggaa agatgattac gttgacacca cccacccggt gagcgacgtt 600
gatctggcgc tgaccggtcc ggcggcgggt agcgcgggtc gttacctgga ccagctgtgg 660
acctggacct gcgaaaacaa atccaacatc gcaagcgttt ggtttgcggc ctctccgggc 720
gctggctgta tgccgacgat ggaaaaagat gctaacccgg ttccggcggc tgcgaccggt 780
aacgttccgg tgatcgcggt gggcggtctg ggtgttggca tcaaagattc cgatccgagc 840
agcgcgttca aaccggaact gccgagcgcc ccggatacca aatgcgttgt tggtctgcac 900
gataacacca acgcggaccg tgattacgat accgttaacc cggaagaaag cgcgctgcgt 960
gctctggtgg gcagcgcgcg ttcccacgtt gaaatctctc agcaggatct gaacgcgacc 1020
tgcccggcgc tgccgcgtta tgacgtgcgc ctgtacgatg cactggcggc gaaactggcg 1080
gctggcgtga aagtgcgtat cgttgtgagc gacccggaaa accgtggcgc ggttggcgtt 1140
ggcggtttct ctcagatcaa atccctgaac gaaatctccg acctgctgcg taaccgtctg 1200
agcctgctgc cgggcggtgc tcagggtgct aaaaccgcta tgtgcggtaa cctgcaactg 1260
gcgaccgcgc gcagcagcga ctctgctaaa tgggctgatg gtaaaccgta cgcgcagcac 1320
cacaaactgg ttagcgttga tgattctgca ttctacatcg gtagcaaaaa cctgtacccg 1380
agctggctgc aagatttcgg ttacatcgtt gaaagcccgg aagcggcgcg tcagctggat 1440
gcggaactgc tggcgccgca gtggaaatac agccaggcga ccgcgacctt cgattacgcg 1500
cgtggcatct gccagggt 1518

Claims (5)

1.一种磷脂酶D突变体，其特征在于，其氨基酸序列为SEQ ID NO:2，SEQ ID NO:3，SEQID NO:4或SEQ ID NO:5。
2.一种编码权利要求1所述磷脂酶D突变体的基因，其特征在于，其核酸序列为SEQ IDNO:7，SEQ ID NO:8，SEQ ID NO:9或SEQ ID NO:10。
3.一种含权利要求2所述基因的重组基因工程菌。
4.一种磷脂酶D重组表达菌株的制备方法，其特征在于，将权利要求2所述基因克隆到表达载体pET-21a、pET28a或pET32a上，转化大肠杆菌SHuffle T7感受态细胞，获得重组基因工程菌，之后将重组基因工程菌发酵，得到高活力磷脂酶D。
5.权利要求1所述磷脂酶D突变体的应用，其特征在于，所述的磷脂酶D突变体应用于磷脂酰丝氨酸的生产中。
CN202110962378.XA 2021-08-20 2021-08-20 一种海洋链霉菌磷脂酶d突变体及其重组表达菌株的制备方法 Active CN113801862B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110962378.XA CN113801862B (zh) 2021-08-20 2021-08-20 一种海洋链霉菌磷脂酶d突变体及其重组表达菌株的制备方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110962378.XA CN113801862B (zh) 2021-08-20 2021-08-20 一种海洋链霉菌磷脂酶d突变体及其重组表达菌株的制备方法

Publications (2)

Publication Number Publication Date
CN113801862A true CN113801862A (zh) 2021-12-17
CN113801862B CN113801862B (zh) 2023-06-20

Family

ID=78941685

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110962378.XA Active CN113801862B (zh) 2021-08-20 2021-08-20 一种海洋链霉菌磷脂酶d突变体及其重组表达菌株的制备方法

Country Status (1)

