CN1145701C - 溶血栓药物重组葡激酶 - Google Patents
溶血栓药物重组葡激酶 Download PDFInfo
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- CN1145701C CN1145701C CNB001126741A CN00112674A CN1145701C CN 1145701 C CN1145701 C CN 1145701C CN B001126741 A CNB001126741 A CN B001126741A CN 00112674 A CN00112674 A CN 00112674A CN 1145701 C CN1145701 C CN 1145701C
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Abstract
溶血栓药物重组葡激酶，公开了一种葡激酶(Sak)基因及其高表达工程菌株，由金黄色葡萄球菌的溶原性转换噬菌体α的DNA，经PCR扩增获得编码Sak基因。所构建的葡激酶工程菌株，其表达量高达25%以上，适于工业化生产。经申请人临床研究表明，重组葡激酶对血纤维蛋白具有特异性的作用，具有溶栓能力强，出血副作用小的特点，使用安全有效。
Description
技术领域
本发明是一种溶血栓药物重组葡激酶，属于生物医学领域的DNA基因工程，涉及一种用于溶解血栓的药物重组葡激酶(Sak)基因及其高表达工程菌株，该表达产物对血纤维蛋白具有很高的降解活性。
技术背景
葡激酶(staphylokinase Sak)是90年代诞生的一种新型生物技术治疗药物，是金黄色葡萄球菌分泌的一种胞外蛋白质，它类似尿激酶(Uk)和链激酶(Sk)，具有溶血栓的特性。目前，临床上治疗血栓的药物主要使用的是尿激酶、链激酶等，存在对血栓的溶解速度慢，溶栓效果差，对构成血栓骨架的血纤维蛋白专一性差，易造成血液系统中正常纤维蛋白原也被降解，从而激活系统纤溶作用引起出血的缺点。德国文献M.G.G(1987，210，P258)和中国专利95111222 94112105 97105988 9810213297103921分别报道了第34位核苷酸序列及其对应氨基酸为GGT Gly的葡激酶(Sak)。文献“Nucleic Acid Research”(1983，11(22)P679)介绍了第11位和第34位核苷酸序列及其对应氨基酸分别为AAG Lys和GGT Gly的葡激酶(Sak)。比利时文献Fibrinolysis(1992，6，P226)报道了第11位核苷酸序列及其对应氨基酸为AAG Lys的葡激酶(Sak)。据“国外医学临床生物化学与检验学分册”(1998年第19第4期156-158)报道了Sako等从金黄色葡萄球菌SФ-C基因克隆了Sak基因并确定了其序列，构建了PMEX-602Sak重组质粒，在tac启动子和两个串连的SD序列控制下，在大肠杆菌中获得高效表达，表达量占菌体的总蛋白10％-15％。“生物工程”1994年12卷2期文章“治疗血栓用药重组葡激酶的高产率生产和纯化”介绍了用发酵罐培养转染大肠杆菌TG1细胞产生葡激酶，其表达量占菌体总蛋白的10％-15％，是不适于工业化生产治疗血栓病的葡激酶。
发明内容
本发明的目的就是找到一种新葡激酶基因序列，构建新的葡激酶基因高表达工程菌株，提高葡激酶基因的表达水平，用于生产人体血栓病治疗的溶血栓药物重组葡激酶。它具有特异地、有选择地、专一地溶解血纤维蛋白凝块作用，能减少出血的副作用，安全有效地溶血栓。
本发明的目的通过以下技术方案来实现：
一种溶血栓药物重组葡激酶，其编码基因可由金黄色葡萄球菌的溶原性转换噬菌体α的DNA，经PCR扩增获得，所述基因的核苷酸全序列及其所编码的所述溶血栓药物重组葡激酶的氨基酸序列如下：TCA AGT TCA TTC GAC AAA GGA AAA TAT AAA AAA GGC GAT GAC GCG AGTSer Ser Ser Pbe Asp Lys Gly Lys Tyr Lys Lys Gly Asp Asp Ala SerTAT TTT GAA CCA ACA GGC CCG TAT TTG ATG GTA AAT GTG ACT GGA GTT GATTyr Phe Glu Pro Thr Gly Pro Tyr Lyr Met Val Asn Val Thr Gly Val AspAGT AAA GGA AAT GAA TTG CTA TCC CCT CAT TAT GTC GAG TTT CCT ATT AAA CCTSer Lys Gly Asn Glu Leu Leu Ser Pro His Tyr Val Glu Phe Pro Ile Lys ProGGG ACT ACA CTT ACA AAA GAA AAA ATT GAA TAC TAT GTC GAA TGG GCA TTA GATGly Thr Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val Glu Trp Ala Leu AspGCG ACA GCA TAT AAA GAG TTT AGA GTA GTT GAA TTA GAT CCA AGC GCA AAG ATCAla Thr Ala Tyr Lys Glu Phe Arg Val Val Glu Leu Asp Pro Ser Ala Lys IleGAA GTC ACT TAT TAT GAT AAG AAT AAG AAA AAA GAA GAA ACG AAG TCT TTC CCTGlu Val Thr Tyr Tyr Asp Lys Asn Lys Lys Lys Glu Glu Thr Lys Ser Phe ProATA ACA GAA AAA GGT TTT GTT GTC CCA GAT TTA TCA GAG CAT ATT AAA AAC CCTIle Thr Glu Lys Gly Phe Val Val Pro Asp Leu Ser Glu His Ile Lys Asn ProGGA TTC AAC TTA ATT ACA AAG GTT GTT ATA GAA AAG AAA TAAGly Phe Asn Leu Ile Thr Lys Val Val Ile Glu Lys Lys End
本发明的重组葡激酶基因的高表达工程菌株，由产葡激酶活性高的噬菌体α的DNA为模板，采用引物I和引物II经PCR扩增，得到含有完整的葡激酶基因DNA小片段，该基因DNA片段与pJLA502载体质粒，分别以BamH I和Nco I酶切；再将酶切后的上述质粒和葡激酶基因DNA小片段，用T4DNA连接酶连接获得含有Sak基因pJLA502质粒DNA，再转化入HB101大肠杆菌细胞内，构建成葡激酶高表达的工程菌株E.coliH152，该工程菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，其保藏号为CGMCC0432。
本发明的高表达工程菌株构建过程中所采用的引物I，其核苷酸序列为：
5′ATCCATGGCCATGCTCAAAAGAAGT 3′
引物II，其核苷酸序列为：
5′CTTATAGAAAAGAAATAAGGATCCCC 3′
本发明相比已有技术具有如下优点：
1.本发明的溶血栓药物重组葡激酶与德国文献M.G.G(1987，210，P258)和中国专利95111222，94112105，97105988，98102132、97103921分别报道的葡激酶在第34位核苷酸序列不同。与文献“Nucleic Acid Research”(1983，11(22)P679)介绍的葡激酶在第11位和第34位核苷酸序列及其对应氨基酸不同。与比利时文献Fibri-nolysis(1992，6，P226)报道葡激酶在第11位核苷酸序列及其对应氨基酸不同，所以本发明的溶血栓药物重组葡激酶是具有与文献报道不同的新的葡激酶基因序列，是一种新型基因序列葡激酶。
2.本发明构建的葡激酶高表达工程菌株H15(中国微生物保藏中心保藏号CGMCC0432)，具有高达25％以上的表达量，高于公认的10％-15％的表达量，是适于工业化生产治疗血栓病的葡激酶。现经临床研究表明这种效果特异的新型分子药物，与尿激酶、链激酶的特性相比，其优点在于：(1)由于其特异地、有选择地、专一地降解构成血栓骨架的血纤维蛋白，减少出血副作用、安全有效。(2)由于其具有较好的纤维蛋白特异性，溶栓能力强。(3)由于葡激酶的分子量只有15.5kD，是链激酶的1/3，因而对血栓的穿透力强。实验表明，在3小时内，50％的溶栓作用需葡激酶2μg/ml，而Sk需12μg/ml，组织纤溶原激活剂t-PA需4.45μg/ml。就此看来，葡激酶的溶栓能力是链激酶的6倍。在治疗血栓过程中，克服了尿激酶(Uk)和链激酶(Sk)对血栓骨架的血纤维蛋白不具亲和性，临床治疗过程中易造成病人全身出血的缺陷，是当今最有前景，最新颖的溶栓药物。近年我国每年有250万人死于心脑血管病，高居死亡类疾病的第一位。我国成人中患高血压病的患者有8,000万人，急需溶栓药物治疗。因此，高效、安全、价格低廉的溶栓药物基因重组葡激酶的出现，将会对广大的心血管疾病患者的治疗带来有益的效果。
附图说明
图1：本发明Sak基因初级克隆流程图
图2：为抗Amp转化子单菌的葡激酶活性检测照片图
图中：1.大肠杆菌C600 2.大肠杆菌转化子-1
3.大肠杆菌转化子-2 4.大肠杆菌转化子-3
图3.转化子抽取的质粒Hind III酶切电泳图
图中：1.大肠杆菌C600(pBR322+Lα3)
2.大肠杆菌C600(pBR322+Lα2)
3.大肠杆菌C600(pBR322)
4.大肠杆菌C600(pBR322+Lα1)
5.αDNA Hind III酶切
6.αDNA Hind III酶切
7.λDNA/BamHI，EcoRI
图4：本发明Sak基因次级克隆的构建
图5：重组葡激酶氨基酸序列
图6：热诱导表达载体构建的示意图
具体实施方式
本发明下面将结合实施例、附图作进一步的详细描述：
实施例1，本发明重组葡激酶基因的来源与初级克隆，如图1所示
