CN113583983A

CN113583983A - 一种融合蛋白或其变体及其在制备骨化二醇中的应用

Info

Publication number: CN113583983A
Application number: CN202010369514.XA
Authority: CN
Inventors: 吴燕; 田振华
Original assignee: Ecolab Biotechnology Shanghai Co Ltd
Current assignee: Ecolab Biotechnology Shanghai Co Ltd
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2021-11-02

Abstract

本发明提供了一种融合蛋白或其变体，其包括K1和RhFR，所述K1的氨基酸序列如SEQ ID NO:1所示，所述RhFR的氨基酸序列如SEQ ID NO:5的第466‑773位的氨基酸所示。本发明还提供了一种所述融合蛋白或其变体的制备方法和应用。将本发明的融合蛋白或其变体应用于催化VD3合成25‑羟基维生素D3(骨化二醇)时无需额外添加电子传递相关蛋白，操作简便，且所述融合蛋白中的电子传递效率高，催化效率高，所得骨化二醇的产量显著提高，且降低了生产的成本，适于工业化生产。

Description

一种融合蛋白或其变体及其在制备骨化二醇中的应用

技术领域

本发明涉及生物技术领域，具体涉及一种融合蛋白或其变体及其基因、其制备方法，以及融合蛋白或其变体在催化VD3制备骨化二醇中的用途。

背景技术

25-羟基维生素D3，又名骨化二醇，是维生素D3(VD3)的活性代谢物，具有较强的生理活性。传统合成25-羟基维生素D3的方法是化学合成法，需要进行多步的基团保护与脱保护，然后经光照反应、开环和异构化的到25-羟基维生素D3。近年来对VD3的微生物转化方面的研究得到了快速的发展，但是转化率低，而提高活性VD3产量的关键之一在于筛选获得转化效率高的关键代谢酶细胞色素P450酶。

细胞色素P450酶广泛存在于原核生物与真核生物中，其介导的单加氧反应通常需要电子传递链铁氧还蛋白与铁氧还蛋白还原酶的参与，且高效的电子传递体系能有效地提高P450的反应效率。也有自洽型P450的报道，反应过程中无需再添加电子传递相关的蛋白。目前，已报道2种自洽型P450，即来源于巨大芽孢杆菌Bacillus megaterium的P450BM3和来源红球菌Rhodococcus的P450 RhFRed，其蛋白结构上包含P450结构域与电子传递结构域，反应过程中无需再添加电子传递相关的蛋白。但目前未见自洽型P450用于催化VD3产生骨化二醇的报道。

Tamura等(BioChemical and Biophysical Research Communications 2009,385,170–175)对Pseudonocardia autotrophica来源的P450酶进行了改造得到了4个位点突变的突变体Vdh-K1(T70R、V156L、E216M和E384R)，并测得用于VD3的催化时Vdh-K1是野生型Vdhwt的活性的21.6倍。但是用于VD3的催化需要电子传递体系的参与，操作繁琐、成本较高，且催化效率仍旧不是很高。Tamura等(ChemBioChem 2013,14,2284-2291)对Pseudonocardiaautotrophica来源的P450酶进行了改造(进行了T107A等突变)，并在红球菌中进行异源表达，通过添加乳酸链球菌素以提高细胞膜的通透性，减少底物VD3传质阻力，最终催化VD3两小时得到的骨化二醇的产量达到573μg/mL，方法中采用了电子传递系统AciFdx和AciFdxR，但产率还有待提高。

因此现有技术中急需一种催化VD3制备骨化二醇时催化效率高、所得产物的产量很高且操作简便、成本较低的方法。

发明内容

本发明所要解决的技术问题是针对现有技术中用细胞色素P450酶催化VD3制备骨化二醇时产率低、成本高、操作繁琐等缺陷，提供了一种融合蛋白或其变体及其基因、其制备方法与用其催化VD3制备骨化二醇的应用。将本发明的融合蛋白或其变体应用于催化VD3合成25-羟基维生素D3(骨化二醇)时无需额外添加电子传递相关蛋白，操作简便，且所述融合蛋白中的电子传递效率高，催化效率高，所得骨化二醇的产量显著提高，且降低了生产的成本，适于工业化生产。

提高P450酶催化VD3产率的方法众多，包括寻求高效的P450酶、优化反应条件等，本发明人经过大量摸索，意外发现将特定种类的P450通过特定的方式改造为自洽型P450酶时，能够显著提高催化VD3制备骨化二醇时的催化效率，并显著提高骨化二醇的产量。

为了解决上述技术问题，本发明第一方面提供了一种融合蛋白或其变体，其包括K1和RhFR，所述K1的氨基酸序列如SEQ ID NO:1所示，所述RhFR的氨基酸序列如SEQ ID NO:5的第466-773位的氨基酸所示。

较佳地，所述融合蛋白从N端至C端依次为K1和RhFR。

较佳地，所述K1和RhFR之间通过连接子(linker)进行连接，所述连接子的氨基酸序列优选如SEQ ID NO:5的第445-465位的氨基酸所示。

较佳地，所述融合蛋白或其变体与分子伴侣共表达，所述分子伴侣优选为Gro7。在本发明某一较佳实施例中，表达所述融合蛋白时，可以加入分子伴侣例如Gro7伴侣蛋白，使得所述融合蛋白与所述分子伴侣共表达。本发明中，所述Gro7伴侣蛋白可以是商购来源的，例如可以是购自Biovector Science Lab,Inc的Gro7。

较佳地，所述变体为在所述RhFR的N端发生氨基酸的插入或缺失；更佳地，所述变体为在所述RhFR的N端发生1-14个氨基酸的插入或缺失；进一步更佳地，所述变体为在所述RhFR的N端发生3-6个氨基酸的插入或缺失。

较佳地，所述融合蛋白或其变体的氨基酸序列如SEQ ID NO:9、SEQ ID NO:11、SEQID NO:13、SEQ ID NO:15、SEQ ID NO:17或SEQ ID NO:19所示。

更佳地，编码所述融合蛋白或其变体的核苷酸序列如SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18或SEQ ID NO:20所示。

为了解决上述技术问题，本发明第二方面提供了一种融合基因，其编码如本发明第一方面所述的融合蛋白或其变体。

为了解决上述技术问题，本发明第三方面提供了一种重组表达载体，所述重组表达载体含有如本发明第二方面所述的融合基因。

较佳地，所述重组表达载体的骨架载体为pET28a。

为了解决上述技术问题，本发明第四方面提供了一种转化体，其包括如本发明第二方面所述的融合基因或者如本发明第三方面所述的重组表达载体。

较佳地，所转化体通过在宿主中导入如本发明第二方面所述的融合基因或者如本发明第三方面所述的重组表达载体获得。

更佳地，所述宿主为大肠杆菌，优选为大肠杆菌E.coli BL21(DE3)细胞。

为了解决上述技术问题，本发明第五方面提供了一种融合蛋白或其变体的制备方法，其包括以下步骤：

(1)获得如本发明第四方面所述的转化体；

(2)筛选所述转化体，表达并纯化所述融合蛋白或其变体。

为了解决上述技术问题，本发明第六方面提供了一种骨化二醇的制备方法，所述制备方法包括以下步骤：在反应溶剂、还原型辅酶NADH/NADPH的存在下，将如本发明第一方面所述的融合蛋白或其变体催化维生素D3进行羟化反应即可。

较佳地，所述维生素D3为助溶剂预溶的维生素D3；所述助溶剂优选包括DMSO、吐温80、Triton X100、甲醇、乙醇、异丙醇和DMF中的一种或多种，例如为乙醇。

较佳地，所述方法还包括在进行所述羟化反应前，在所述反应溶剂中加入环糊精的步骤，所述环糊精例如可以为羟丙基-β-环糊精的步骤；所述羟丙基-β-环糊精占反应体系的质量体积百分比优选为0.05％-0.4％，例如0.25％。

较佳地，所述反应的温度为20～33℃，例如为22℃、25℃、28℃或30℃。本发明人实验过程中发现，不在此温度范围内时，所得产物的产量会有所降低。

较佳地，所述反应的pH为6.0～8.0，例如为7.4。本发明人在实验过程中发现，pH不在本发明规定的范围内时，所得产物的产量会有所降低。

较佳地，所述维生素D3的浓度为1g/L-10g/L，例如为1g/L、2g/L、3g/L、4g/L、5g/L、6g/L、7g/L、8g/L、9g/L或10g/L。本发明人实验过程中发现，VD3浓度过高，无法溶解或者可能底物抑制酶活，反应不完全，浓度过低，产量也很低。

较佳地，所述NADH/NADPH与所述维生素D3的摩尔比为0.001:1～2:1，例如0.2:1。

较佳地，所述的制备方法还包括以下步骤：在脱氢酶以及供氢体的存在下，将氧化型辅酶NAD⁺/NADP⁺进行还原反应，得到所述的还原型辅酶NADH/NADPH即可；

更佳地，所述的脱氢酶为葡萄糖脱氢酶、醇脱氢酶或甲酸脱氢酶；和/或，所述的供氢体为葡萄糖、异丙醇或甲酸盐；

进一步更佳地，当所述的脱氢酶为醇脱氢酶时，所述的供氢体为异丙醇；当所述的脱氢酶为葡萄糖脱氢酶时，所述的供氢体为葡萄糖；当所述的脱氢酶为甲酸脱氢酶时，所述的供氢体为甲酸盐。

为了解决上述技术问题，本发明第七方面提供了一种如本发明第一方面所述的融合蛋白或其变体、如本发明第二方面的融合基因、如本发明第三方面所述的重组表达载体、或如本发明第四方面所述的转化体在制备骨化二醇中的应用。

在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。

本发明所用试剂和原料均市售可得。

本发明的积极进步效果在于：将本发明的融合蛋白或其变体应用于催化VD3合成25-羟基维生素D3(骨化二醇)时无需额外添加电子传递相关蛋白，操作简便，且所述融合蛋白中的电子传递效率高，催化效率高，所得骨化二醇的产量显著提高，且降低了生产的成本，适于工业化生产。在本发明某一较佳实施例中，本发明的融合蛋白或其变体催化VD3时，所得骨化二醇的产量高达4.427g/L。

附图说明

图1为实施例6中K1-RhFR-I6-Gro7的检测图谱。

图2为VD3底物对照品的图谱结果。

图3为骨化二醇对照品的图谱结果。

具体实施方式

下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。

产物的HPLC分析方法

色谱条件：色谱柱：Poroshell EC-C18(4.0μm,4.6×150mm)；检测波长：265nm；流速：1mL/min；柱温：35℃；进样体积：10μL。梯度洗脱程序如下：0-8min，H2O：乙腈＝85％：15％；8-20min，H2O：乙腈＝0：100％；20-21min，H2O：乙腈＝85％：15％；21-27min，H2O：乙腈＝85％：15％。

pET28a购买自Novagen公司；DpnI酶、NdeI酶、HindIII酶购买自Thermo Fisher公司；ExnaseⅡ酶购买至南京诺唯赞生物科技有限公司；E.coli BL21(DE3)感受态细胞购买自北京鼎国昌盛生物技术有限责任公司；NAD+购买自深圳邦泰生物工程有限公司；维生素D3、质粒提取试剂盒购买自生工生物工程(上海)股份有限公司。

实施例1融合蛋白酶菌株的构建

1.1蛋白序列分析

本实施例将能催化VD3的P450酶Vdh-K1与P450 BM3和P450 RhFRed的还原区域进行融合，构建自洽型P450融合蛋白，具体步骤如下：

根据编码NCBI上已报道的细胞色素P450酶SEQ ID NO:1、3、5的基因序列SEQ IDNO:2、4、6全基因合成各基因。合成基因的公司为苏州金唯智生物科技有限公司(苏州工业园区星湖街218号生物纳米科技园C3楼)。各基因信息如表1所示。

表1

利用Discovery studio软件分析BM3蛋白的氨基酸序列，发现N端的第1-459位氨基酸为蛋白的P450结构域；第460-479位氨基酸为P450结构域与电子传递结构域的Linker；第480-1048位氨基酸为蛋白的电子传递结构域。

利用Discovery studio软件分析RhFRed蛋白的氨基酸序列，发现N端的第24-444位氨基酸为蛋白的P450结构域；第445-465位氨基酸为P450结构域与电子传递结构域的Linker(连接子)；第466-773位氨基酸为蛋白的电子传递结构域。

1.2K1、FdR、Fdx蛋白工程菌株以及融合蛋白工程菌株构建

将实施例1.1合成的K1、BM3、RhFRed以及合成的电子传递系统AciFdR、AciFdx(如下表2所示)基因分别连pET28a，酶切位点NdeI&HindIII，将酶连好的载体转化至宿主E.coli BL21(DE3)感受态细胞，分别得到含有K1、BM3、RhFRed、AciFdR、AciFdx的工程菌株。利用上海生工的质粒提取试剂盒提取质粒，分别得到pET28a-K1、pET28a-BM3、pET28a-RhFRed、pET28a-FdR、pET28a-Fdx质粒。

将所得pET28a-K1、pET28a-FdR质粒分别与Gro7伴侣质粒共转E.coli BL21，得到BL21-pET28a-K1-Gro7和BL21-pET28a-FdR-Gro7工程菌。将pET28a-Fdx质粒转化BL21，得到BL21-pET28a-Fdx工程菌。

表2

酶编号	基因来源	NCBI登录号
			AciFdx	Acinetobacter sp.OC4	BAE78451.1
AciFdR	Acinetobacter sp.OC4	BAE78453.1

以原质粒pET28a-K1为模板，K1-Rh-F1、K1-Rh-R1为引物，扩增目的片段ΔK1-1。以pET28a-RhFRed质粒为模板，RhFR-F1、RhFR-R1为引物，扩增5’端与ΔK1-1的3’端具有15bP同源臂，且3’端与ΔK1-1的5’端具有15bP同源臂的载体片段RhFR。将PCR产物用Dpn1消化，37℃，2小时。反应完成后ΔK1-1和RhFR用重组酶ExnaseⅡ进行重组，37℃，0.5小时，重组产物转化至BL21感受态细胞，涂布在含有50μg/mL卡那霉素的LB培养基，37℃培养过夜，得到BL21-pET28a-K1-RhFR转化子，即BL21-pET28a-K1-RhFR工程菌。挑取BL21-pET28a-K1-RhFR转化子接种至含50μg/mL卡那霉素的5ml LB液体培养基中，37℃震荡培养6h，提取pET28a-K1-RhFR质粒，将pET28a-K1-RhFR质粒与Gro7伴侣质粒共转BL21，得到BL21-pET28a-K1-RhFR-Gro7工程菌。引物及其序列如表2所示。

以原质粒pET28a-K1为模板，K1-BM3-F1、K1-BM3-R1为引物，扩增目的片段ΔK1-2。以pET28a-BM3质粒为模板，BM3R-F1、BM3R-R1为引物，扩增5’端与ΔK1-2的3’端具有15bP同源臂，且3’端与K1-2的5’端具有15bP同源臂的载体片段BM3R。将PCR产物用Dpn1消化，37℃，2小时。反应完成后ΔK1-2和BM3R用重组酶ExnaseⅡ进行重组，37℃，0.5小时，重组产物转化至BL21感受态细胞，涂布在含有50μg/mL卡那霉素的LB培养基，37℃培养过夜，得到BL21-pET28a-K1-BM3R转化子，即BL21-pET28a-K1-BM3R工程菌。挑取BL21-pET28a-K1-BM3R转化子接种至含50μg/mL卡那霉素的5ml LB液体培养基中，37℃震荡培养6h，提取pET28a-K1-BM3R质粒，将pET28a-K1-BM3R质粒与Gro7伴侣质粒(购自Biovector Science Lab,Inc)共转BL21，得到BL21-pET28a-K1-BM3R-Gro7工程菌。引物及其序列如表3所示。

表3

实施例2融合蛋白酶的制备

分别将实施例1中构建好的BL21-pET28a-K1-Gro7、BL21-pET28a-FdR-Gro7、BL21-pET28a-Fdx工程菌以及融合蛋白工程菌BL21-pET28a-K1-RhFR、BL21-pET28a-K1-RhFR-Gro7、BL21-pET28a-K1-BM3R、BL21-pET28a-K1-BM3R-Gro7的单菌落接种至含50μg/ml卡那霉素与50μg/ml氯霉素的5ml LB液体培养基中，37℃震荡培养12h。按2v/v％接种量转接至50ml同样含50μg/ml卡那霉素与50μg/ml氯霉素的新鲜LB液体培养基中，37℃震荡至OD600达到0.8左右时，加入IPTG至其终浓度为0.5mM，22℃诱导培养22h。培养结束后，将培养液10000rpm离心10min，弃上清液，收集菌体(即菌泥)，置于-20℃冰箱中保存，待用。

分别将各融合蛋白的菌泥用100mM PBS7.4以1:4(W/V)的比例进行均质，均质液用4000rpm离心20min，弃沉淀。上清用2‰的PEI进行絮凝，4000rpm离心20min，上清即为K1-Gro7、AciFdR-Gro7、AciFdx蛋白以及融合蛋白K1-BM3R、K1-BM3R-Gro7、K1-RhFR、K1-RhFR-Gro7的粗酶液。

实施例3融合蛋白P450活性测定

采用CO差光谱法测定K1融合蛋白P450浓度。

测定方法：分别取待测样品(即实施例2中的融合蛋白粗酶液)1mL于2根10mL离心管，标为对照管、样品管。将样品拿至通风橱，先取一根离心管装适量水，将CO管道插入水中，调节三通阀至CO的出气速度约为1秒一个气泡。对照管与样品管分别加入1mg连二亚硫酸钠粉末，反复颠倒使连二亚硫酸钠溶解完全并混合均匀。分别将对照管与样品管液体转移至比色皿中，于紫外分光光度计上扫描400-500nm的吸光值。

酶浓度计算：

C_P450＝(ΔA450-ΔA490)/(ε₄₅₀·L)

其中：

C_P450，所测样品中P450酶的浓度，单位nmol/mL；

ΔA450，A450_样品-A450_对照的差值；

ΔA490，A490_样品-A490_对照的差值；

ε₄₅₀，P450摩尔吸光系数，为0.091mL/nmol^-1·cm^-1。

L，光程，1cm。

测定结果如下表4所示：

表4

上述所得融合蛋白均具有P450活性，说明融合蛋白构建成功。

实施例4葡萄糖脱氢酶(GDH)基因的获取和表达

根据来源于枯草芽胞杆菌(Bacillμs sμbtilis)168(NCBI登录号为NP_388275.1)的葡萄糖脱氢酶基因序列，全合成葡萄糖脱氢酶基因。

葡萄糖脱氢酶基因连pET21a，酶切位点NdeI&HindIII，将酶连好的载体转化至宿主E.coli BL21(DE3)感受态细胞，得到含有葡萄糖脱氢酶基因的工程菌株。将含有葡萄糖脱氢酶基因的工程菌在经平皿划线活化后，挑单菌落接种至含100μg/ml氨苄青霉素的5mlLB液体培养基中，37℃震荡培养12h。按2％(v/v)接种量转接至50ml同样含100μg/ml氨苄青霉素的新鲜LB液体培养基中，37℃震荡至OD₆₀₀达到0.8左右时，加入IPTG至其终浓度为0.5mM，18℃诱导培养16h。培养结束后，将培养液10000rpm离心10min，弃上清液，收集菌体(即得到葡萄糖脱氢酶菌泥)，置于-20℃冰箱中保存，待用。

实施例5融合蛋白酶体外催化VD3

底物VD3(购自上海德默医药科技有限公司)用乙醇配制成浓度为50g/L的母液，并加入25％的羟丙基-β-环糊精助溶。使用实施例2中的粗酶液进行体外酶催化反应，反应体系如表5所示。

表5

①加入蛋白粗酶液为K1-Gro7粗酶液的反应体系

②加入蛋白粗酶液为各融合蛋白的反应体系

28℃下，反应14h，随后取样100μL，加入500μL乙醇与400μL乙腈，12000rpm离心3min，上清液过膜除杂后进行HPLC检测。检测结果如下表6所示：

表6

其中，K1-RhFR和K1-RhFR-Gro7的催化能力高于K1-BM3R和K1-BM3R-Gro7，故后续针对融合蛋白K1-RhFR进行Linker的优化。

实施例6融合蛋白Linker优化

为了进一步提高融合蛋白K1-RhFR的活性，设计引物将融合蛋白上K1-RhFR的天然Linker延长或缩短。I3、I6表示在天然Linker的N端分别插入3个、6个氨基酸；D3、D6表示在天然Linker的N端分别删除3个、6个氨基酸；I14表示在N端插入14个氨基酸，具体序列见表7。

表7

以pET28a-K1-RhFR为模板，以I3F、I3R为引物进行PCR，将PCR产物用Dpn1消化，37℃，2小时。反应完成后PCR产物转化至BL21感受态细胞，涂布在含有50μg/mL卡那霉素的LB培养基，37℃培养过夜，得到BL21-pET28a-K1-RhFR-I3转化子。挑取BL21-pET28a-K1-RhFR-I3转化子接种至含50μg/mL卡那霉素的5ml LB液体培养基中，37℃震荡培养6h，提取pET28a-K1-RhFR-I3质粒，将pET28a-K1-RhFR-I3质粒与Gro7伴侣质粒共转BL21，得到BL21-pET28a-K1-RhFR-I3-Gro7工程菌。引物及其序列如表3所示。

采用上述同样的方法，分别获得BL21-pET28a-K1-RhFR-I6-Gro7、BL21-pET28a-K1-RhFR-D3-Gro7、BL21-pET28a-K1-RhFR-D6-Gro7、BL21-pET28a-K1-RhFR-I14-Gro7工程菌，即带有不同Linker长度的工程菌。

按照实施例2同样的方法，得到下表8中融合蛋白酶粗酶液。

按照实施例5同样的方法，将所得融合蛋白酶粗酶液用于体外催化VD3，结果如下表7所示。其中以K1-RhFR-I6-Gro7为例，催化后所得产物的检测图谱如图1所示，保留时间19.050min为VD3，10.884min为骨化二醇。图2为VD3对照品(购自上海德默医药科技有限公司)的图谱，保留时间为19.020min，图3为骨化二醇对照品(购自国家标准物质网)的图谱，保留时间为10.920min。可见，该实施例中VD3与产物骨化二醇的出峰时间与各自对照品的出峰时间基本一致，该实施例制备得到骨化二醇，其他粗酶液的结果也与K1-RhFR-I6-Gro7一致，所得产物中VD3和骨化二醇的出峰时间与各自对照品的出峰时间均基本一致。

表8

SEQUENCE LISTING

<110> 上海弈柯莱生物医药科技有限公司

<120> 一种融合蛋白或其变体及其在制备骨化二醇中的应用

<130> P19014220C

<160> 38

<170> PatentIn version 3.5

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Asn Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu

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Asp Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg

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Gly Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser

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Met Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu

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Asp Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys

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Glu Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn

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Val Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys

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Ser Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu

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Leu Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile

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Pro Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly

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Leu Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu

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Ala His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln

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Tyr Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met

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Ala Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu

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Leu Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr

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Met Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser

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Glu Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile

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Ser Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser

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Val Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile

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Ala Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys

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Phe Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu

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atggtcggac cgggaacagg cgtcgcgccg tttagaggct ttgtgcaggc gcgcaaacag 2760

ctaaaagaac aaggacagtc acttggagaa gcacatttat acttcggctg ccgttcacct 2820

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cgatacgcaa aagacgtgtg ggctggg 3147

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Met Ser Ala Ser Val Pro Ala Ser Ala Pro Ala Cys Pro Val Asp His

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Ala Ala Leu Ala Gly Gly Cys Pro Val Ser Ala Asn Ala Ala Ala Phe

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Asp Pro Phe Gly Ser Ala Tyr Gln Thr Asp Pro Ala Glu Ser Leu Arg

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Trp Ser Arg Asp Glu Glu Pro Val Phe Tyr Ser Pro Glu Leu Gly Tyr

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Trp Val Val Thr Arg Tyr Glu Asp Val Lys Ala Val Phe Arg Asp Asn

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Ile Leu Phe Ser Pro Ala Ile Ala Leu Glu Lys Ile Thr Pro Val Ser

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Ala Glu Ala Thr Ala Thr Leu Ala Arg Tyr Asp Tyr Ala Met Ala Arg

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Thr Leu Val Asn Glu Asp Glu Pro Ala His Met Pro Arg Arg Arg Ala

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Leu Met Asp Pro Phe Thr Pro Lys Glu Leu Ala His His Glu Ala Met

130 135 140

Val Arg Arg Leu Thr Arg Glu Tyr Val Asp Arg Phe Val Glu Ser Gly

145 150 155 160

Lys Ala Asp Leu Val Asp Glu Met Leu Trp Glu Val Pro Leu Thr Val

165 170 175

Ala Leu His Phe Leu Gly Val Pro Glu Glu Asp Met Ala Thr Met Arg

180 185 190

Lys Tyr Ser Ile Ala His Thr Val Asn Thr Trp Gly Arg Pro Ala Pro

195 200 205

Glu Glu Gln Val Ala Val Ala Glu Ala Val Gly Arg Phe Trp Gln Tyr

210 215 220

Ala Gly Thr Val Leu Glu Lys Met Arg Gln Asp Pro Ser Gly His Gly

225 230 235 240

Trp Met Pro Tyr Gly Ile Arg Lys Gln Arg Glu Met Pro Asp Val Val

245 250 255

Thr Asp Ser Tyr Leu His Ser Met Met Met Ala Gly Ile Val Ala Ala

260 265 270

His Glu Thr Thr Ala Asn Ala Ser Ala Asn Ala Phe Lys Leu Leu Leu

275 280 285

Glu Asn Arg Ala Val Trp Glu Glu Ile Cys Ala Asp Pro Ser Leu Ile

290 295 300

Pro Asn Ala Val Glu Glu Cys Leu Arg His Ser Gly Ser Val Ala Ala

305 310 315 320

Trp Arg Arg Val Ala Thr Ala Asp Thr Arg Ile Gly Asp Val Asp Ile

325 330 335

Pro Ala Gly Ala Lys Leu Leu Val Val Asn Ala Ser Ala Asn His Asp

340 345 350

Glu Arg His Phe Glu Arg Pro Asp Glu Phe Asp Ile Arg Arg Pro Asn

355 360 365

Ser Ser Asp His Leu Thr Phe Gly Tyr Gly Ser His Gln Cys Met Gly

370 375 380

Lys Asn Leu Ala Arg Met Glu Met Gln Ile Phe Leu Glu Glu Leu Thr

385 390 395 400

Thr Arg Leu Pro His Met Glu Leu Val Pro Asp Gln Glu Phe Thr Tyr

405 410 415

Leu Pro Asn Thr Ser Phe Arg Gly Pro Asp His Val Trp Val Gln Trp

420 425 430

Asp Pro Gln Ala Asn Pro Glu Arg Thr Asp Pro Ala Val Leu His Arg

435 440 445

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

450 455 460

Thr Val Thr Val Glu Arg Leu Asp Arg Ile Ala Asp Asp Val Leu Arg

465 470 475 480

Leu Val Leu Arg Asp Ala Gly Gly Lys Thr Leu Pro Thr Trp Thr Pro

485 490 495

Gly Ala His Ile Asp Leu Asp Leu Gly Ala Leu Ser Arg Gln Tyr Ser

500 505 510

Leu Cys Gly Ala Pro Asp Ala Pro Ser Tyr Glu Ile Ala Val His Leu

515 520 525

Asp Pro Glu Ser Arg Gly Gly Ser Arg Tyr Ile His Glu Gln Leu Glu

530 535 540

Val Gly Ser Pro Leu Arg Met Arg Gly Pro Arg Asn His Phe Ala Leu

545 550 555 560

Asp Pro Gly Ala Glu His Tyr Val Phe Val Ala Gly Gly Ile Gly Ile

565 570 575

Thr Pro Val Leu Ala Met Ala Asp His Ala Arg Ala Arg Gly Trp Ser

580 585 590

Tyr Glu Leu His Tyr Cys Gly Arg Asn Arg Ser Gly Met Ala Tyr Leu

595 600 605

Glu Arg Val Ala Gly His Gly Asp Arg Ala Ala Leu His Val Ser Glu

610 615 620

Glu Gly Thr Arg Ile Asp Leu Ala Ala Leu Leu Ala Glu Pro Ala Pro

625 630 635 640

Gly Val Gln Ile Tyr Ala Cys Gly Pro Gly Arg Leu Leu Ala Gly Leu

645 650 655

Glu Asp Ala Ser Arg Asn Trp Pro Asp Gly Ala Leu His Val Glu His

660 665 670

Phe Thr Ser Ser Leu Ala Ala Leu Asp Pro Asp Val Glu His Ala Phe

675 680 685

Asp Leu Glu Leu Arg Asp Ser Gly Leu Thr Val Arg Val Glu Pro Thr

690 695 700

Gln Thr Val Leu Asp Ala Leu Arg Ala Asn Asn Ile Asp Val Pro Ser

705 710 715 720

Asp Cys Glu Glu Gly Leu Cys Gly Ser Cys Glu Val Ala Val Leu Asp

725 730 735

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

740 745 750

Ala Asn Arg Gln Met Met Thr Cys Cys Ser Arg Ala Cys Gly Asp Arg

755 760 765

Leu Ala Leu Arg Leu

770

<210> 6

<211> 2319

<212> DNA

<213> Rhodococcus

<400> 6

atgagtgcat cagttccggc gtcggcgccg gcgtgtcccg tcgaccacgc ggccctggcg 60

ggcggctgcc cggtgtcggc gaacgccgcg gcgttcgatc cgttcggttc cgcgtaccag 120

accgatccgg ccgagtcgct gcgctggtcc cgcgacgagg agccggtgtt ctacagcccc 180

gaactcggct actgggtcgt cacccggtac gaggatgtga aggcggtgtt ccgcgacaac 240

atcctgttct cgccggcgat cgcgctggag aagatcactc ccgtctcggc ggaggccacc 300

gccaccctcg cccggtacga ctacgccatg gcccggaccc tcgtgaacga ggacgagccc 360

gcccacatgc cgcgccgccg cgcgctcatg gatccgttca ccccgaagga actggcgcac 420

cacgaggcga tggtgcgacg gctcacgcgc gaatacgtcg accgcttcgt cgaatccggc 480

aaggccgacc tggtggacga gatgctgtgg gaggttccgc tcaccgtcgc cctgcacttc 540

ctcggcgtgc cggaggagga catggcgacg atgcgcaagt actcgatcgc gcacaccgtg 600

aacacctggg gccgccccgc gcccgaggag caggtggccg tcgccgaggc ggtcggcagg 660

ttctggcagt acgcgggcac ggtgctcgag aagatgcggc aggacccgtc gggacacggc 720

tggatgccct acgggatccg caagcagcgg gagatgccgg acgtcgtcac cgactcctac 780

ctgcactcga tgatgatggc cggcatcgtc gccgcgcacg agaccacggc caacgcgtcc 840

gcgaacgcgt tcaagctgct gctcgagaac cgcgcggtgt gggaggagat ctgcgcggat 900

ccgtcgctga tccccaacgc cgtcgaggag tgcctgcgcc actccgggtc cgtggcggcg 960

tggcgacggg tggccaccgc cgacacccgc atcggcgacg tcgacatccc cgccggcgcc 1020

aagctgctcg tcgtcaacgc gtccgccaac cacgacgagc gccacttcga gcgccccgac 1080

gagttcgaca tccggcgccc gaactcgagc gaccatctca ccttcgggta cggcagccac 1140

cagtgcatgg gcaagaacct ggcccgcatg gagatgcaga tcttcctcga ggaactcacc 1200

acgcggcttc cccacatgga actcgtaccc gatcaggagt tcacctacct gccgaatacg 1260

tccttccgcg gacccgacca cgtgtgggtg cagtgggatc cgcaggcgaa tcccgagcgc 1320

accgatcctg ctgtgctgca ccggcatcaa ccggtcacca tcggagaacc cgccgcccgg 1380

gcggtgtccc gcaccgtcac cgtcgagcgc ctggaccgga tcgccgacga cgtgctgcgc 1440

ctcgtcctgc gcgacgccgg cggaaagaca ttacccacgt ggactcccgg cgcccatatc 1500

gacctcgacc tcggcgcgct gtcgcgccag tactccctgt gcggcgcgcc cgatgcgccg 1560

agctacgaga ttgccgtgca cctggatccc gagagccgcg gcggttcgcg ctacatccac 1620

gaacagctcg aggtgggaag cccgctccgg atgcgcggcc ctcggaacca tttcgcgctc 1680

gaccccggcg ccgagcacta cgtgttcgtc gccggcggca tcggcatcac cccagtcctg 1740

gccatggccg accacgcccg cgcccggggg tggagctacg aactgcacta ctgcggccga 1800

aaccgttccg gcatggccta tctcgagcgt gtcgccgggc acggtgaccg ggccgccctg 1860

cacgtgtccg aggaaggcac ccggatcgac ctcgccgccc tcctcgccga gcccgccccc 1920

ggcgtccaga tctacgcgtg cgggcccggg cggctgctcg ccggactcga ggacgcgagc 1980

cggaactggc ccgacggggc gctgcacgtc gagcacttca cctcgtccct cgcggcgctc 2040

gatccggacg tcgagcacgc cttcgacctc gaactgcgtg actcggggct gaccgtgcgg 2100

gtcgaaccca cccagaccgt cctcgacgcg ttgcgcgcca acaacatcga cgtgcccagc 2160

gactgcgagg aaggcctctg cggctcgtgc gaggtcgccg tcctcgacgg cgaggtcgac 2220

catcgcgaca cggtgctgac caaggccgag cgggcggcga accggcagat gatgacctgc 2280

tgctcgcgtg cctgtggcga ccggctggcc ctgcgactc 2319

<210> 7

<211> 993

<212> PRT

<213> Artificial Sequence

<220>

<223> K1-BM3R氨基酸序列

<400> 7

Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly

1 5 10 15

Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu

20 25 30

Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu

35 40 45

Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser

50 55 60

Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro

65 70 75 80

Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr

85 90 95

Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn

100 105 110

Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly

115 120 125

Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln

130 135 140

Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp

145 150 155 160

Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro

165 170 175

Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser

180 185 190

Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser

195 200 205

Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu

210 215 220

Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr

225 230 235 240

Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro

245 250 255

Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala

260 265 270

Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile

275 280 285

Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala

290 295 300

Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp

305 310 315 320

Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly

325 330 335

Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu

340 345 350

Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg

355 360 365

Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg

370 375 380

Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro

385 390 395 400

Arg Ser Ala Pro Ser Pro Ser Thr Glu Gln Ser Ala Lys Lys Val Arg

405 410 415

Lys Lys Ala Glu Asn Ala His Asn Thr Pro Leu Leu Val Leu Tyr Gly

420 425 430

Ser Asn Met Gly Thr Ala Glu Gly Thr Ala Arg Asp Leu Ala Asp Ile

435 440 445

Ala Met Ser Lys Gly Phe Ala Pro Gln Val Ala Thr Leu Asp Ser His

450 455 460

Ala Gly Asn Leu Pro Arg Glu Gly Ala Val Leu Ile Val Thr Ala Ser

465 470 475 480

Tyr Asn Gly His Pro Pro Asp Asn Ala Lys Gln Phe Val Asp Trp Leu

485 490 495

Asp Gln Ala Ser Ala Asp Glu Val Lys Gly Val Arg Tyr Ser Val Phe

500 505 510

Gly Cys Gly Asp Lys Asn Trp Ala Thr Thr Tyr Gln Lys Val Pro Ala

515 520 525

Phe Ile Asp Glu Thr Leu Ala Ala Lys Gly Ala Glu Asn Ile Ala Asp

530 535 540

Arg Gly Glu Ala Asp Ala Ser Asp Asp Phe Glu Gly Thr Tyr Glu Glu

545 550 555 560

Trp Arg Glu His Met Trp Ser Asp Val Ala Ala Tyr Phe Asn Leu Asp

565 570 575

Ile Glu Asn Ser Glu Asp Asn Lys Ser Thr Leu Ser Leu Gln Phe Val

580 585 590

Asp Ser Ala Ala Asp Met Pro Leu Ala Lys Met His Gly Ala Phe Ser

595 600 605

Thr Asn Val Val Ala Ser Lys Glu Leu Gln Gln Pro Gly Ser Ala Arg

610 615 620

Ser Thr Arg His Leu Glu Ile Glu Leu Pro Lys Glu Ala Ser Tyr Gln

625 630 635 640

Glu Gly Asp His Leu Gly Val Ile Pro Arg Asn Tyr Glu Gly Ile Val

645 650 655

Asn Arg Val Thr Ala Arg Phe Gly Leu Asp Ala Ser Gln Gln Ile Arg

660 665 670

Leu Glu Ala Glu Glu Glu Lys Leu Ala His Leu Pro Leu Ala Lys Thr

675 680 685

Val Ser Val Glu Glu Leu Leu Gln Tyr Val Glu Leu Gln Asp Pro Val

690 695 700

Thr Arg Thr Gln Leu Arg Ala Met Ala Ala Lys Thr Val Cys Pro Pro

705 710 715 720

His Lys Val Glu Leu Glu Ala Leu Leu Glu Lys Gln Ala Tyr Lys Glu

725 730 735

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

740 745 750

Pro Ala Cys Glu Met Lys Phe Ser Glu Phe Ile Ala Leu Leu Pro Ser

755 760 765

Ile Arg Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Pro Arg Val Asp Glu

770 775 780

Lys Gln Ala Ser Ile Thr Val Ser Val Val Ser Gly Glu Ala Trp Ser

785 790 795 800

Gly Tyr Gly Glu Tyr Lys Gly Ile Ala Ser Asn Tyr Leu Ala Glu Leu

805 810 815

Gln Glu Gly Asp Thr Ile Thr Cys Phe Ile Ser Thr Pro Gln Ser Glu

820 825 830

Phe Thr Leu Pro Lys Asp Pro Glu Thr Pro Leu Ile Met Val Gly Pro

835 840 845

Gly Thr Gly Val Ala Pro Phe Arg Gly Phe Val Gln Ala Arg Lys Gln

850 855 860

Leu Lys Glu Gln Gly Gln Ser Leu Gly Glu Ala His Leu Tyr Phe Gly

865 870 875 880

Cys Arg Ser Pro His Glu Asp Tyr Leu Tyr Gln Glu Glu Leu Glu Asn

885 890 895

Ala Gln Ser Glu Gly Ile Ile Thr Leu His Thr Ala Phe Ser Arg Met

900 905 910

Pro Asn Gln Pro Lys Thr Tyr Val Gln His Val Met Glu Gln Asp Gly

915 920 925

Lys Lys Leu Ile Glu Leu Leu Asp Gln Gly Ala His Phe Tyr Ile Cys

930 935 940

Gly Asp Gly Ser Gln Met Ala Pro Ala Val Glu Ala Thr Leu Met Lys

945 950 955 960

Ser Tyr Ala Asp Val His Gln Val Ser Glu Ala Asp Ala Arg Leu Trp

965 970 975

Leu Gln Gln Leu Glu Glu Lys Gly Arg Tyr Ala Lys Asp Val Trp Ala

980 985 990

Gly

<210> 8

<211> 2979

<212> DNA

<213> Artificial Sequence

<220>

<223> K1-BM3R核苷酸序列

<400> 8

atggctctga ccaccaccgg taccgaacag cacgacctgt tctctggtac cttctggcag 60

aacccgcacc cggcttacgc tgctctgcgt gctgaagacc cggttcgtaa actggctctg 120

ccggacggtc cggtttggct gctgacccgt tacgctgacg ttcgtgaagc tttcgttgac 180

ccgcgtctgt ctaaagactg gcgtcaccgt ctgccggaag accagcgtgc tgacatgccg 240

gctaccccga ccccgatgat gatcctgatg gacccgccgg accacacccg tctgcgtaaa 300

ctggttggtc gttctttcac cgttcgtcgt atgaacgaac tggaaccgcg tatcaccgaa 360

atcgctgacg gtctgctggc tggtctgccg accgacggtc cggttgacct gatgcgtgaa 420

tacgctttcc agatcccggt tcaggttatc tgcgaactgc tgggtctgcc ggctgaagac 480

cgtgacgact tctctgcttg gtcttctgtt ctggttgacg actctccggc tgacgacaaa 540

aacgctgcta tgggtaaact gcacggttac ctgtctgacc tgctggaacg taaacgtacc 600

gaaccggacg acgctctgct gtcttctctg ctggctgttt ctgacatgga cggtgaccgt 660

ctgtctcagg aagaactggt tgctatggct atgctgctgc tgatcgctgg tcacgaaacc 720

accgttaacc tgatcggtaa cggtgttctg gctctgctga cccacccgga ccagcgtaaa 780

ctgctggctg aagacccgtc tctgatctct tctgctgttg aagaattcct gcgtttcgac 840

tctccggttt ctcaggctcc gatccgtttc accgctgaag acgttaccta ctctggtgtt 900

accatcccgg ctggtgaaat ggttatgctg ggtctggctg ctgctaaccg tgacgctgac 960

tggatgccgg aaccggaccg tctggacatc acccgtgacg cttctggtgg tgttttcttc 1020

ggtcacggta tccacttctg cctgggtgct cagctggctc gtctggaagg tcgtgttgct 1080

atcggtcgtc tgttcgctga ccgtccggaa ctggctctgg ctgttggtct ggacgaactg 1140

gtttaccgtc gttctaccct ggttcgtggt ctgtctcgta tgccggttac catgggtccg 1200

cgttctgctc cgtctccgtc taccgaacag tctgctaaaa aagttcgtaa aaaagctgaa 1260

aacgctcaca acaccccgct gctggttctg tacggttcta acatgggtac cgctgaaggt 1320

accgctcgtg acctggctga catcgctatg tctaaaggtt tcgctccgca ggttgctacc 1380

ctggactctc acgctggtaa cctgccgcgt gaaggtgctg ttctgatcgt taccgcttct 1440

tacaacggtc acccgccgga caacgctaaa cagttcgttg actggctgga ccaggcttct 1500

gctgacgaag ttaaaggtgt tcgttactct gttttcggtt gcggtgacaa aaactgggct 1560

accacctacc agaaagttcc ggctttcatc gacgaaaccc tggctgctaa aggtgctgaa 1620

aacatcgctg accgtggtga agctgacgct tctgacgact tcgaaggtac ctacgaagaa 1680

tggcgtgaac acatgtggtc tgacgttgct gcttacttca acctggacat cgaaaactct 1740

gaagacaaca aatctaccct gtctctgcag ttcgttgact ctgctgctga catgccgctg 1800

gctaaaatgc acggtgcttt ctctaccaac gttgttgctt ctaaagaact gcagcagccg 1860

ggttctgctc gttctacccg tcacctggaa atcgaactgc cgaaagaagc ttcttaccag 1920

gaaggtgacc acctgggtgt tatcccgcgt aactacgaag gtatcgttaa ccgtgttacc 1980

gctcgtttcg gtctggacgc ttctcagcag atccgtctgg aagctgaaga agaaaaactg 2040

gctcacctgc cgctggctaa aaccgtttct gttgaagaac tgctgcagta cgttgaactg 2100

caggacccgg ttacccgtac ccagctgcgt gctatggctg ctaaaaccgt ttgcccgccg 2160

cacaaagttg aactggaagc tctgctggaa aaacaggctt acaaagaaca ggttctggct 2220

aaacgtctga ccatgctgga actgctggaa aaatacccgg cttgcgaaat gaaattctct 2280

gaattcatcg ctctgctgcc gtctatccgt ccgcgttact actctatctc ttcttctccg 2340

cgtgttgacg aaaaacaggc ttctatcacc gtttctgttg tttctggtga agcttggtct 2400

ggttacggtg aatacaaagg tatcgcttct aactacctgg ctgaactgca ggaaggtgac 2460

accatcacct gcttcatctc taccccgcag tctgaattca ccctgccgaa agacccggaa 2520

accccgctga tcatggttgg tccgggtacc ggtgttgctc cgttccgtgg tttcgttcag 2580

gctcgtaaac agctgaaaga acagggtcag tctctgggtg aagctcacct gtacttcggt 2640

tgccgttctc cgcacgaaga ctacctgtac caggaagaac tggaaaacgc tcagtctgaa 2700

ggtatcatca ccctgcacac cgctttctct cgtatgccga accagccgaa aacctacgtt 2760

cagcacgtta tggaacagga cggtaaaaaa ctgatcgaac tgctggacca gggtgctcac 2820

ttctacatct gcggtgacgg ttctcagatg gctccggctg ttgaagctac cctgatgaaa 2880

tcttacgctg acgttcacca ggtttctgaa gctgacgctc gtctgtggct gcagcagctg 2940

gaagaaaaag gtcgttacgc taaagacgtt tgggctggt 2979

<210> 9

<211> 732

<212> PRT

<213> Artificial Sequence

<220>

<223> K1-RhFR氨基酸序列

<400> 9

Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly

1 5 10 15

Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu

20 25 30

Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu

35 40 45

Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser

50 55 60

Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro

65 70 75 80

Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr

85 90 95

Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn

100 105 110

Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly

115 120 125

Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln

130 135 140

Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp

145 150 155 160

Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro

165 170 175

Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser

180 185 190

Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser

195 200 205

Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu

210 215 220

Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr

225 230 235 240

Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro

245 250 255

Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala

260 265 270

Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile

275 280 285

Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala

290 295 300

Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp

305 310 315 320

Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly

325 330 335

Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu

340 345 350

Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg

355 360 365

Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg

370 375 380

Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro

385 390 395 400

Arg Ser Ala Val Leu His Arg His Gln Pro Val Thr Ile Gly Glu Pro

405 410 415

Ala Ala Arg Ala Val Ser Arg Thr Val Thr Val Glu Arg Leu Asp Arg

420 425 430

Ile Ala Asp Asp Val Leu Arg Leu Val Leu Arg Asp Ala Gly Gly Lys

435 440 445

Thr Leu Pro Thr Trp Thr Pro Gly Ala His Ile Asp Leu Asp Leu Gly

450 455 460

Ala Leu Ser Arg Gln Tyr Ser Leu Cys Gly Ala Pro Asp Ala Pro Ser

465 470 475 480

Tyr Glu Ile Ala Val His Leu Asp Pro Glu Ser Arg Gly Gly Ser Arg

485 490 495

Tyr Ile His Glu Gln Leu Glu Val Gly Ser Pro Leu Arg Met Arg Gly

500 505 510

Pro Arg Asn His Phe Ala Leu Asp Pro Gly Ala Glu His Tyr Val Phe

515 520 525

Val Ala Gly Gly Ile Gly Ile Thr Pro Val Leu Ala Met Ala Asp His

530 535 540

Ala Arg Ala Arg Gly Trp Ser Tyr Glu Leu His Tyr Cys Gly Arg Asn

545 550 555 560

Arg Ser Gly Met Ala Tyr Leu Glu Arg Val Ala Gly His Gly Asp Arg

565 570 575

Ala Ala Leu His Val Ser Glu Glu Gly Thr Arg Ile Asp Leu Ala Ala

580 585 590

Leu Leu Ala Glu Pro Ala Pro Gly Val Gln Ile Tyr Ala Cys Gly Pro

595 600 605

Gly Arg Leu Leu Ala Gly Leu Glu Asp Ala Ser Arg Asn Trp Pro Asp

610 615 620

Gly Ala Leu His Val Glu His Phe Thr Ser Ser Leu Ala Ala Leu Asp

625 630 635 640

Pro Asp Val Glu His Ala Phe Asp Leu Glu Leu Arg Asp Ser Gly Leu

645 650 655

Thr Val Arg Val Glu Pro Thr Gln Thr Val Leu Asp Ala Leu Arg Ala

660 665 670

Asn Asn Ile Asp Val Pro Ser Asp Cys Glu Glu Gly Leu Cys Gly Ser

675 680 685

Cys Glu Val Ala Val Leu Asp Gly Glu Val Asp His Arg Asp Thr Val

690 695 700

Leu Thr Lys Ala Glu Arg Ala Ala Asn Arg Gln Met Met Thr Cys Cys

705 710 715 720

Ser Arg Ala Cys Gly Asp Arg Leu Ala Leu Arg Leu

725 730

<210> 10

<211> 2196

<212> DNA

<213> Artificial Sequence

<220>

<223> K1-RhFR核苷酸序列

<400> 10

atggctctga ccaccaccgg taccgaacag cacgacctgt tctctggtac cttctggcag 60

aacccgcacc cggcttacgc tgctctgcgt gctgaagacc cggttcgtaa actggctctg 120

ccggacggtc cggtttggct gctgacccgt tacgctgacg ttcgtgaagc tttcgttgac 180

ccgcgtctgt ctaaagactg gcgtcaccgt ctgccggaag accagcgtgc tgacatgccg 240

gctaccccga ccccgatgat gatcctgatg gacccgccgg accacacccg tctgcgtaaa 300

ctggttggtc gttctttcac cgttcgtcgt atgaacgaac tggaaccgcg tatcaccgaa 360

atcgctgacg gtctgctggc tggtctgccg accgacggtc cggttgacct gatgcgtgaa 420

tacgctttcc agatcccggt tcaggttatc tgcgaactgc tgggtctgcc ggctgaagac 480

cgtgacgact tctctgcttg gtcttctgtt ctggttgacg actctccggc tgacgacaaa 540

aacgctgcta tgggtaaact gcacggttac ctgtctgacc tgctggaacg taaacgtacc 600

gaaccggacg acgctctgct gtcttctctg ctggctgttt ctgacatgga cggtgaccgt 660

ctgtctcagg aagaactggt tgctatggct atgctgctgc tgatcgctgg tcacgaaacc 720

accgttaacc tgatcggtaa cggtgttctg gctctgctga cccacccgga ccagcgtaaa 780

ctgctggctg aagacccgtc tctgatctct tctgctgttg aagaattcct gcgtttcgac 840

tctccggttt ctcaggctcc gatccgtttc accgctgaag acgttaccta ctctggtgtt 900

accatcccgg ctggtgaaat ggttatgctg ggtctggctg ctgctaaccg tgacgctgac 960

tggatgccgg aaccggaccg tctggacatc acccgtgacg cttctggtgg tgttttcttc 1020

ggtcacggta tccacttctg cctgggtgct cagctggctc gtctggaagg tcgtgttgct 1080

atcggtcgtc tgttcgctga ccgtccggaa ctggctctgg ctgttggtct ggacgaactg 1140

gtttaccgtc gttctaccct ggttcgtggt ctgtctcgta tgccggttac catgggtccg 1200

cgttctgctg ttctgcaccg tcaccagccg gttaccatcg gtgaaccggc tgctcgtgct 1260

gtttctcgta ccgttaccgt tgaacgtctg gaccgtatcg ctgacgacgt tctgcgtctg 1320

gttctgcgtg acgctggtgg taaaaccctg ccgacctgga ccccgggtgc tcacatcgac 1380

ctggacctgg gtgctctgtc tcgtcagtac tctctgtgcg gtgctccgga cgctccgtct 1440

tacgaaatcg ctgttcacct ggacccggaa tctcgtggtg gttctcgtta catccacgaa 1500

cagctggaag ttggttctcc gctgcgtatg cgtggtccgc gtaaccactt cgctctggac 1560

ccgggtgctg aacactacgt tttcgttgct ggtggtatcg gtatcacccc ggttctggct 1620

atggctgacc acgctcgtgc tcgtggttgg tcttacgaac tgcactactg cggtcgtaac 1680

cgttctggta tggcttacct ggaacgtgtt gctggtcacg gtgaccgtgc tgctctgcac 1740

gtttctgaag aaggtacccg tatcgacctg gctgctctgc tggctgaacc ggctccgggt 1800

gttcagatct acgcttgcgg tccgggtcgt ctgctggctg gtctggaaga cgcttctcgt 1860

aactggccgg acggtgctct gcacgttgaa cacttcacct cttctctggc tgctctggac 1920

ccggacgttg aacacgcttt cgacctggaa ctgcgtgact ctggtctgac cgttcgtgtt 1980

gaaccgaccc agaccgttct ggacgctctg cgtgctaaca acatcgacgt tccgtctgac 2040

tgcgaagaag gtctgtgcgg ttcttgcgaa gttgctgttc tggacggtga agttgaccac 2100

cgtgacaccg ttctgaccaa agctgaacgt gctgctaacc gtcagatgat gacctgctgc 2160

tctcgtgctt gcggtgaccg tctggctctg cgtctg 2196

<210> 11

<211> 735

<212> PRT

<213> Artificial Sequence

<220>

<223> K1-RhFR-I3氨基酸序列

<400> 11

Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly

1 5 10 15

Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu

20 25 30

Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu

35 40 45

Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser

50 55 60

Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro

65 70 75 80

Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr

85 90 95

Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn

100 105 110

Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly

115 120 125

Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln

130 135 140

Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp

145 150 155 160

Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro

165 170 175

Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser

180 185 190

Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser

195 200 205

Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu

210 215 220

Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr

225 230 235 240

Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro

245 250 255

Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala

260 265 270

Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile

275 280 285

Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala

290 295 300

Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp

305 310 315 320

Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly

325 330 335

Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu

340 345 350

Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg

355 360 365

Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg

370 375 380

Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro

385 390 395 400

Arg Ser Ala Gly Gly Ser Val Leu His Arg His Gln Pro Val Thr Ile

405 410 415

Gly Glu Pro Ala Ala Arg Ala Val Ser Arg Thr Val Thr Val Glu Arg

420 425 430

Leu Asp Arg Ile Ala Asp Asp Val Leu Arg Leu Val Leu Arg Asp Ala

435 440 445

Gly Gly Lys Thr Leu Pro Thr Trp Thr Pro Gly Ala His Ile Asp Leu

450 455 460

Asp Leu Gly Ala Leu Ser Arg Gln Tyr Ser Leu Cys Gly Ala Pro Asp

465 470 475 480

Ala Pro Ser Tyr Glu Ile Ala Val His Leu Asp Pro Glu Ser Arg Gly

485 490 495

Gly Ser Arg Tyr Ile His Glu Gln Leu Glu Val Gly Ser Pro Leu Arg

500 505 510

Met Arg Gly Pro Arg Asn His Phe Ala Leu Asp Pro Gly Ala Glu His

515 520 525

Tyr Val Phe Val Ala Gly Gly Ile Gly Ile Thr Pro Val Leu Ala Met

530 535 540

Ala Asp His Ala Arg Ala Arg Gly Trp Ser Tyr Glu Leu His Tyr Cys

545 550 555 560

Gly Arg Asn Arg Ser Gly Met Ala Tyr Leu Glu Arg Val Ala Gly His

565 570 575

Gly Asp Arg Ala Ala Leu His Val Ser Glu Glu Gly Thr Arg Ile Asp

580 585 590

Leu Ala Ala Leu Leu Ala Glu Pro Ala Pro Gly Val Gln Ile Tyr Ala

595 600 605

Cys Gly Pro Gly Arg Leu Leu Ala Gly Leu Glu Asp Ala Ser Arg Asn

610 615 620

Trp Pro Asp Gly Ala Leu His Val Glu His Phe Thr Ser Ser Leu Ala

625 630 635 640

Ala Leu Asp Pro Asp Val Glu His Ala Phe Asp Leu Glu Leu Arg Asp

645 650 655

Ser Gly Leu Thr Val Arg Val Glu Pro Thr Gln Thr Val Leu Asp Ala

660 665 670

Leu Arg Ala Asn Asn Ile Asp Val Pro Ser Asp Cys Glu Glu Gly Leu

675 680 685

Cys Gly Ser Cys Glu Val Ala Val Leu Asp Gly Glu Val Asp His Arg

690 695 700

Asp Thr Val Leu Thr Lys Ala Glu Arg Ala Ala Asn Arg Gln Met Met

705 710 715 720

Thr Cys Cys Ser Arg Ala Cys Gly Asp Arg Leu Ala Leu Arg Leu

725 730 735

<210> 12

<211> 2205

<212> DNA

<213> Artificial Sequence

<220>

<223> K1-RhFR-I3核苷酸序列

<400> 12

atggcactga ccaccaccgg taccgaacag catgacctgt ttagcggtac cttttggcag 60

aatccgcatc cggcgtatgc agcactgcgt gcagaagatc cggttcgtaa actggcactg 120

ccggatggtc cggtgtggct gctgacccgt tatgcagatg ttcgtgaagc atttgttgat 180

ccgcgtctga gtaaagattg gcgtcatcgt ctgccggaag atcagcgtgc cgatatgccg 240

gcaaccccga ccccgatgat gattctgatg gacccgccgg atcatacacg tttacgtaaa 300

ctggttggtc gtagttttac cgttcgtcgt atgaatgaac tggaaccgcg tattaccgaa 360

attgcagatg gtctgctggc aggtctgccg accgatggtc cggttgatct gatgcgtgaa 420

tatgcatttc agattccggt tcaggttata tgtgaactgc tgggtctgcc ggcagaagat 480

cgtgatgatt tttcagcatg gtcaagtgtg ctggttgatg attctccggc agatgataaa 540

aatgccgcaa tgggtaaact gcatggttat ctgtcagatc tgctggaacg taaacgtacc 600

gaaccggatg atgcactgct gagtagcctg ctggcggttt ctgatatgga tggtgatcgt 660

ctgtctcagg aagaactggt tgcaatggca atgctgctgc tgattgcagg tcatgaaacc 720

accgttaatc tgattggtaa tggtgtgctg gcactgctga cccatccgga tcagcgtaaa 780

ctgttagctg aagatccgag tctgattagc tcagcagttg aagaatttct gcgttttgat 840

tctccggtta gccaggcacc gatccgtttt accgctgaag atgttaccta tagtggtgtt 900

accattccgg caggtgaaat ggttatgctg ggtctggcag cagcaaatcg cgatgcagat 960

tggatgccgg aaccggatcg tctggatatt acccgtgatg caagtggtgg tgttttcttt 1020

ggtcatggta ttcatttttg tctgggtgcg cagctggcac gtctggaagg tcgtgtggca 1080

attggtcgtc tgtttgcaga tcgtccggaa ctggcactgg cagttggtct ggatgaactg 1140

gtgtatcgtc gtagcaccct ggttcgtggt ctgagtagga tgccggtgac aatgggtccg 1200

cgttcagcag gcggaagtgt gctgcaccgg catcaaccgg tcaccatcgg agaacccgcc 1260

gcccgggcgg tgtcccgcac cgtcaccgtc gagcgcctgg accggatcgc cgacgacgtg 1320

ctgcgcctcg tcctgcgcga cgccggcgga aagacattac ccacgtggac tcccggcgcc 1380

catatcgacc tcgacctcgg cgcgctgtcg cgccagtact ccctgtgcgg cgcgcccgat 1440

gcgccgagct acgagattgc cgtgcacctg gatcccgaga gccgcggcgg ttcgcgctac 1500

atccacgaac agctcgaggt gggaagcccg ctccggatgc gcggccctcg gaaccatttc 1560

gcgctcgacc ccggcgccga gcactacgtg ttcgtcgccg gcggcatcgg catcacccca 1620

gtcctggcca tggccgacca cgcccgcgcc cgggggtgga gctacgaact gcactactgc 1680

ggccgaaacc gttccggcat ggcctatctc gagcgtgtcg ccgggcacgg tgaccgggcc 1740

gccctgcacg tgtccgagga aggcacccgg atcgacctcg ccgccctcct cgccgagccc 1800

gcccccggcg tccagatcta cgcgtgcggg cccgggcggc tgctcgccgg actcgaggac 1860

gcgagccgga actggcccga cggggcgctg cacgtcgagc acttcacctc gtccctcgcg 1920

gcgctcgatc cggacgtcga gcacgccttc gacctcgaac tgcgtgactc ggggctgacc 1980

gtgcgggtcg aacccaccca gaccgtcctc gacgcgttgc gcgccaacaa catcgacgtg 2040

cccagcgact gcgaggaagg cctctgcggc tcgtgcgagg tcgccgtcct cgacggcgag 2100

gtcgaccatc gcgacacggt gctgaccaag gccgagcggg cggcgaaccg gcagatgatg 2160

acctgctgct cgcgtgcctg tggcgaccgg ctggccctgc gactc 2205

<210> 13

<211> 738

<212> PRT

<213> Artificial Sequence

<220>

<223> K1-RhFR-I6氨基酸序列

<400> 13

Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly

1 5 10 15

Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu

20 25 30

Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu

35 40 45

Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser

50 55 60

Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro

65 70 75 80

Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr

85 90 95

Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn

100 105 110

Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly

115 120 125

Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln

130 135 140

Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp

145 150 155 160

Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro

165 170 175

Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser

180 185 190

Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser

195 200 205

Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu

210 215 220

Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr

225 230 235 240

Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro

245 250 255

Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala

260 265 270

Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile

275 280 285

Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala

290 295 300

Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp

305 310 315 320

Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly

325 330 335

Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu

340 345 350

Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg

355 360 365

Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg

370 375 380

Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro

385 390 395 400

Arg Ser Ala Gly Gly Ser Gly Gly Ser Val Leu His Arg His Gln Pro

405 410 415

Val Thr Ile Gly Glu Pro Ala Ala Arg Ala Val Ser Arg Thr Val Thr

420 425 430

Val Glu Arg Leu Asp Arg Ile Ala Asp Asp Val Leu Arg Leu Val Leu

435 440 445

Arg Asp Ala Gly Gly Lys Thr Leu Pro Thr Trp Thr Pro Gly Ala His

450 455 460

Ile Asp Leu Asp Leu Gly Ala Leu Ser Arg Gln Tyr Ser Leu Cys Gly

465 470 475 480

Ala Pro Asp Ala Pro Ser Tyr Glu Ile Ala Val His Leu Asp Pro Glu

485 490 495

Ser Arg Gly Gly Ser Arg Tyr Ile His Glu Gln Leu Glu Val Gly Ser

500 505 510

Pro Leu Arg Met Arg Gly Pro Arg Asn His Phe Ala Leu Asp Pro Gly

515 520 525

Ala Glu His Tyr Val Phe Val Ala Gly Gly Ile Gly Ile Thr Pro Val

530 535 540

Leu Ala Met Ala Asp His Ala Arg Ala Arg Gly Trp Ser Tyr Glu Leu

545 550 555 560

His Tyr Cys Gly Arg Asn Arg Ser Gly Met Ala Tyr Leu Glu Arg Val

565 570 575

Ala Gly His Gly Asp Arg Ala Ala Leu His Val Ser Glu Glu Gly Thr

580 585 590

Arg Ile Asp Leu Ala Ala Leu Leu Ala Glu Pro Ala Pro Gly Val Gln

595 600 605

Ile Tyr Ala Cys Gly Pro Gly Arg Leu Leu Ala Gly Leu Glu Asp Ala

610 615 620

Ser Arg Asn Trp Pro Asp Gly Ala Leu His Val Glu His Phe Thr Ser

625 630 635 640

Ser Leu Ala Ala Leu Asp Pro Asp Val Glu His Ala Phe Asp Leu Glu

645 650 655

Leu Arg Asp Ser Gly Leu Thr Val Arg Val Glu Pro Thr Gln Thr Val

660 665 670

Leu Asp Ala Leu Arg Ala Asn Asn Ile Asp Val Pro Ser Asp Cys Glu

675 680 685

Glu Gly Leu Cys Gly Ser Cys Glu Val Ala Val Leu Asp Gly Glu Val

690 695 700

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

705 710 715 720

Gln Met Met Thr Cys Cys Ser Arg Ala Cys Gly Asp Arg Leu Ala Leu

725 730 735

Arg Leu

<210> 14

<211> 2214

<212> DNA

<213> Artificial Sequence

<220>

<223> K1-RhFR-I6

<400> 14

atggcactga ccaccaccgg taccgaacag catgacctgt ttagcggtac cttttggcag 60

aatccgcatc cggcgtatgc agcactgcgt gcagaagatc cggttcgtaa actggcactg 120

ccggatggtc cggtgtggct gctgacccgt tatgcagatg ttcgtgaagc atttgttgat 180

ccgcgtctga gtaaagattg gcgtcatcgt ctgccggaag atcagcgtgc cgatatgccg 240

gcaaccccga ccccgatgat gattctgatg gacccgccgg atcatacacg tttacgtaaa 300

ctggttggtc gtagttttac cgttcgtcgt atgaatgaac tggaaccgcg tattaccgaa 360

attgcagatg gtctgctggc aggtctgccg accgatggtc cggttgatct gatgcgtgaa 420

tatgcatttc agattccggt tcaggttata tgtgaactgc tgggtctgcc ggcagaagat 480

cgtgatgatt tttcagcatg gtcaagtgtg ctggttgatg attctccggc agatgataaa 540

aatgccgcaa tgggtaaact gcatggttat ctgtcagatc tgctggaacg taaacgtacc 600

gaaccggatg atgcactgct gagtagcctg ctggcggttt ctgatatgga tggtgatcgt 660

ctgtctcagg aagaactggt tgcaatggca atgctgctgc tgattgcagg tcatgaaacc 720

accgttaatc tgattggtaa tggtgtgctg gcactgctga cccatccgga tcagcgtaaa 780

ctgttagctg aagatccgag tctgattagc tcagcagttg aagaatttct gcgttttgat 840

tctccggtta gccaggcacc gatccgtttt accgctgaag atgttaccta tagtggtgtt 900

accattccgg caggtgaaat ggttatgctg ggtctggcag cagcaaatcg cgatgcagat 960

tggatgccgg aaccggatcg tctggatatt acccgtgatg caagtggtgg tgttttcttt 1020

ggtcatggta ttcatttttg tctgggtgcg cagctggcac gtctggaagg tcgtgtggca 1080

attggtcgtc tgtttgcaga tcgtccggaa ctggcactgg cagttggtct ggatgaactg 1140

gtgtatcgtc gtagcaccct ggttcgtggt ctgagtagga tgccggtgac aatgggtccg 1200

cgttcagcag gcggaagtgg cggaagtgtg ctgcaccggc atcaaccggt caccatcgga 1260

gaacccgccg cccgggcggt gtcccgcacc gtcaccgtcg agcgcctgga ccggatcgcc 1320

gacgacgtgc tgcgcctcgt cctgcgcgac gccggcggaa agacattacc cacgtggact 1380

cccggcgccc atatcgacct cgacctcggc gcgctgtcgc gccagtactc cctgtgcggc 1440

gcgcccgatg cgccgagcta cgagattgcc gtgcacctgg atcccgagag ccgcggcggt 1500

tcgcgctaca tccacgaaca gctcgaggtg ggaagcccgc tccggatgcg cggccctcgg 1560

aaccatttcg cgctcgaccc cggcgccgag cactacgtgt tcgtcgccgg cggcatcggc 1620

atcaccccag tcctggccat ggccgaccac gcccgcgccc gggggtggag ctacgaactg 1680

cactactgcg gccgaaaccg ttccggcatg gcctatctcg agcgtgtcgc cgggcacggt 1740

gaccgggccg ccctgcacgt gtccgaggaa ggcacccgga tcgacctcgc cgccctcctc 1800

gccgagcccg cccccggcgt ccagatctac gcgtgcgggc ccgggcggct gctcgccgga 1860

ctcgaggacg cgagccggaa ctggcccgac ggggcgctgc acgtcgagca cttcacctcg 1920

tccctcgcgg cgctcgatcc ggacgtcgag cacgccttcg acctcgaact gcgtgactcg 1980

gggctgaccg tgcgggtcga acccacccag accgtcctcg acgcgttgcg cgccaacaac 2040

atcgacgtgc ccagcgactg cgaggaaggc ctctgcggct cgtgcgaggt cgccgtcctc 2100

gacggcgagg tcgaccatcg cgacacggtg ctgaccaagg ccgagcgggc ggcgaaccgg 2160

cagatgatga cctgctgctc gcgtgcctgt ggcgaccggc tggccctgcg actc 2214

<210> 15

<211> 729

<212> PRT

<213> Artificial Sequence

<220>

<223> K1-RhFR-D3氨基酸序列

<400> 15

Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly

1 5 10 15

Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu

20 25 30

Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu

35 40 45

Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser

50 55 60

Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro

65 70 75 80

Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr

85 90 95

Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn

100 105 110

Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly

115 120 125

Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln

130 135 140

Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp

145 150 155 160

Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro

165 170 175

Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser

180 185 190

Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser

195 200 205

Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu

210 215 220

Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr

225 230 235 240

Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro

245 250 255

Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala

260 265 270

Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile

275 280 285

Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala

290 295 300

Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp

305 310 315 320

Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly

325 330 335

Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu

340 345 350

Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg

355 360 365

Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg

370 375 380

Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro

385 390 395 400

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

405 410 415

Ala Val Ser Arg Thr Val Thr Val Glu Arg Leu Asp Arg Ile Ala Asp

420 425 430

Asp Val Leu Arg Leu Val Leu Arg Asp Ala Gly Gly Lys Thr Leu Pro

435 440 445

Thr Trp Thr Pro Gly Ala His Ile Asp Leu Asp Leu Gly Ala Leu Ser

450 455 460

Arg Gln Tyr Ser Leu Cys Gly Ala Pro Asp Ala Pro Ser Tyr Glu Ile

465 470 475 480

Ala Val His Leu Asp Pro Glu Ser Arg Gly Gly Ser Arg Tyr Ile His

485 490 495

Glu Gln Leu Glu Val Gly Ser Pro Leu Arg Met Arg Gly Pro Arg Asn

500 505 510

His Phe Ala Leu Asp Pro Gly Ala Glu His Tyr Val Phe Val Ala Gly

515 520 525

Gly Ile Gly Ile Thr Pro Val Leu Ala Met Ala Asp His Ala Arg Ala

530 535 540

Arg Gly Trp Ser Tyr Glu Leu His Tyr Cys Gly Arg Asn Arg Ser Gly

545 550 555 560

Met Ala Tyr Leu Glu Arg Val Ala Gly His Gly Asp Arg Ala Ala Leu

565 570 575

His Val Ser Glu Glu Gly Thr Arg Ile Asp Leu Ala Ala Leu Leu Ala

580 585 590

Glu Pro Ala Pro Gly Val Gln Ile Tyr Ala Cys Gly Pro Gly Arg Leu

595 600 605

Leu Ala Gly Leu Glu Asp Ala Ser Arg Asn Trp Pro Asp Gly Ala Leu

610 615 620

His Val Glu His Phe Thr Ser Ser Leu Ala Ala Leu Asp Pro Asp Val

625 630 635 640

Glu His Ala Phe Asp Leu Glu Leu Arg Asp Ser Gly Leu Thr Val Arg

645 650 655

Val Glu Pro Thr Gln Thr Val Leu Asp Ala Leu Arg Ala Asn Asn Ile

660 665 670

Asp Val Pro Ser Asp Cys Glu Glu Gly Leu Cys Gly Ser Cys Glu Val

675 680 685

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

690 695 700

Ala Glu Arg Ala Ala Asn Arg Gln Met Met Thr Cys Cys Ser Arg Ala

705 710 715 720

Cys Gly Asp Arg Leu Ala Leu Arg Leu

725

<210> 16

<211> 2187

<212> DNA

<213> Artificial Sequence

<220>

<223> K1-RhFR-D3核苷酸序列

<400> 16

atggcactga ccaccaccgg taccgaacag catgacctgt ttagcggtac cttttggcag 60

aatccgcatc cggcgtatgc agcactgcgt gcagaagatc cggttcgtaa actggcactg 120

ccggatggtc cggtgtggct gctgacccgt tatgcagatg ttcgtgaagc atttgttgat 180

ccgcgtctga gtaaagattg gcgtcatcgt ctgccggaag atcagcgtgc cgatatgccg 240

gcaaccccga ccccgatgat gattctgatg gacccgccgg atcatacacg tttacgtaaa 300

ctggttggtc gtagttttac cgttcgtcgt atgaatgaac tggaaccgcg tattaccgaa 360

attgcagatg gtctgctggc aggtctgccg accgatggtc cggttgatct gatgcgtgaa 420

tatgcatttc agattccggt tcaggttata tgtgaactgc tgggtctgcc ggcagaagat 480

cgtgatgatt tttcagcatg gtcaagtgtg ctggttgatg attctccggc agatgataaa 540