Country Link
CN (1) CN113801862B (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112662644A (zh) * 2021-01-19 2021-04-16 华南理工大学 一种甘油磷酸二酯磷酸二酯酶突变体及其应用
CN112662645A (zh) * 2021-01-19 2021-04-16 华南理工大学 一种鞘磷脂酶d突变体及其应用
CN114525266A (zh) * 2022-02-22 2022-05-24 华南理工大学 一种来源于南极细菌的磷脂酶d突变体及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040110180A1 (en) * 2001-04-06 2004-06-10 Shirley Recipon Kinases and phosphatases
US7829682B1 (en) * 2003-04-30 2010-11-09 Incyte Corporation Human β-adrenergic receptor kinase nucleic acid molecule
CN108118041A (zh) * 2017-12-29 2018-06-05 华南理工大学 一种磷脂酶d突变体、重组基因工程菌及其制备方法和应用
CN110129300A (zh) * 2016-06-02 2019-08-16 天津科技大学 一种新型磷脂酶d
CN112899256A (zh) * 2021-01-29 2021-06-04 华南理工大学 一种来源于南极细菌的耐低温磷脂酶d及其制备方法和应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040110180A1 (en) * 2001-04-06 2004-06-10 Shirley Recipon Kinases and phosphatases
US7829682B1 (en) * 2003-04-30 2010-11-09 Incyte Corporation Human β-adrenergic receptor kinase nucleic acid molecule
CN110129300A (zh) * 2016-06-02 2019-08-16 天津科技大学 一种新型磷脂酶d
CN110218712A (zh) * 2016-06-02 2019-09-10 天津科技大学 磷脂酶d突变体及其在制备磷脂酸、磷脂酰丝氨酸中的应用
CN108118041A (zh) * 2017-12-29 2018-06-05 华南理工大学 一种磷脂酶d突变体、重组基因工程菌及其制备方法和应用
CN112899256A (zh) * 2021-01-29 2021-06-04 华南理工大学 一种来源于南极细菌的耐低温磷脂酶d及其制备方法和应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FANGHUA WANG ET AL.: "Crystal Structure of a Phospholipase D from the Plant-Associated Bacteria Serratia plymuthica Strain AS9 Reveals a Unique Arrangement of Catalytic Pocket", INT J MOL SCI *
冯小娜 等: "蛋白质工程改造磷脂酶D提高磷脂酰丝氨酸产量", 食品工业科技 *
董自星 等: "微生物磷脂酶D的克隆表达研究进展", 食品工业科技 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112662644A (zh) * 2021-01-19 2021-04-16 华南理工大学 一种甘油磷酸二酯磷酸二酯酶突变体及其应用
CN112662645A (zh) * 2021-01-19 2021-04-16 华南理工大学 一种鞘磷脂酶d突变体及其应用
CN112662645B (zh) * 2021-01-19 2022-04-22 华南理工大学 一种鞘磷脂酶d突变体及其应用
CN112662644B (zh) * 2021-01-19 2022-04-22 华南理工大学 一种甘油磷酸二酯磷酸二酯酶突变体及其应用
CN114525266A (zh) * 2022-02-22 2022-05-24 华南理工大学 一种来源于南极细菌的磷脂酶d突变体及其应用
CN114525266B (zh) * 2022-02-22 2023-06-20 华南理工大学 一种来源于南极细菌的磷脂酶d突变体及其应用

Also Published As

Publication number Publication date
CN113801862B (zh) 2023-06-20

Similar Documents

Publication Publication Date Title
CN113801862B (zh) 一种海洋链霉菌磷脂酶d突变体及其重组表达菌株的制备方法
CN108118041B (zh) 一种磷脂酶d突变体、重组基因工程菌及其制备方法和应用
CN114945663B (zh) 耐高温逆转录酶突变体及其应用
CN110229805B (zh) 一种通过序列一致性制备的谷氨酸脱羧酶突变体及其应用
CN107267471B (zh) 双功能谷胱甘肽合成酶突变体、核苷酸序列及其制备方法和应用
CN112795546B (zh) 一种具有高逆转录效率的耐高温逆转录酶突变体及其应用
CN112795550B (zh) 耐高温逆转录酶突变体
CN113430181B (zh) 一种来源亚洲象肠道宏基因组的细菌漆酶及其基因
CN115161303A (zh) 一种磷脂酶突变体及其合成甘油磷脂的方法
CN114507658A (zh) 酶共表达系统及其在合成唾液酸的应用
CN113604453B (zh) 一种海洋链霉菌磷脂酶d突变体和应用
CN112359032B (zh) 突变型酯酶及其应用、重组载体及其制备方法和应用、重组工程菌及其应用
CN107916257B (zh) T1脂肪酶突变体和应用
CN113249349B (zh) 突变型醇脱氢酶、重组载体及其制备方法和应用
CN115247158B (zh) 一种甘油磷酸氧化酶突变体及其筛选方法、制备方法和应用
CN112662645B (zh) 一种鞘磷脂酶d突变体及其应用
CN112301014B (zh) 一种热稳定性提高的酯酶突变体及其应用
CN114480329B (zh) 高效率mmlv酶突变体
CN114480334B (zh) 用于新冠病毒检测的逆转录酶突变体
CN114480337B (zh) 逆转录酶突变体及逆转录方法
CN108220268B (zh) 一种禾谷镰胞菌脂肪酶突变体及其编码基因和工程菌
CN114250206B (zh) 甲基转移酶突变体、重组载体、重组工程菌及其应用
CN114525266B (zh) 一种来源于南极细菌的磷脂酶d突变体及其应用
CN114480335B (zh) 逆转录酶及逆转录检测试剂
CN114480338B (zh) 用于核酸检测的逆转录酶突变体

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant