CN108623672A - 新的egf-a和b的组合突变体制备及其在生物药物中的应用 - Google Patents
新的egf-a和b的组合突变体制备及其在生物药物中的应用 Download PDFInfo
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Abstract
本发明提供了新的人LDL受体EGF(AB)突变体，以及突变体与人免疫球蛋白Fc片段融合的制备方法。本发明的EGF(AB)突变体比EGF‑A突变体具有更好的生物学活性；融合Fc片段的EGF(AB)突变体比EGF(AB)突变体具有更长的半衰期，可用于治疗高胆固醇血症、动脉粥样硬化等心血管疾病以及通过降低血浆PCSK9水平而受益的其它疾病。
Description
技术领域
本发明属于生物技术领域，涉及低密度脂蛋白受体(low-density lipoproteinreceptor,LDLR)胞外域表皮生长因子前体同源域A和B(Epidermal growth factorprecursor homology domain A and B,EGF-AB)的突变体及其融合蛋白，制备方法和治疗高胆固醇血症、动脉粥样硬化等心血管疾病以及通过降低血浆PCSK9水平而受益的其它疾病的应用。
背景技术
LDL受体是一种跨膜细胞表面糖蛋白，包含5个功能结构域：配体结合域、表皮生长因子前体同源域、O-连接糖域、跨膜域和胞浆域。LDLR的配体结合域与表皮生长因子前体同源域均位于胞外，前者可与载脂蛋白ApoB、载脂蛋白ApoE、低密度脂蛋白(LDL)和β极低密度脂蛋白(β-VLDL)结合，并形成配体与受体的复合体内吞入胞，因而在胆固醇代谢过程中起了关键作用。表皮生长因子前体同源域因与表皮生长因子(epithelial growth factor,EGF)前体胞外域有33-35％的结构同源性而得名，其EGF-A区与前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin 9,PCSK9)的催化活性有关，PCSK9通过其催化结构域与EGF-A结合，并导致LDLR降解或降低其摄取LDL的能力。而EGF-B区具有钙结合特性，有助于功能域之间的稳定。至今，全球范围内已近700余种LDLR的缺陷类型与家族性高胆固醇血症(FH)发生相关，其中10％的缺陷为LDLR的EGF-A或EGF-B发生的缺陷(http://www.ucl.ac.uk/fh)。EGF-A是影响PCSK9催化活性的重要片段，体外功能研究发现，对EGF-A 区的位点突变，可使其结合PCSK9的能力减弱或增强，如对EGF-A的Leu318实施不同电荷的氨基酸置换，与野生型EGF-A相比，只有精氨酸置换显著增强EGF-A突变体与PCSK9的结合能力(Gu等，Journal of Lipid Research.2013；54:3345-3357)；EGF-A区Cys308的突变(C308Y)是中国和波兰的高胆固醇血症患者中的突变类型(Zhang等，J Lipid Res.2003 Oct；44(10):1850-8；-Miarka等,Mol Biol Rep.2012 May；39(5):5181-6.)，其具有结合PCSK9的能力。中国专利公开了C308Y与其它突变位点的串联形式，以及在制备治疗高胆固醇血症中的用途。但我们最近发现，EGF-A区Cys308的另一突变类型(C308T)也有显著结合PCSK9的能力；我们进一步还发现，EGF-B或EGF-B突变体可显著增强包含(C308T)或其它突变位点的EGF-A突变体结合PCSK9的能力，抑制PCSK9介导的LDLR降解，恢复肝细胞表面LDLR对LDL的摄取能力；而且EGF-AB突变体明显优于野生型EGF-AB。
发明内容
本发明的目的是提供新的EGF(AB)突变体及其与Fc的可溶性融合蛋白，所提供的突变体是在LDL受体胞外域的第295位天冬酰胺(Asn)由丝氨酸(Ser)取代(或置换)、第308位半胱胺酸(Cys)由苏氨酸(Thr)取代(或置换)、第318位亮氨酸(Leu)由精氨酸(Arg)取代(或置换)、第334位异亮氨酸(Ile)由缬氨酸(Val)取代(或置换)、第346位亮氨酸(Leu)由组氨酸(His)取代(或置换)的任意一位以上氨基酸发生取代(或置换)的EGF-AB片段。
本发明提供了EGF-AB突变体的氨基酸序列表如SEQ ID NO：1、3、5、7、9、11、13、15、17、19、21所述；
本发明提供的EGF(AB)突变体融合蛋白，为所述EGF(AB)突变体通过如下方式与人IgG Fc片段融合：EGF(AB)—连接肽—Fc或EGF(AB)—Fc；EGF(AB)—连接肽—Fc—连接肽—EGF(AB)或EGF(AB)—Fc—EGF(AB)；(EGF(AB)—连接肽)n—Fc—(连接肽—EGF(AB))n或EGF(AB)n—Fc—EGF(AB)n，其中，n取1～3整数，连接肽序列为：GGGSGGGSGGGSGGGS。其具体序列如下：
IgG1 Fc的氨基酸如序列表中的SEQ ID NO：23所述。
IgG2 Fc的氨基酸如序列表中的SEQ ID NO：25所述。
IgG3 Fc的氨基酸如序列表中的SEQ ID NO：27所述。
IgG4 Fc的氨基酸如序列表中的SEQ ID NO：29所述。
mEGF(AB)15-Fc的氨基酸如序列表中的SEQ ID NO：31所述；
mEGF(AB)16-Fc的氨基酸如序列表中的SEQ ID NO：33所述；
mEGF(AB)19-Fc的氨基酸如序列表中的SEQ ID NO：35所述；
mEGF(AB)52-Fc的氨基酸如序列表中的SEQ ID NO：37所述；
mEGF(AB)55-Fc的氨基酸如序列表中的SEQ ID NO：39所述；
2mEGF(AB)55-Fc的氨基酸如序列表中的SEQ ID NO：41所述；
2mEGF(AB)56-Fc的氨基酸如序列表中的SEQ ID NO：43所述；
编码所述上述融合蛋白核苷酸序列如下：
IgG1 Fc的核苷酸如序列表中的SEQ ID NO：24所述。
IgG2 Fc的核苷酸如序列表中的SEQ ID NO：26所述。
IgG3 Fc的核苷酸如序列表中的SEQ ID NO：28所述。
IgG4 Fc的核苷酸如序列表中的SEQ ID NO：30所述。
mEGF(AB)15-Fc的核苷酸如序列表中的SEQ ID NO：32所述；
mEGF(AB)16-Fc的核苷酸如序列表中的SEQ ID NO：34所述；
mEGF(AB)19-Fc的核苷酸如序列表中的SEQ ID NO：36所述；
mEGF(AB)52-Fc的核苷酸如序列表中的SEQ ID NO：38所述；
mEGF(AB)55-Fc的核苷酸如序列表中的SEQ ID NO：40所述；
2mEGF(AB)55-Fc的核苷酸如序列表中的SEQ ID NO：42所述；
2mEGF(AB)56-Fc的核苷酸如序列表中的SEQ ID NO：44所述；
本发明同时提供了上述核酸序列的表达载体，其能在所转染的宿主细胞中复制表达。
含有表达上述突变体或其融合蛋白的宿主细胞，可以为中国仓鼠卵巢(CHO)细胞及其亚系或人胚肾293细胞及其亚系。
本发明还提供了上述EGF-AB突变体或其融合蛋白及制备用于治疗高胆固醇血症，以及通过降低血浆PCSK9水平而受益的其它疾病的用途。
附图说明
图1本发明EGF(AB)多肽或融合蛋白对PCSK9介导LDLR降解的影响。1为EGF(AB)wt组,2为EGF(AB)wt-Fc组,3为mEGF(AB)55-Fc组,4为2mEGF(AB)55-Fc组,5为2mEGF(AB)56-Fc组。
图2本发明EGF(AB)多肽或融合蛋白对HepG2细胞摄取LDL的影响。
具体实施方式
提供以下实施例对本发明作进一步阐释。应当理解，这些实施例仅用于说明本发明而非对本发明有任何限制。本领域技术人员在本说明书的启示下对本发明实施中所作的任何变动都将落在权利要求书的范围内。
实施例1多肽及其融合蛋白的制备
1.质粒构建
1.1基因和引物合成
表达序列通过下述合成的基因和引物重组至表达载体。基因和引物由上海生工生物工程有限公司合成，合成基因序列重组至质粒载体pUC19中，命名为pUC19-mEGF(A)16、pUC19-EGF(AB)wt、pUC19-mEGF(AB)15、pUC19-mEGF(AB)16、pUC19-mEGF(AB)19、pUC19-mEGF(AB)52、pUC19-mEGF(AB)55、pUC19-2mEGF(AB)55、pUC19-2mEGF(AB)56。
合成融合蛋白基因片段:
EGF(A)wt：
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTGCAATGACCTTAAGATCGGCTACGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCCACCTCCTCCACCACCTCTGATTAATTAAGCG
mEGF(A)16：
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTACAATGACCTTAAGATCGGCTACGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGC
EGF(AB)wt：
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTGCAATGACCTTAAGATCGGCTACGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATATCGATGAGTGTCAGGATCCCGACACCTGCAGCCAGCTCTGCGTGAACCTGGAGGGTGGCTACAAGTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCCACACGAAGGCCTGCAAGCCACCTCCTCCACCACCTCTGATTAATTAAGCG
mEGF(AB)15：
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCACCAATGACCTTAAGATCGGCTACGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATGTCGATGAGTGTCAGGATCCCGACACCTGCAGCCAGCACTGCGTGAACCTGGAGGGTGGCTACAAGTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCCACACGAAGGCCTGCAAG
mEGF(AB)16：
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCTACAATGACCTTAAGATCGGCTACGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATGTCGATGAGTGTCAGGATCCCGACACCTGCAGCCAGCACTGCGTGAACCTGGAGGGTGGCTACAAGTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCCACACGAAGGCCTGCAAG
mEGF(AB)19：
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTGGGACCAGCGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCACCAATGACCTTAAGATCGGCTACGAGTGCCTGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATGTCGATGAGTGTCAGGATCCCGACACCTGCAGCCAGCACTGCGTGAACCTGGAGGGTGGCTACAAGTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCCACACGAAGGCCTGCAAG
mEGF(AB)52：
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTGGGACCAACGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCACCAATGACCTTAAGATCGGCTACGAGTGCCGGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATGTCGATGAGTGTCAGGATCCCGACACCTGCAGCCAGCACTGCGTGAACCTGGAGGGTGGCTACAAGTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCCACACGAAGGCCTGCAAG
mEGF(AB)55：
GCAGATCTCCTAGGGCCACCATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCTGGGACCAGCGAATGCTTGGACAACAACGGCGGCTGTTCCCACGTCACCAATGACCTTAAGATCGGCTACGAGTGCCGGTGCCCCGACGGCTTCCAGCTGGTGGCCCAGCGAAGATGCGAAGATGTCGATGAGTGTCAGGATCCCGACACCTGCAGCCAGCACTGCGTGAACCTGGAGGGTGGCTACAAGTGCCAGTGTGAGGAAGGCTTCCAGCTGGACCCCCACACGAAGGCCTGCAAG
合成引物：
P367F：GCAGATCTCCTAGGGCCACCATGGCCTGGATGAT
P367R：TACCGCCACCTCCGGACCCTCCGCCACCCTTGCAGGCCTT
P369F：AGGAGGCGGATCCGGTGGAGGCGGATCCGGGACCAGCGAATGCTT
P369R：ATCCGCCTCCTCCGGAGCCGCCACCGCCCTTGCAGGCCTTCGT
P370F：TCCGGAGGTGGCGGTAGCGGCGGTGGGGGATCCGCTGAGTCCAAGTATG
P370R1：CGCTTAATTAATCAGCCCAGGCTCAGGGA
P370R2：GATCCGCCTCCTCCGGAGCCGCCACCGCCGCCCAGGCTCAGGGACA
P371R：CGCTTAATTAATCACTTGCAGGCCTTCGT
1.2基因片段的扩增
利用特异引物通过PCR方法扩增获得部分基因片段。PCR反应体系1(总体积50μl)：Phusion酶0.5μl、5×Buffer 10μl、dNTP 1μl、引物1μl、模板1μl，最后用双蒸水补至50μl；反应条件：98℃预变性30s，98℃10s、55℃30s、72℃30s，30个循环，72℃延伸5min。基因产物以琼脂糖凝胶电泳检测。
表1引物及模板信息1
利用特异引物通过PCR方法扩增获得目的基因片段。PCR反应体系2(总体积50μl)：Phusion酶0.5μl、5×Buffer 10μl、dNTP 1μl、引物1μl、模板1μl，最后用双蒸水补至50μl；反应条件：98℃预变性30s，98℃10s、55℃30s、72℃60s，30个循环，72℃延伸5min。基因产物以琼脂糖凝胶电泳检测。
表2引物及模板信息2
1.3载体和基因片段的酶切处理
对pUC19-mEGF(A)wt质粒、pUC19-mEGF(AB)wt质粒、pCHO1.0质粒和mEGF(A)16-Fc、mEGF(AB)wt-Fc、mEGF(AB)15-Fc、mEGF(AB)16-Fc、mEGF(AB)19-Fc、mEGF(AB)52-Fc、mEGF(AB)55-Fc、2mEGF(AB)55-Fc和2mEGF(AB)56-Fc目的基因片段分别进行双酶切处理，酶切体系在1.5ml离心管中建立如下成分：上述质粒或目的基因片段40μl、Cutsmart 10μl、AvrⅡ和Pac I(A/P)各5μl、灭菌水50μl，混匀后37℃反应过夜，利用QIAGEN产物纯化试剂盒回收。
1.4重组质粒的连接转化
在T4连接酶的作用下，将上述已酶切回收获得的质粒pCHO1.0(AvrⅡ和Pac I，A/P)分别与酶切的mEGF(A)wt(A/P)、mEGF(AB)wt(A/P)、mEGF(A)16-Fc(A/P)、mEGF(AB)wt-Fc(A/P)、mEGF(AB)15-Fc(A/P)、mEGF(AB)16-Fc(A/P)、mEGF(AB)19-Fc(A/P)、mEGF(AB)52-Fc(A/P)、mEGF(AB)55-Fc(A/P)、2mEGF(AB)55-Fc(A/P)和2mEGF(AB)56-Fc(A/P)基因片段连接。连接反应在1.5mlEP管中建立如下体系：pCHO1.0(A/P)1μl、酶切目的基因片段分别7μl、10×T4 Buffer 1μl、T4 DNA ligase 1μl，混匀后于室温(20℃左右)下反应4h以上，连接产物转化至Top10大肠杆菌感受态细胞中，涂于2YT(AMP)平板培养基上，37℃恒温培养箱静止孵育过夜，平板编号如下：
pCHO-EGF(A)wt、pCHO-EGF(AB)wt、pCHO-mEGF(A)16-Fc、pCHO-mEGF(AB)wt-Fc、pCHO-mEGF(AB)15-Fc、pCHO-mEGF(AB)16-Fc、pCHO-mEGF(AB)19-Fc、pCHO-mEGF(AB)52-Fc、pCHO-mEGF(AB)55-Fc、pCHO-2mEGF(AB)55-Fc和pCHO-2mEGF(AB)56-Fc。
1.5重组质粒的菌落PCR筛选
从上述菌落中挑取数个重组单菌落经过培养后分别进行PCR鉴定。菌液PCR反应体系(总体积20μl)：2×Taq HS 10μl、菌液模板3μl、上下游引物(P367F和367R)各0.5μl、最后用双蒸水补至20μL，反应条件：95℃3min，94℃60s、53℃60s、72℃30s，30个循环；72℃延伸5min。琼脂糖凝胶电泳鉴定分析结果。
1.6重组质粒的酶切鉴定
通过菌落PCR鉴定正确后再进行酶切鉴定。首先进行重组菌的质粒提取，然后进行酶切，酶切体系是在1.5ml EP管中加入如下成分：质粒7μl、Cutsmart 1μl、AvrⅡ和Pac I(A/P)各0.5μl、补灭菌水至10μl，混匀后于37℃下反应4h。琼脂糖凝胶电泳分析结果。
1.7重组质粒的测序鉴定
将通过菌落PCR和酶切鉴定正确的重组质粒再送至基因公司进行测序鉴定。经鉴定序列正确后，采用QIAGEN Plasmid Midi Kit大量提取表达质粒，并保存备用。
1.8质粒转染及细胞筛选
采用宿主细胞CHO-S或DG44，按照FreedomTM CHO-STM Kit试剂盒说明书分别进行质粒转染。转入质粒的细胞分别置于摇瓶培养(37℃、5％CO2、110rpm/min)至48h，细胞计数仪检测细胞活率。
转染48h后进行梯度加压筛选：10P/100M，20P/200M(P＝10μg/mL Puromycin，M＝nM MTX)；30P/500M，50P/1000M，获得初筛细胞；再以有限稀释法进行单克隆筛选，检测目的蛋白表达水平筛选获得优选克隆，并扩大培养。
1.9蛋白表达和纯化
1.9.1重组融合蛋白表达纯化
1)重组融合蛋白亲和捕获：采用Mabselect SuRe填料柱捕获重组融合蛋白。具体步骤为：将优化克隆进行扩大培养，离心收集细胞上清，并经0.45μm囊式滤器过滤去除杂质。将层析柱与AKTA层析***相连，流动相A为：20mM磷酸盐，含150mM氯化钠，pH 7.2；流动相B为：200mM甘氨酸盐酸，pH 3.2；流速10ml/min，检测波长214nm和280nm。先用流动相A平衡亲和柱5-10CV，然后进行上样，目的蛋白结合于亲和介质上，。上样结束后，采用流动相A进行清洗层析柱3-5CV，最后用流动相B对其目的蛋白进行洗脱，收集洗脱液用1M Tris(pH9.0)调节pH值至中性；将洗脱样品浓缩换液至20mM Tris-HCl缓冲液，pH7.4。
2)重组融合蛋白精细纯化：采用Sepharose High Performance Q填料柱精纯重组融合蛋白。具体步骤为：将层析柱与AKTA层析***相连。流动相A为20mM Tris-HCl，pH7.4；流动相B为A+0.035M氯化钠，pH7.4；流动相C为A+0.215M氯化钠，pH7.4；流速10ml/min，检测波长214nm和280nm。先用流动相A平衡层析柱5-10CV，将亲和洗脱样品上样，再用缓冲液B冲洗3CV，缓冲液C进行洗脱，洗脱组分即为重组融合蛋白。
1.9.2重组多肽表达纯化
1)重组多肽亲和捕获：采用Ni Sepharose 6FF填料柱捕获重组融合蛋白。具体步骤为：将优化克隆进行扩大培养，离心收集细胞上清液并用0.45μm囊式滤器过滤。将层析柱与AKTA层析***相连；流动相A为：20mM磷酸盐，含350mM氯化钠、20mM咪唑，pH 7.4；流动相B为：A+500mM咪唑；流速10mL/min，检测波长214nm和280nm。先用流动相A平衡亲和柱5-10CV，对已收集的细胞上清液进行上样，目的蛋白结合于介质上，。上样结束后，采用流动相A进行清洗层析柱3-5CV，最后用流动相B对其目的蛋白进行洗脱。将所收集的洗脱样品浓缩换液至20mM Tris-HCl缓冲液，pH7.4。
2)重组多肽精细纯化：采用Sepharose High Performance Q填料柱精纯重组融合蛋白。具体步骤为：将层析柱与AKTA层析***相连。流动相A为20mM Tris-HCl，pH7.4；流动相B为A+0.02M氯化钠，pH7.4；流动相C为A+0.185M氯化钠，pH7.4；流速10mL/min，检测波长214nm和280nm。先用流动相A平衡层析柱5-10CV，将亲和样品上样，再用缓冲液B冲洗3CV，缓冲液C进行洗脱，洗脱组分即为重组多肽。
实施例2重组EGF(AB)突变体及其融合蛋白与PCSK9亲和力
重组多肽或融合蛋白与PCSK9的结合亲和力通过生物膜干涉法测定在Otect QK(Fortebio)上测量。SA sensor传感器(Fortebio，货号18-5063)被固化在含0.5％BSA和1mMCaCl2的TrisHCl pH 7.4缓冲液中的PCSK9，在相同缓冲液中进行洗涤，并且将其转移到含有在相同缓冲液中的浓度为0～1μM的EGF(A/AB)多肽及其融合蛋白的孔中。将针对仅含有缓冲液的参比孔的信号从所有结合数据中减去。亲和力KD通过拟合为稳态算法使用Octet软件获得，数据以mean±SD表示。
概括在表3中的结合动力学参数显示：与EGF(A)wt多肽相比，mEGF(A)16-Fc、EGF(AB)wt、EGF(AB)wt-Fc与PCS以及EGF(AB)突变体融合蛋白与PCSK9的结合亲和力明显增加；其中mEGF(AB)55-Fc、2mEGF(AB)55-Fc、2mEGF(AB)56-Fc与PCSK9的亲和力相对最佳。说明本发明所提供的EGF(AB)突变体融合蛋白能够不同程度地增加EGF(A)与PCSK9的亲和力。
表3 EGF(A/AB)多肽及EGF(A/AB)突变体融合蛋白与PCSK9亲和力检测
实施例3重组EGF(AB)突变体及其融合蛋白竞争抑制PCSK9与LDLR的结合
重组多肽或融合蛋白抑制PCSK9与LDLR结合的活性通过竞争ELISA检测。具体讲，将1μg/ml的重组人LDLR(R&D公司)以碳酸盐缓冲液(pH9.6)包被于96孔酶标板，4℃孵育过夜；将10nM生物素标记的PCSK9与不同摩尔浓度的EGF多肽突变体或融合蛋白(0.046-8100nM)等体积混匀，室温孵育1h；然后加至预先包被重组人LDLR的酶标板中，37℃孵育2h；藕联有辣根过氧化物酶的链亲和素检测结合于LDLR的生物素标记PCSK9；计算EGF多肽突变体或融合蛋白竞争抑制PCSK9与LDLR结合的半数有效浓度(IC50)。测试结果见表4，本发明提供的EGF(AB)突变体融合蛋白可明显抑制PCSK9与LDLR的结合，其中mEGF(AB)55-Fc、2mEGF(AB)55-Fc、2mEGF(AB)56-Fc作用较佳。
表4 EGF突变体及其融合蛋白竞争抑制PCSK9与LDLR的结合检测
表3 EGF(A/AB)多肽及EGF(A/AB)突变体融合蛋白抑制PCSK9与LDLR的结合
实施例3重组EGF(AB)突变体及其融合蛋白对PCSK9降解LDLR作用的影响
人肝癌细胞HepG2培养至对数生长期，用PBS洗细胞一次，经胰酶消化制成细胞悬液并调整细胞密度为1×105cells/ml，接种细胞于48孔培养板(500μl/孔)，。于37℃、5％CO2继续培养24h。去除细胞培养上清，每孔加入500μl无血清培养基，继续培养16h；弃去上清，，加入PCSK9(终浓度25μg/ml)，同时分别加入不同摩尔浓度的重组多肽或融合蛋白(0.001～2μM)，于37℃5％CO2孵育4h；再加入LDL(终浓度为6μg/ml)孵育3h后，弃去上清，用PBS冲洗细胞3次。加入200μl异丙醇37℃孵育30min，充分萃取细胞摄取的LDL，转移150μl萃取液至96黑色酶标板中，测定荧光强度，激发波长488nm，检测波长520nm。
另取细胞以1×104cells/ml，接种细胞于激光共聚焦细胞培养皿(1ml/孔)。置培养板于37℃、5％CO2培养箱中，继续培养24h。更换为无血清培养基继续培养16h；加入PCSK9(终浓度25μg/ml)，同时分别加入EGF(AB)多肽或融合蛋白(0.01～1μM)，置于37℃5％CO2培养箱孵育24h，用PBS冲洗，收获细胞，采用免疫荧光技术观察LDL-R的表达或降解，并进行荧光强度定量分析。
实验结果显示，用本发明所提供的EGF(AB)多肽突变体融合蛋白均明显抑制PCSK9介导的LDLR降解，其抑制作用明显优于EGF(AB)野生型多肽及其融合蛋白；与此同时，EGF(AB)多肽突变体融合蛋白也可拮抗PCSK9所致的LDL摄取减少，明显增加肝细胞对LDL的摄取量，其作用也明显优于EGF(AB)野生型多肽及其融合蛋白。多肽及融合蛋白半数抑制浓度(IC50)分别为：(EGF(AB)wt,1196白半数抑制浓度)、(EGF(AB)wt-Fc,707.0.0-Fc,)、(mEGF(AB)55-Fc,498.5±19.28nM)、(2mEGF(AB)55-Fc,276.3±14.51nM)、(2mEGF(AB)56-Fc,241.1±12.74nM)。
实施例4重组EGF(AB)突变体及其融合蛋白在大鼠体内的半衰期
取健康雄性SD大鼠，随机分为EGF(AB)wt、EGF(AB)wt-Fc、mEGF(AB)55-Fc、2mEGF(AB)55-Fc、2mEGF(AB)56-Fc组，每组5只。各组大鼠依据体重分别皮下注射给予相应药物(5mg/kg)。于给药前和给药后不同时间点眼眶静脉取血250μl，制备血清，ELISA方法检测血清中各蛋白的含量。
由表4结果显示，大鼠皮下注射同剂量的EGF(AB)多肽及其突变体融合蛋白后，本发明EGF(AB)突变体融合蛋白半衰期较EGF(AB)野生型多肽的半衰期明显延长，并且2mEGF(AB)56-Fc的半衰期可长达71.6h。
表4 EGF(AB)及其突变体融合蛋白在大鼠血液中的代谢参数比较
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His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 24
<211> 681
<212> DNA
<213> 人工序列
<400> 24
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540
gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 600
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660
ctctccctgt ctccgggtaa a 681
<210> 25
<211> 228
<212> PRT
<213> 人工序列
<400> 25
Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
1 5 10 15
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
35 40 45
His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
50 55 60
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
65 70 75 80
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
85 90 95
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro
100 105 110
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
130 135 140
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
145 150 155 160
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175
Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
180 185 190
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
195 200 205
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
210 215 220
Ser Pro Gly Lys
225
<210> 26
<211> 684
<212> DNA
<213> 人工序列
<400> 26
gagcgcaaat gttgtgtcga gtgcccaccg tgcccagcac cacctgtggc aggaccgtca 60
gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120
acgtgcgtgg tggtggacgt gagccacgaa gaccccgagg tccagttcaa ctggtacgtg 180
gacggcgtgg aggtgcataa tgccaagaca aagccacggg aggagcagtt caacagcacg 240
ttccgtgtgg tcagcgtcct caccgttgtg caccaggact ggctgaacgg caaggagtac 300
aagtgcaagg tctccaacaa aggcctccca gcccccatcg agaaaaccat ctccaaaacc 360
aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 420
aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg 480
gagtgggaga gcaatgggca gccggagaac aactacaaga ccacacctcc catgctggac 540
tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600
gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660
agcctctccc tgtctccggg taaa 684
<210> 27
<211> 264
<212> PRT
<213> 人工序列
<400> 27
Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys
1 5 10 15
Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro
20 25 30
Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala
35 40 45
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
50 55 60
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
65 70 75 80
Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val
85 90 95
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
100 105 110
Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His Gln
115 120 125
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
130 135 140
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
145 150 155 160
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
165 170 175
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
180 185 190
Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr
195 200 205
Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
210 215 220
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe
225 230 235 240
Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys
245 250 255
Ser Leu Ser Leu Ser Pro Gly Lys
260
<210> 28
<211> 792
<212> DNA
<213> 人工序列
<400> 28
gagctcaaaa ccccacttgg tgacacaact cacacatgcc cacggtgccc agagcccaaa 60
tcttgtgaca cacctccccc gtgcccacgg tgcccagagc ccaaatcttg tgacacacct 120
cccccatgcc cacggtgccc agcacctgaa ctcctgggag gaccgtcagt cttcctcttc 180
cccccaaaac ccaaggatac ccttatgatt tcccggaccc ctgaggtcac gtgcgtggtg 240
gtggacgtga gccacgaaga ccccgaggtc cagttcaagt ggtacgtgga cggcgtggag 300
gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgtt ccgtgtggtc 360
agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 420
tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaaaccaa aggacagccc 480
cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc 540
agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 600
agcgggcagc cggagaacaa ctacaacacc acgcctccca tgctggactc cgacggctcc 660
ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacatcttc 720
tcatgctccg tgatgcatga ggctctgcac aaccgcttca cgcagaagag cctctccctg 780
tctccgggta aa 792
<210> 29
<211> 229
<212> PRT
<213> 人工序列
<400> 29
Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
1 5 10 15
Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
20 25 30
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
35 40 45
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
50 55 60
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
65 70 75 80
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
85 90 95
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
100 105 110
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
115 120 125
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
130 135 140
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
145 150 155 160
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
165 170 175
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
180 185 190
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
195 200 205
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
210 215 220
Ser Leu Ser Leu Gly
225
<210> 30
<211> 687
<212> DNA
<213> 人工序列
<400> 30
gctgagtcca agtatggccc tccctgccct ccttgccctg ctcctgaggc tgctggaggc 60
cctagcgtgt tcctgttccc ccctaagcct aaggacaccc tgatgatttc ccggaccccc 120
gaggtgacct gtgtggtggt ggatgtgtcc caggaggacc ctgaagtgca gttcaactgg 180
tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggaaga gcagttcaac 240
agcacctaca gggtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 300
gagtacaagt gcaaggtgag caataagggc ctgccctcct ccatcgagaa gaccatttcc 360
aaggccaagg gccagcccag ggaaccccag gtgtacaccc tccctcccag ccaggaggag 420
atgaccaaga accaggtgtc cctgacctgc ctggtgaaag gcttctaccc ctccgacatt 480
gccgtcgagt gggaaagcaa cggccagccc gagaacaatt acaagaccac accccccgtg 540
ctggacagcg atggcagctt tttcctgtac tccaggctga ccgtcgacaa gtccaggtgg 600
caggagggca acgtcttctc ctgctccgtg atgcatgagg ccctgcacaa ccactacacc 660
cagaagtccc tgtccctgag cctgggc 687
<210> 31
<211> 324
<212> PRT
<213> 人工序列
<400> 31
Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
85 90 95
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
100 105 110
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
130 135 140
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
145 150 155 160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
165 170 175
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
180 185 190
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
195 200 205
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly
<210> 32
<211> 975
<212> DNA
<213> 人工序列
<400> 32
gggaccaacg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgcctgtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggtggcggag ggtccggagg tggcggtagc ggcggtgggg gatccgctga gtccaagtat 300
ggccctccct gccctccttg ccctgctcct gaggctgctg gaggccctag cgtgttcctg 360
ttccccccta agcctaagga caccctgatg atttcccgga cccccgaggt gacctgtgtg 420
gtggtggatg tgtcccagga ggaccctgaa gtgcagttca actggtacgt ggacggcgtg 480
gaggtgcaca acgccaagac caagccccgg gaagagcagt tcaacagcac ctacagggtg 540
gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 600
gtgagcaata agggcctgcc ctcctccatc gagaagacca tttccaaggc caagggccag 660
cccagggaac cccaggtgta caccctccct cccagccagg aggagatgac caagaaccag 720
gtgtccctga cctgcctggt gaaaggcttc tacccctccg acattgccgt cgagtgggaa 780
agcaacggcc agcccgagaa caattacaag accacacccc ccgtgctgga cagcgatggc 840
agctttttcc tgtactccag gctgaccgtc gacaagtcca ggtggcagga gggcaacgtc 900
ttctcctgct ccgtgatgca tgaggccctg cacaaccact acacccagaa gtccctgtcc 960
ctgagcctgg gctga 975
<210> 33
<211> 324
<212> PRT
<213> 人工序列
<400> 33
Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Tyr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
85 90 95
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
100 105 110
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
130 135 140
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
145 150 155 160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
165 170 175
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
180 185 190
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
195 200 205
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly
<210> 34
<211> 975
<212> DNA
<213> 人工序列
<400> 34
gggaccaacg aatgcttgga caacaacggc ggctgttccc acgtctacaa tgaccttaag 60
atcggctacg agtgcctgtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggtggcggag ggtccggagg tggcggtagc ggcggtgggg gatccgctga gtccaagtat 300
ggccctccct gccctccttg ccctgctcct gaggctgctg gaggccctag cgtgttcctg 360
ttccccccta agcctaagga caccctgatg atttcccgga cccccgaggt gacctgtgtg 420
gtggtggatg tgtcccagga ggaccctgaa gtgcagttca actggtacgt ggacggcgtg 480
gaggtgcaca acgccaagac caagccccgg gaagagcagt tcaacagcac ctacagggtg 540
gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 600
gtgagcaata agggcctgcc ctcctccatc gagaagacca tttccaaggc caagggccag 660
cccagggaac cccaggtgta caccctccct cccagccagg aggagatgac caagaaccag 720
gtgtccctga cctgcctggt gaaaggcttc tacccctccg acattgccgt cgagtgggaa 780
agcaacggcc agcccgagaa caattacaag accacacccc ccgtgctgga cagcgatggc 840
agctttttcc tgtactccag gctgaccgtc gacaagtcca ggtggcagga gggcaacgtc 900
ttctcctgct ccgtgatgca tgaggccctg cacaaccact acacccagaa gtccctgtcc 960
ctgagcctgg gctga 975
<210> 35
<211> 324
<212> PRT
<213> 人工序列
<400> 35
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
85 90 95
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
100 105 110
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
130 135 140
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
145 150 155 160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
165 170 175
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
180 185 190
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
195 200 205
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly
<210> 36
<211> 975
<212> DNA
<213> 人工序列
<400> 36
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgcctgtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggtggcggag ggtccggagg tggcggtagc ggcggtgggg gatccgctga gtccaagtat 300
ggccctccct gccctccttg ccctgctcct gaggctgctg gaggccctag cgtgttcctg 360
ttccccccta agcctaagga caccctgatg atttcccgga cccccgaggt gacctgtgtg 420
gtggtggatg tgtcccagga ggaccctgaa gtgcagttca actggtacgt ggacggcgtg 480
gaggtgcaca acgccaagac caagccccgg gaagagcagt tcaacagcac ctacagggtg 540
gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 600
gtgagcaata agggcctgcc ctcctccatc gagaagacca tttccaaggc caagggccag 660
cccagggaac cccaggtgta caccctccct cccagccagg aggagatgac caagaaccag 720
gtgtccctga cctgcctggt gaaaggcttc tacccctccg acattgccgt cgagtgggaa 780
agcaacggcc agcccgagaa caattacaag accacacccc ccgtgctgga cagcgatggc 840
agctttttcc tgtactccag gctgaccgtc gacaagtcca ggtggcagga gggcaacgtc 900
ttctcctgct ccgtgatgca tgaggccctg cacaaccact acacccagaa gtccctgtcc 960
ctgagcctgg gctga 975
<210> 37
<211> 324
<212> PRT
<213> 人工序列
<400> 37
Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
85 90 95
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
100 105 110
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
130 135 140
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
145 150 155 160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
165 170 175
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
180 185 190
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
195 200 205
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly
<210> 38
<211> 975
<212> DNA
<213> 人工序列
<400> 38
gggaccaacg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgccggtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggtggcggag ggtccggagg tggcggtagc ggcggtgggg gatccgctga gtccaagtat 300
ggccctccct gccctccttg ccctgctcct gaggctgctg gaggccctag cgtgttcctg 360
ttccccccta agcctaagga caccctgatg atttcccgga cccccgaggt gacctgtgtg 420
gtggtggatg tgtcccagga ggaccctgaa gtgcagttca actggtacgt ggacggcgtg 480
gaggtgcaca acgccaagac caagccccgg gaagagcagt tcaacagcac ctacagggtg 540
gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 600
gtgagcaata agggcctgcc ctcctccatc gagaagacca tttccaaggc caagggccag 660
cccagggaac cccaggtgta caccctccct cccagccagg aggagatgac caagaaccag 720
gtgtccctga cctgcctggt gaaaggcttc tacccctccg acattgccgt cgagtgggaa 780
agcaacggcc agcccgagaa caattacaag accacacccc ccgtgctgga cagcgatggc 840
agctttttcc tgtactccag gctgaccgtc gacaagtcca ggtggcagga gggcaacgtc 900
ttctcctgct ccgtgatgca tgaggccctg cacaaccact acacccagaa gtccctgtcc 960
ctgagcctgg gctga 975
<210> 39
<211> 324
<212> PRT
<213> 人工序列
<400> 39
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
85 90 95
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
100 105 110
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
130 135 140
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
145 150 155 160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
165 170 175
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
180 185 190
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
195 200 205
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly
<210> 40
<211> 975
<212> DNA
<213> 人工序列
<400> 40
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgccggtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggtggcggag ggtccggagg tggcggtagc ggcggtgggg gatccgctga gtccaagtat 300
ggccctccct gccctccttg ccctgctcct gaggctgctg gaggccctag cgtgttcctg 360
ttccccccta agcctaagga caccctgatg atttcccgga cccccgaggt gacctgtgtg 420
gtggtggatg tgtcccagga ggaccctgaa gtgcagttca actggtacgt ggacggcgtg 480
gaggtgcaca acgccaagac caagccccgg gaagagcagt tcaacagcac ctacagggtg 540
gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 600
gtgagcaata agggcctgcc ctcctccatc gagaagacca tttccaaggc caagggccag 660
cccagggaac cccaggtgta caccctccct cccagccagg aggagatgac caagaaccag 720
gtgtccctga cctgcctggt gaaaggcttc tacccctccg acattgccgt cgagtgggaa 780
agcaacggcc agcccgagaa caattacaag accacacccc ccgtgctgga cagcgatggc 840
agctttttcc tgtactccag gctgaccgtc gacaagtcca ggtggcagga gggcaacgtc 900
ttctcctgct ccgtgatgca tgaggccctg cacaaccact acacccagaa gtccctgtcc 960
ctgagcctgg gctga 975
<210> 41
<211> 419
<212> PRT
<213> 人工序列
<400> 41
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
85 90 95
Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr Asn
100 105 110
Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln Leu
115 120 125
Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro Asp
130 135 140
Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys Gln
145 150 155 160
Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys Gly
165 170 175
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu
180 185 190
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
195 200 205
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
210 215 220
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
225 230 235 240
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
245 250 255
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
260 265 270
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
275 280 285
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
290 295 300
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
305 310 315 320
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
325 330 335
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
340 345 350
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
355 360 365
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
370 375 380
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
385 390 395 400
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
405 410 415
Ser Leu Gly
<210> 42
<211> 1260
<212> DNA
<213> 人工序列
<400> 42
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgccggtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggcggtggcg gctccggagg aggcggatcc ggtggaggcg gatccgggac cagcgaatgc 300
ttggacaaca acggcggctg ttcccacgtc accaatgacc ttaagatcgg ctacgagtgc 360
cggtgccccg acggcttcca gctggtggcc cagcgaagat gcgaagatgt cgatgagtgt 420
caggatcccg acacctgcag ccagcactgc gtgaacctgg agggtggcta caagtgccag 480
tgtgaggaag gcttccagct ggacccccac acgaaggcct gcaagggtgg cggagggtcc 540
ggaggtggcg gtagcggcgg tgggggatcc gctgagtcca agtatggccc tccctgccct 600
ccttgccctg ctcctgaggc tgctggaggc cctagcgtgt tcctgttccc ccctaagcct 660
aaggacaccc tgatgatttc ccggaccccc gaggtgacct gtgtggtggt ggatgtgtcc 720
caggaggacc ctgaagtgca gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc 780
aagaccaagc cccgggaaga gcagttcaac agcacctaca gggtggtgag cgtgctgacc 840
gtgctgcacc aggactggct gaacggcaaa gagtacaagt gcaaggtgag caataagggc 900
ctgccctcct ccatcgagaa gaccatttcc aaggccaagg gccagcccag ggaaccccag 960
gtgtacaccc tccctcccag ccaggaggag atgaccaaga accaggtgtc cctgacctgc 1020
ctggtgaaag gcttctaccc ctccgacatt gccgtcgagt gggaaagcaa cggccagccc 1080
gagaacaatt acaagaccac accccccgtg ctggacagcg atggcagctt tttcctgtac 1140
tccaggctga ccgtcgacaa gtccaggtgg caggagggca acgtcttctc ctgctccgtg 1200
atgcatgagg ccctgcacaa ccactacacc cagaagtccc tgtccctgag cctgggctga 1260
<210> 43
<211> 419
<212> PRT
<213> 人工序列
<400> 43
Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser His Val Thr
1 5 10 15
Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp Gly Phe Gln
20 25 30
Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys Gln Asp Pro
35 40 45
Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly Tyr Lys Cys
50 55 60
Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys Ala Cys Lys
65 70 75 80
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
85 90 95
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
100 105 110
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
115 120 125
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
130 135 140
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
145 150 155 160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
165 170 175
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
180 185 190
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
195 200 205
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
325 330 335
Gly Gly Ser Gly Thr Ser Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser
340 345 350
His Val Thr Asn Asp Leu Lys Ile Gly Tyr Glu Cys Arg Cys Pro Asp
355 360 365
Gly Phe Gln Leu Val Ala Gln Arg Arg Cys Glu Asp Val Asp Glu Cys
370 375 380
Gln Asp Pro Asp Thr Cys Ser Gln His Cys Val Asn Leu Glu Gly Gly
385 390 395 400
Tyr Lys Cys Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys
405 410 415
Ala Cys Lys
<210> 44
<211> 1260
<212> DNA
<213> 人工序列
<400> 44
gggaccagcg aatgcttgga caacaacggc ggctgttccc acgtcaccaa tgaccttaag 60
atcggctacg agtgccggtg ccccgacggc ttccagctgg tggcccagcg aagatgcgaa 120