aatgccgcaa tgggtaaact gcatggttat ctgtcagatc tgctggaacg taaacgtacc 600

gaaccggatg atgcactgct gagtagcctg ctggcggttt ctgatatgga tggtgatcgt 660

ctgtctcagg aagaactggt tgcaatggca atgctgctgc tgattgcagg tcatgaaacc 720

accgttaatc tgattggtaa tggtgtgctg gcactgctga cccatccgga tcagcgtaaa 780

ctgttagctg aagatccgag tctgattagc tcagcagttg aagaatttct gcgttttgat 840

tctccggtta gccaggcacc gatccgtttt accgctgaag atgttaccta tagtggtgtt 900

accattccgg caggtgaaat ggttatgctg ggtctggcag cagcaaatcg cgatgcagat 960

tggatgccgg aaccggatcg tctggatatt acccgtgatg caagtggtgg tgttttcttt 1020

ggtcatggta ttcatttttg tctgggtgcg cagctggcac gtctggaagg tcgtgtggca 1080

attggtcgtc tgtttgcaga tcgtccggaa ctggcactgg cagttggtct ggatgaactg 1140

gtgtatcgtc gtagcaccct ggttcgtggt ctgagtagga tgccggtgac aatgggtccg 1200

cgttcagcac ggcatcaacc ggtcaccatc ggagaacccg ccgcccgggc ggtgtcccgc 1260

accgtcaccg tcgagcgcct ggaccggatc gccgacgacg tgctgcgcct cgtcctgcgc 1320

gacgccggcg gaaagacatt acccacgtgg actcccggcg cccatatcga cctcgacctc 1380

ggcgcgctgt cgcgccagta ctccctgtgc ggcgcgcccg atgcgccgag ctacgagatt 1440

gccgtgcacc tggatcccga gagccgcggc ggttcgcgct acatccacga acagctcgag 1500

gtgggaagcc cgctccggat gcgcggccct cggaaccatt tcgcgctcga ccccggcgcc 1560

gagcactacg tgttcgtcgc cggcggcatc ggcatcaccc cagtcctggc catggccgac 1620

cacgcccgcg cccgggggtg gagctacgaa ctgcactact gcggccgaaa ccgttccggc 1680

atggcctatc tcgagcgtgt cgccgggcac ggtgaccggg ccgccctgca cgtgtccgag 1740

gaaggcaccc ggatcgacct cgccgccctc ctcgccgagc ccgcccccgg cgtccagatc 1800

tacgcgtgcg ggcccgggcg gctgctcgcc ggactcgagg acgcgagccg gaactggccc 1860

gacggggcgc tgcacgtcga gcacttcacc tcgtccctcg cggcgctcga tccggacgtc 1920

gagcacgcct tcgacctcga actgcgtgac tcggggctga ccgtgcgggt cgaacccacc 1980

cagaccgtcc tcgacgcgtt gcgcgccaac aacatcgacg tgcccagcga ctgcgaggaa 2040

ggcctctgcg gctcgtgcga ggtcgccgtc ctcgacggcg aggtcgacca tcgcgacacg 2100

gtgctgacca aggccgagcg ggcggcgaac cggcagatga tgacctgctg ctcgcgtgcc 2160

tgtggcgacc ggctggccct gcgactc 2187

<210> 17

<211> 726

<212> PRT

<213> Artificial Sequence

<220>

<223> K1-RhFR-D6氨基酸序列

<400> 17

Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly

1 5 10 15

Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu

20 25 30

Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu

35 40 45

Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser

50 55 60

Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro

65 70 75 80

Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr

85 90 95

Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn

100 105 110

Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly

115 120 125

Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln

130 135 140

Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp

145 150 155 160

Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro

165 170 175

Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser

180 185 190

Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser

195 200 205

Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu

210 215 220

Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr

225 230 235 240

Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro

245 250 255

Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala

260 265 270

Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile

275 280 285

Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala

290 295 300

Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp

305 310 315 320

Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly

325 330 335

Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu

340 345 350

Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg

355 360 365

Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg

370 375 380

Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro

385 390 395 400

Arg Ser Ala Pro Val Thr Ile Gly Glu Pro Ala Ala Arg Ala Val Ser

405 410 415

Arg Thr Val Thr Val Glu Arg Leu Asp Arg Ile Ala Asp Asp Val Leu

420 425 430

Arg Leu Val Leu Arg Asp Ala Gly Gly Lys Thr Leu Pro Thr Trp Thr

435 440 445

Pro Gly Ala His Ile Asp Leu Asp Leu Gly Ala Leu Ser Arg Gln Tyr

450 455 460

Ser Leu Cys Gly Ala Pro Asp Ala Pro Ser Tyr Glu Ile Ala Val His

465 470 475 480

Leu Asp Pro Glu Ser Arg Gly Gly Ser Arg Tyr Ile His Glu Gln Leu

485 490 495

Glu Val Gly Ser Pro Leu Arg Met Arg Gly Pro Arg Asn His Phe Ala

500 505 510

Leu Asp Pro Gly Ala Glu His Tyr Val Phe Val Ala Gly Gly Ile Gly

515 520 525

Ile Thr Pro Val Leu Ala Met Ala Asp His Ala Arg Ala Arg Gly Trp

530 535 540

Ser Tyr Glu Leu His Tyr Cys Gly Arg Asn Arg Ser Gly Met Ala Tyr

545 550 555 560

Leu Glu Arg Val Ala Gly His Gly Asp Arg Ala Ala Leu His Val Ser

565 570 575

Glu Glu Gly Thr Arg Ile Asp Leu Ala Ala Leu Leu Ala Glu Pro Ala

580 585 590

Pro Gly Val Gln Ile Tyr Ala Cys Gly Pro Gly Arg Leu Leu Ala Gly

595 600 605

Leu Glu Asp Ala Ser Arg Asn Trp Pro Asp Gly Ala Leu His Val Glu

610 615 620

His Phe Thr Ser Ser Leu Ala Ala Leu Asp Pro Asp Val Glu His Ala

625 630 635 640

Phe Asp Leu Glu Leu Arg Asp Ser Gly Leu Thr Val Arg Val Glu Pro

645 650 655

Thr Gln Thr Val Leu Asp Ala Leu Arg Ala Asn Asn Ile Asp Val Pro

660 665 670

Ser Asp Cys Glu Glu Gly Leu Cys Gly Ser Cys Glu Val Ala Val Leu

675 680 685

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

690 695 700

Ala Ala Asn Arg Gln Met Met Thr Cys Cys Ser Arg Ala Cys Gly Asp

705 710 715 720

Arg Leu Ala Leu Arg Leu

725

<210> 18

<211> 2178

<212> DNA

<213> Artificial Sequence

<220>

<223> K1-RhFR-D6核苷酸序列

<400> 18

atggcactga ccaccaccgg taccgaacag catgacctgt ttagcggtac cttttggcag 60

aatccgcatc cggcgtatgc agcactgcgt gcagaagatc cggttcgtaa actggcactg 120

ccggatggtc cggtgtggct gctgacccgt tatgcagatg ttcgtgaagc atttgttgat 180

ccgcgtctga gtaaagattg gcgtcatcgt ctgccggaag atcagcgtgc cgatatgccg 240

gcaaccccga ccccgatgat gattctgatg gacccgccgg atcatacacg tttacgtaaa 300

ctggttggtc gtagttttac cgttcgtcgt atgaatgaac tggaaccgcg tattaccgaa 360

attgcagatg gtctgctggc aggtctgccg accgatggtc cggttgatct gatgcgtgaa 420

tatgcatttc agattccggt tcaggttata tgtgaactgc tgggtctgcc ggcagaagat 480

cgtgatgatt tttcagcatg gtcaagtgtg ctggttgatg attctccggc agatgataaa 540

aatgccgcaa tgggtaaact gcatggttat ctgtcagatc tgctggaacg taaacgtacc 600

gaaccggatg atgcactgct gagtagcctg ctggcggttt ctgatatgga tggtgatcgt 660

ctgtctcagg aagaactggt tgcaatggca atgctgctgc tgattgcagg tcatgaaacc 720

accgttaatc tgattggtaa tggtgtgctg gcactgctga cccatccgga tcagcgtaaa 780

ctgttagctg aagatccgag tctgattagc tcagcagttg aagaatttct gcgttttgat 840

tctccggtta gccaggcacc gatccgtttt accgctgaag atgttaccta tagtggtgtt 900

accattccgg caggtgaaat ggttatgctg ggtctggcag cagcaaatcg cgatgcagat 960

tggatgccgg aaccggatcg tctggatatt acccgtgatg caagtggtgg tgttttcttt 1020

ggtcatggta ttcatttttg tctgggtgcg cagctggcac gtctggaagg tcgtgtggca 1080

attggtcgtc tgtttgcaga tcgtccggaa ctggcactgg cagttggtct ggatgaactg 1140

gtgtatcgtc gtagcaccct ggttcgtggt ctgagtagga tgccggtgac aatgggtccg 1200

cgttcagcac cggtcaccat cggagaaccc gccgcccggg cggtgtcccg caccgtcacc 1260

gtcgagcgcc tggaccggat cgccgacgac gtgctgcgcc tcgtcctgcg cgacgccggc 1320

ggaaagacat tacccacgtg gactcccggc gcccatatcg acctcgacct cggcgcgctg 1380

tcgcgccagt actccctgtg cggcgcgccc gatgcgccga gctacgagat tgccgtgcac 1440

ctggatcccg agagccgcgg cggttcgcgc tacatccacg aacagctcga ggtgggaagc 1500

ccgctccgga tgcgcggccc tcggaaccat ttcgcgctcg accccggcgc cgagcactac 1560

gtgttcgtcg ccggcggcat cggcatcacc ccagtcctgg ccatggccga ccacgcccgc 1620

gcccgggggt ggagctacga actgcactac tgcggccgaa accgttccgg catggcctat 1680

ctcgagcgtg tcgccgggca cggtgaccgg gccgccctgc acgtgtccga ggaaggcacc 1740

cggatcgacc tcgccgccct cctcgccgag cccgcccccg gcgtccagat ctacgcgtgc 1800

gggcccgggc ggctgctcgc cggactcgag gacgcgagcc ggaactggcc cgacggggcg 1860

ctgcacgtcg agcacttcac ctcgtccctc gcggcgctcg atccggacgt cgagcacgcc 1920

ttcgacctcg aactgcgtga ctcggggctg accgtgcggg tcgaacccac ccagaccgtc 1980

ctcgacgcgt tgcgcgccaa caacatcgac gtgcccagcg actgcgagga aggcctctgc 2040

ggctcgtgcg aggtcgccgt cctcgacggc gaggtcgacc atcgcgacac ggtgctgacc 2100

aaggccgagc gggcggcgaa ccggcagatg atgacctgct gctcgcgtgc ctgtggcgac 2160

cggctggccc tgcgactc 2178

<210> 19

<211> 746

<212> PRT

<213> Artificial Sequence

<220>

<223> K1-RhFR-I14氨基酸序列

<400> 19

Met Ala Leu Thr Thr Thr Gly Thr Glu Gln His Asp Leu Phe Ser Gly

1 5 10 15

Thr Phe Trp Gln Asn Pro His Pro Ala Tyr Ala Ala Leu Arg Ala Glu

20 25 30

Asp Pro Val Arg Lys Leu Ala Leu Pro Asp Gly Pro Val Trp Leu Leu

35 40 45

Thr Arg Tyr Ala Asp Val Arg Glu Ala Phe Val Asp Pro Arg Leu Ser

50 55 60

Lys Asp Trp Arg His Arg Leu Pro Glu Asp Gln Arg Ala Asp Met Pro

65 70 75 80

Ala Thr Pro Thr Pro Met Met Ile Leu Met Asp Pro Pro Asp His Thr

85 90 95

Arg Leu Arg Lys Leu Val Gly Arg Ser Phe Thr Val Arg Arg Met Asn

100 105 110

Glu Leu Glu Pro Arg Ile Thr Glu Ile Ala Asp Gly Leu Leu Ala Gly

115 120 125

Leu Pro Thr Asp Gly Pro Val Asp Leu Met Arg Glu Tyr Ala Phe Gln

130 135 140

Ile Pro Val Gln Val Ile Cys Glu Leu Leu Gly Leu Pro Ala Glu Asp

145 150 155 160

Arg Asp Asp Phe Ser Ala Trp Ser Ser Val Leu Val Asp Asp Ser Pro

165 170 175

Ala Asp Asp Lys Asn Ala Ala Met Gly Lys Leu His Gly Tyr Leu Ser

180 185 190

Asp Leu Leu Glu Arg Lys Arg Thr Glu Pro Asp Asp Ala Leu Leu Ser

195 200 205

Ser Leu Leu Ala Val Ser Asp Met Asp Gly Asp Arg Leu Ser Gln Glu

210 215 220