gatgtcgatg agtgtcagga tcccgacacc tgcagccagc actgcgtgaa cctggagggt 180
ggctacaagt gccagtgtga ggaaggcttc cagctggacc cccacacgaa ggcctgcaag 240
ggtggcggag ggtccggagg tggcggtagc ggcggtgggg gatccgctga gtccaagtat 300
ggccctccct gccctccttg ccctgctcct gaggctgctg gaggccctag cgtgttcctg 360
ttccccccta agcctaagga caccctgatg atttcccgga cccccgaggt gacctgtgtg 420
gtggtggatg tgtcccagga ggaccctgaa gtgcagttca actggtacgt ggacggcgtg 480
gaggtgcaca acgccaagac caagccccgg gaagagcagt tcaacagcac ctacagggtg 540
gtgagcgtgc tgaccgtgct gcaccaggac tggctgaacg gcaaagagta caagtgcaag 600
gtgagcaata agggcctgcc ctcctccatc gagaagacca tttccaaggc caagggccag 660
cccagggaac cccaggtgta caccctccct cccagccagg aggagatgac caagaaccag 720
gtgtccctga cctgcctggt gaaaggcttc tacccctccg acattgccgt cgagtgggaa 780
agcaacggcc agcccgagaa caattacaag accacacccc ccgtgctgga cagcgatggc 840
agctttttcc tgtactccag gctgaccgtc gacaagtcca ggtggcagga gggcaacgtc 900
ttctcctgct ccgtgatgca tgaggccctg cacaaccact acacccagaa gtccctgtcc 960
ctgagcctgg gcggcggtgg cggctccgga ggaggcggat ccggtggagg cggatccggg 1020
accagcgaat gcttggacaa caacggcggc tgttcccacg tcaccaatga ccttaagatc 1080
ggctacgagt gccggtgccc cgacggcttc cagctggtgg cccagcgaag atgcgaagat 1140
gtcgatgagt gtcaggatcc cgacacctgc agccagcact gcgtgaacct ggagggtggc 1200
tacaagtgcc agtgtgagga aggcttccag ctggaccccc acacgaaggc ctgcaagtga 1260
Claims (9)
1.新的结合PCSK9的EGF(AB)突变体，其由LDL受体胞外域EGF-A区第294-319位中的任意氨基酸和EGF-B区第333-347位中的任意氨基酸取代(或置换)获得。
2.权利要求1所述的EGF(AB)突变体，其特征在于，LDLR胞外域EGF-A区第295位的天冬酰胺(Asn)由丝氨酸(Ser)取代(N295S)，第308位半胱胺酸(Cys)由苏氨酸(Thr)或酪氨酸(Tyr)取代(C308T或C308Y)，第318位亮氨酸(Leu)由精氨酸(Arg)取代(L318R)；胞外域EGF-B区第334位异亮氨酸(Ile)由缬氨酸(Val)取代(I334V)，第346位亮氨酸(Leu)由组氨酸(His)取代(L346H)。
3.权利要求2所述的EGF(AB)突变体，其特征在于，是由选自如下任一形式的组成：
N295S-I334V-L346H；
C308T-I334V-L346H或C308Y-I334V-L346H；
L318R-I334V-L346H；
N295S-C308T-I334V-L346H或N295S-C308Y-I334V-L346H；
N295S-L318R-I334V-L346H；
C308T-L318R-I334V-L346H或C308Y-L318R-I334V-L346H；
N295S-C308T-L318R-I334V-L346H或N295S-C308Y-L318R-I334V-L346H。
4.权利要求1所述的EGF(AB)突变体，其由选自如下任一项的氨基酸序列：
a)SEQ ID NO：1
GTSECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLDPHTKACK
b)SEQ ID NO：3
GTNECLDNNGGCSHVTNDLKIGYECLCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLDPHTKACK
c)SEQ ID NO：5
GTNECLDNNGGCSHVYNDLKIGYECLCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLDPHTKACK
d)SEQ ID NO：7
GTNECLDNNGGCSHVCNDLKIGYECRCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLDPHTKACK
e)SEQ ID NO：9
GTSECLDNNGGCSHVTNDLKIGYECLCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLDPHTKACK
f)SEQ ID NO：11
GTSECLDNNGGCSHVYNDLKIGYECLCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLDPHTKACK
g)SEQ ID NO：13
GTSECLDNNGGCSHVCNDLKIGYECRCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLDPHTKACK
h)SEQ ID NO：15
GTNECLDNNGGCSHVTNDLKIGYECRCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLDPHTKACK
i)SEQ ID NO：17
GTNECLDNNGGCSHVYNDLKIGYECRCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLDPHTKACK
j)SEQ ID NO：19
GTSECLDNNGGCSHVTNDLKIGYECRCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLDPHTKACK
k)SEQ ID NO：21
GTSECLDNNGGCSHVYNDLKIGYECRCPDGFQLVAQRRCEDVDECQDPDTCSQHCVNLEGGYKCQCEEGFQLDPHTKACK。
5.编码权利要求4(a-k)所述的EGF(AB)突变体的核苷酸序列，如SEQ ID NO：2、4、6、8、10、12、14、16、、18、20、22。
6.权利要求4(a-k)所述的任一项EGF(AB)突变体与人免疫球蛋白Fc片段的融合蛋白，其特征是所述的EGF(AB)突变体通过适当的连接肽与Fc片段连接或直接与Fc片段连接，选自由以下组成的连接方式：
EGF(AB)—连接肽—Fc
或:EGF(AB)—Fc
EGF(AB)—连接肽—Fc—连接肽—EGF(AB)
或:EGF(AB)—Fc—EGF(AB)
[EGF(AB)]n—连接肽—Fc—连接肽—[EGF(AB)]n
或:EGF(AB)n—Fc—EGF(AB)n
其中，n取1～3整数，连接肽由3-16个氨基酸组成。
7.权利要求4所述的EGF(AB)突变体与人免疫球蛋白Fc片段的融合蛋白，其特征是所述的免疫球蛋白是由选自人IgG1-4的Fc片段。
8.制备基因工程EGF(AB)突变体融合蛋白的方法，其特征在于所述的方法包括如下部分：
a)以人肝细胞作为模板，获取人LDL受体的EGF(AB)基因；
b)采用基因合成方法(氨基酸取代或置换方法)
c)构建真核细胞表达载体，其包含所述任一项EGF(AB)突变体基因或至少一种连接Fc的基因序列；
d)表达质粒定点整合至CHO细胞系基因组或293细胞系基因组，表达所述EGF(AB)突变体或连接Fc的融合蛋白；
e)收集表达上清，经亲和捕获或(和)离子交换层析获得所述的EGF(AB)突变体或其融合蛋白。
9.权利要求1-4所述的EGF(AB)突变体或其融合蛋白在制备用于治疗高胆固醇血症、动脉粥样硬化等心血管疾病以及通过降低血浆PCSK9水平而受益的其他疾病的用途。
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| CN109504737A (zh) * | 2018-11-23 | 2019-03-22 | 黄河科技学院 | 一种体外筛选pcsk9抑制剂的方法 |
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| CN113106103A (zh) * | 2021-04-05 | 2021-07-13 | 华中科技大学同济医学院附属协和医院 | Ldlr基因突变体及其应用 |
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| CN113106103A (zh) * | 2021-04-05 | 2021-07-13 | 华中科技大学同济医学院附属协和医院 | Ldlr基因突变体及其应用 |
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