Glu Leu Val Ala Met Ala Met Leu Leu Leu Ile Ala Gly His Glu Thr

225 230 235 240

Thr Val Asn Leu Ile Gly Asn Gly Val Leu Ala Leu Leu Thr His Pro

245 250 255

Asp Gln Arg Lys Leu Leu Ala Glu Asp Pro Ser Leu Ile Ser Ser Ala

260 265 270

Val Glu Glu Phe Leu Arg Phe Asp Ser Pro Val Ser Gln Ala Pro Ile

275 280 285

Arg Phe Thr Ala Glu Asp Val Thr Tyr Ser Gly Val Thr Ile Pro Ala

290 295 300

Gly Glu Met Val Met Leu Gly Leu Ala Ala Ala Asn Arg Asp Ala Asp

305 310 315 320

Trp Met Pro Glu Pro Asp Arg Leu Asp Ile Thr Arg Asp Ala Ser Gly

325 330 335

Gly Val Phe Phe Gly His Gly Ile His Phe Cys Leu Gly Ala Gln Leu

340 345 350

Ala Arg Leu Glu Gly Arg Val Ala Ile Gly Arg Leu Phe Ala Asp Arg

355 360 365

Pro Glu Leu Ala Leu Ala Val Gly Leu Asp Glu Leu Val Tyr Arg Arg

370 375 380

Ser Thr Leu Val Arg Gly Leu Ser Arg Met Pro Val Thr Met Gly Pro

385 390 395 400

Arg Ser Ala Glu Leu Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu

405 410 415

Asn Val Leu His Arg His Gln Pro Val Thr Ile Gly Glu Pro Ala Ala

420 425 430

Arg Ala Val Ser Arg Thr Val Thr Val Glu Arg Leu Asp Arg Ile Ala

435 440 445

Asp Asp Val Leu Arg Leu Val Leu Arg Asp Ala Gly Gly Lys Thr Leu

450 455 460

Pro Thr Trp Thr Pro Gly Ala His Ile Asp Leu Asp Leu Gly Ala Leu

465 470 475 480

Ser Arg Gln Tyr Ser Leu Cys Gly Ala Pro Asp Ala Pro Ser Tyr Glu

485 490 495

Ile Ala Val His Leu Asp Pro Glu Ser Arg Gly Gly Ser Arg Tyr Ile

500 505 510

His Glu Gln Leu Glu Val Gly Ser Pro Leu Arg Met Arg Gly Pro Arg

515 520 525

Asn His Phe Ala Leu Asp Pro Gly Ala Glu His Tyr Val Phe Val Ala

530 535 540

Gly Gly Ile Gly Ile Thr Pro Val Leu Ala Met Ala Asp His Ala Arg

545 550 555 560

Ala Arg Gly Trp Ser Tyr Glu Leu His Tyr Cys Gly Arg Asn Arg Ser

565 570 575

Gly Met Ala Tyr Leu Glu Arg Val Ala Gly His Gly Asp Arg Ala Ala

580 585 590

Leu His Val Ser Glu Glu Gly Thr Arg Ile Asp Leu Ala Ala Leu Leu

595 600 605

Ala Glu Pro Ala Pro Gly Val Gln Ile Tyr Ala Cys Gly Pro Gly Arg

610 615 620

Leu Leu Ala Gly Leu Glu Asp Ala Ser Arg Asn Trp Pro Asp Gly Ala

625 630 635 640

Leu His Val Glu His Phe Thr Ser Ser Leu Ala Ala Leu Asp Pro Asp

645 650 655

Val Glu His Ala Phe Asp Leu Glu Leu Arg Asp Ser Gly Leu Thr Val

660 665 670

Arg Val Glu Pro Thr Gln Thr Val Leu Asp Ala Leu Arg Ala Asn Asn

675 680 685

Ile Asp Val Pro Ser Asp Cys Glu Glu Gly Leu Cys Gly Ser Cys Glu

690 695 700

Val Ala Val Leu Asp Gly Glu Val Asp His Arg Asp Thr Val Leu Thr

705 710 715 720

Lys Ala Glu Arg Ala Ala Asn Arg Gln Met Met Thr Cys Cys Ser Arg

725 730 735

Ala Cys Gly Asp Arg Leu Ala Leu Arg Leu

740 745

<210> 20

<211> 2238

<212> DNA

<213> Artificial Sequence

<220>

<223> K1-RhFR-I14核苷酸序列

<400> 20

atggcactga ccaccaccgg taccgaacag catgacctgt ttagcggtac cttttggcag 60

aatccgcatc cggcgtatgc agcactgcgt gcagaagatc cggttcgtaa actggcactg 120

ccggatggtc cggtgtggct gctgacccgt tatgcagatg ttcgtgaagc atttgttgat 180

ccgcgtctga gtaaagattg gcgtcatcgt ctgccggaag atcagcgtgc cgatatgccg 240

gcaaccccga ccccgatgat gattctgatg gacccgccgg atcatacacg tttacgtaaa 300

ctggttggtc gtagttttac cgttcgtcgt atgaatgaac tggaaccgcg tattaccgaa 360

attgcagatg gtctgctggc aggtctgccg accgatggtc cggttgatct gatgcgtgaa 420

tatgcatttc agattccggt tcaggttata tgtgaactgc tgggtctgcc ggcagaagat 480

cgtgatgatt tttcagcatg gtcaagtgtg ctggttgatg attctccggc agatgataaa 540

aatgccgcaa tgggtaaact gcatggttat ctgtcagatc tgctggaacg taaacgtacc 600

gaaccggatg atgcactgct gagtagcctg ctggcggttt ctgatatgga tggtgatcgt 660

ctgtctcagg aagaactggt tgcaatggca atgctgctgc tgattgcagg tcatgaaacc 720

accgttaatc tgattggtaa tggtgtgctg gcactgctga cccatccgga tcagcgtaaa 780

ctgttagctg aagatccgag tctgattagc tcagcagttg aagaatttct gcgttttgat 840

tctccggtta gccaggcacc gatccgtttt accgctgaag atgttaccta tagtggtgtt 900

accattccgg caggtgaaat ggttatgctg ggtctggcag cagcaaatcg cgatgcagat 960

tggatgccgg aaccggatcg tctggatatt acccgtgatg caagtggtgg tgttttcttt 1020

ggtcatggta ttcatttttg tctgggtgcg cagctggcac gtctggaagg tcgtgtggca 1080

attggtcgtc tgtttgcaga tcgtccggaa ctggcactgg cagttggtct ggatgaactg 1140

gtgtatcgtc gtagcaccct ggttcgtggt ctgagtagga tgccggtgac aatgggtccg 1200

cgttcagcag aactgcagag tgcaaaaaaa gttcgtaaaa aagcagaaaa tgtgctgcac 1260

cggcatcaac cggtcaccat cggagaaccc gccgcccggg cggtgtcccg caccgtcacc 1320

gtcgagcgcc tggaccggat cgccgacgac gtgctgcgcc tcgtcctgcg cgacgccggc 1380

ggaaagacat tacccacgtg gactcccggc gcccatatcg acctcgacct cggcgcgctg 1440

tcgcgccagt actccctgtg cggcgcgccc gatgcgccga gctacgagat tgccgtgcac 1500

ctggatcccg agagccgcgg cggttcgcgc tacatccacg aacagctcga ggtgggaagc 1560

ccgctccgga tgcgcggccc tcggaaccat ttcgcgctcg accccggcgc cgagcactac 1620

gtgttcgtcg ccggcggcat cggcatcacc ccagtcctgg ccatggccga ccacgcccgc 1680

gcccgggggt ggagctacga actgcactac tgcggccgaa accgttccgg catggcctat 1740

ctcgagcgtg tcgccgggca cggtgaccgg gccgccctgc acgtgtccga ggaaggcacc 1800

cggatcgacc tcgccgccct cctcgccgag cccgcccccg gcgtccagat ctacgcgtgc 1860

gggcccgggc ggctgctcgc cggactcgag gacgcgagcc ggaactggcc cgacggggcg 1920

ctgcacgtcg agcacttcac ctcgtccctc gcggcgctcg atccggacgt cgagcacgcc 1980

ttcgacctcg aactgcgtga ctcggggctg accgtgcggg tcgaacccac ccagaccgtc 2040

ctcgacgcgt tgcgcgccaa caacatcgac gtgcccagcg actgcgagga aggcctctgc 2100

ggctcgtgcg aggtcgccgt cctcgacggc gaggtcgacc atcgcgacac ggtgctgacc 2160

aaggccgagc gggcggcgaa ccggcagatg atgacctgct gctcgcgtgc ctgtggcgac 2220

cggctggccc tgcgactc 2238

<210> 21

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> K1-Rh-F1

<400> 21

accggctggc cctgcgactc taaaagcttg cggccgcact 40

<210> 22

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> K1-Rh-R1

<400> 22

ggttgatgcc ggtgcagcac tgctgaacgc ggacccattg 40

<210> 23

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> RhFR-F1

<400> 23

caatgggtcc gcgttcagca gtgctgcacc ggcatcaacc 40

<210> 24

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> RhFR-R1

<400> 24

agtgcggccg caagctttta gagtcgcagg gccagccggt 40

<210> 25

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> K1-BM3-F1

<400> 25

caaaagacgt gtgggctggg taaaagcttg cggccgcact 40

<210> 26

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> K1-BM3-R1

<400> 26

tgttcagtgc taggtgaagg tgctgaacgc ggacccattg 40

<210> 27

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> BM3R-F1

<400> 27

caatgggtcc gcgttcagca ccttcaccta gcactgaaca 40

<210> 28

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> BM3R-R1

<400> 28

agtgcggccg caagctttta cccagcccac acgtcttttg 40

<210> 29

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> I3-F

<400> 29

ccgcgttcag caggcggaag tgtgctgcac cgg 33

<210> 30

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> I3-R

<400> 30

ccggtgcagc acacttccgc ctgctgaacg cgg 33

<210> 31

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> I6-F

<400> 31

gcgttcagca ggcggaagtg gcggaagtgt gctgcacc 38

<210> 32

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> I6-R

<400> 32

ggtgcagcac acttccgcca cttccgcctg ctgaacgc 38

<210> 33

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> D3-F

<400> 33

ggtccgcgtt cagcacggca tcaaccggtc 30

<210> 34

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> D3-R

<400> 34

gaccggttga tgccgtgctg aacgcggacc 30

<210> 35

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> D6-F

<400> 35

ggtccgcgtt cagcaccggt caccatcgga 30

<210> 36

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> D6-R

<400> 36

tccgatggtg accggtgctg aacgcggacc 30

<210> 37

<211> 72

<212> DNA

<213> Artificial Sequence

<220>

<223> I14-F

<400> 37

atgccggtgc agcacatttt ctgctttttt acgaactttt tttgcactct gcagttctgc 60

tgaacgcgga cc 72

<210> 38

<211> 72

<212> DNA

<213> Artificial Sequence

<220>

<223> I14-R

<400> 38

ggtccgcgtt cagcagaact gcagagtgca aaaaaagttc gtaaaaaagc agaaaatgtg 60

ctgcaccggc at 72

Claims

1.一种融合蛋白或其变体，其特征在于，其包括K1和RhFR，所述K1的氨基酸序列如SEQID NO:1所示，所述RhFR的氨基酸序列如SEQ ID NO:5的第466-773位的氨基酸所示。

2.如权利要求1所述的融合蛋白或其变体，其特征在于，所述融合蛋白从N端至C端依次为K1和RhFR；

和/或，所述K1和RhFR之间通过连接子进行连接，所述连接子的氨基酸序列优选如SEQID NO:5的第445-465位的氨基酸所示；

和/或，所述融合蛋白或其变体与分子伴侣共表达，所述分子伴侣优选为Gro7。

3.如权利要求1或2所述的融合蛋白或其变体，其特征在于，所述变体为在所述RhFR的N端发生氨基酸的插入或缺失，优选在所述RhFR的N端发生1-14个更优选发生3-6个氨基酸的插入或缺失；

较佳地，所述融合蛋白或其变体的氨基酸序列如SEQ ID NO:9、SEQ ID NO:11、SEQ IDNO:13、SEQ ID NO:15、SEQ ID NO:17或SEQ ID NO:19所示；

4.一种融合基因，其特征在于，其编码如权利要求1～3任一项所述的融合蛋白或其变体。

5.一种重组表达载体，其特征在于，所述重组表达载体含有如权利要求4所述的融合基因；

较佳地，所述重组表达载体的骨架载体为pET28a。

6.一种转化体，其特征在于，其包括如权利要求4所述的融合基因或者如权利要求5所述的重组表达载体；

较佳地，所转化体通过在宿主中导入所述融合基因或者所述重组表达载体获得，所述宿主优选为大肠杆菌，更优选为大肠杆菌E.coli BL21(DE3)细胞。

7.一种融合蛋白或其变体的制备方法，其包括以下步骤：

(1)获得如权利要求6所述的转化体；

(2)筛选所述转化体，表达并纯化所述融合蛋白或其变体。

8.一种骨化二醇的制备方法，其特征在于，所述制备方法包括以下步骤：在反应溶剂、还原型辅酶NADH/NADPH的存在下，将如权利要求1～3任一项所述的融合蛋白或其变体催化维生素D3进行羟化反应即可；

较佳地：

所述维生素D3为助溶剂预溶的维生素D3；所述助溶剂优选包括DMSO、吐温80、TritonX100、甲醇、乙醇、异丙醇和DMF中的一种或多种，例如为乙醇；

和/或，所述方法还包括在进行所述羟化反应前，在所述反应溶剂中加入环糊精的步骤，所述环糊精例如为羟丙基-β-环糊精；所述羟丙基-β-环糊精占反应体系的质量体积百分比优选为0.05％-0.4％，例如0.25％；

和/或，所述反应的温度为20～33℃，例如为22℃、25℃、28℃或30℃；

和/或，所述反应的pH为6.0～8.0，例如为7.4；

和/或，所述维生素D3的浓度为1g/L-10g/L，例如为2g/L、3g/L、4g/L、5g/L、6g/L、7g/L、8g/L或9g/L；

和/或，所述NADH/NADPH与所述维生素D3的摩尔比为0.001:1～2:1，例如0.2:1。

9.如权利要求8所述的方法，其特征在于，所述的制备方法还包括以下步骤：在脱氢酶以及供氢体的存在下，将氧化型辅酶NAD⁺/NADP⁺进行还原反应，得到所述的NADH/NADPH即可；

较佳地，所述的脱氢酶为葡萄糖脱氢酶、醇脱氢酶或甲酸脱氢酶；和/或，所述的供氢体为葡萄糖、异丙醇或甲酸盐；

更佳地，当所述的脱氢酶为醇脱氢酶时，所述的供氢体为异丙醇；当所述的脱氢酶为葡萄糖脱氢酶时，所述的供氢体为葡萄糖；当所述的脱氢酶为甲酸脱氢酶时，所述的供氢体为甲酸盐。

10.一种如权利要求1～3任一项所述的融合蛋白或其变体、如权利要求4所述的融合基因、如权利要求5所述的重组表达载体、或如权利要求6所述的转化体在制备骨化二醇中的应用。