从收集的179株金葡菌中筛选葡激酶阳性菌株，选取其葡激酶活性强的66号菌株，用丝裂霉素诱导前噬菌体，使其裂碎，从裂解液中分离携带葡激酶基因组的溶原转换噬菌体α，抽取噬菌体的DNA，经Hind III内切酶酶切，用鸟枪法和质粒pBR322连接，转化大肠杆菌EcoliC600构建流程如下。筛选得到三株携带外源基因的转化子(如图2所示)。其质粒pSak-1经酶切电泳，其中一株具有葡激酶产生能力(如图3所示)。与标准分子量比较，表明含有葡激酶基因约为2.7Kb片段。
实施例2，本发明重组葡激酶基因次级克隆的构建，如图4所示：
在初级克隆的基础上，用EcoR I酶切与质粒PUCBM20为载体的连接，得到质粒PUC-E，将PUC-E质粒用Hind III酶切。回收4.7Kb片段环化，得到2.1Kb的次级克隆。
实施例3，葡激酶次级克隆基因片段的序列测定：
次级克隆PUC-E用EcoR I切下约有2100bp一片段，转移到MBmp18噬菌体上用Sanger双脱氧链终止法进行，序列中型号为AppledBiostens373。在此，2001bp的序列中发现一个读码结构(1028-1517)，它编码一个163个氨基酸的蛋白质，其分子量为18.476KD，包含了信号肽及编号136个氨基酸的葡激酶序列(如图5a、图5b、图5c所示)。
实施例4：两端引物设计
根据测序结果，设计合成引物I和引物II：
5′ATCCATGGCCATGCTCAAAAGAAGT 3′
5′CTTATAGAAAAGAAATAAGGATCCCC 3′
实施例5：葡激酶表达工程菌株的构建引物合成后，从MV1190(PUC-E)菌体中抽取DNA片段作为DNA模板，进行PCR扩增，得到Sak基因。将基因片段通过内切酶位点BamH I和Nco I酶切，再与表达载体pJLA502连接(如图6所示)，转化大肠杆菌HB101，涂布在含Amp的LB平板上挑出Sak活力最强的菌株命名为EcoliH15(中国微生物保藏号CGMCC0432)。
实施例6：工程菌株用培养液振荡培养，并补以营养液，调整pH并控制溶氧量，保温一段时间，离心收集菌体。收集到的菌体经冻融、加入PBS，离心、两步柱层析法纯化，其纯化度可达99％以上。
工程菌构建步骤如下：
1.葡激酶溶原转换噬菌体的分离：将实验室收藏的金黄色葡萄球菌，在经56℃热处理过的人血浆平板37℃培养过夜，筛选具有产生透明圈能力的菌株，筛选到的葡激酶阳性菌株，在液体中培养诱导分离葡激酶溶原转换噬菌体，分离到一株溶原性转换噬菌体。
2.Hind III酶切噬菌体DNA，连接到质粒pBR322上，转化大肠杆菌C600，筛选到一株产葡激酶的大肠杆菌转化子，经分析，其外源DNA段为2.72Kb，合成其两端引物，通过PCR扩增，获得葡激酶基因。
3.再将葡激酶基因连接到载体pJLA502转化入大肠杆菌HB101，从而获得了热诱导葡激酶高表达菌株。
生产更多的葡激酶只要将工程菌株经振荡培养→冻融提取→两步纯化工艺重复循环即可。
Claims (2)
1.一种溶血栓药物重组葡激酶，其特征在于：编码所述溶血栓药物重组葡激酶的基因可由金黄色葡萄球菌的溶原性转换噬菌体α的DNA经PCR扩增获得，所述基因的核苷酸全序列及其所编码的所述溶血栓药物重组葡激酶的氨基酸序列如下：TCA AGT TCA TTC GAC AAA GGA AAA TAT AAA AAA GGC GAT GAC GCG AGTSer Ser Ser Phe Asp Lys Gly Lys Tyr Lys Lys Gly Asp Asp Ala SerTAT TTT GAA CCA ACA GGC CCG TAT TTG ATG GTA AAT GTG ACT GGA GTT GATTyr Phe Glu Pro Thr Gly Pro Tyr Leu Met Val Asn Val Thr Gly Val AspAGT AAA GGA AAT GAA TTG CTA TCC CCT CAT TAT GTC GAG TTT CCT ATT AAA CCTSer Lys Gly Asn Glu Leu Leu Ser Pro His Tyr Val Glu Phe Pro Ile Lys ProGGG ACT ACA CTT ACA AAA GAA AAA ATT GAA TAC TAT GTC GAA TGG GCA TTA GATGly Thr Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val Glu Trp Ala Leu AspGCG ACA GCA TAT AAA GAG TTT AGA GTA GTT GAA TTA GAT CCA AGC GCA AAG ATCAla Thr Ala Tyr Lys Glu Phe Arg Val Val Glu Leu Asp Pro Ser Ala Lys IleGAA GTC ACT TAT TAT GAT AAG AAT AAG AAA AAA GAA GAA ACG AAG TCT TTC CCTGlu Val Thr Tyr Tyr Asp Lys Asn Lys Lys Lys Glu Glu Thr Lys Ser Phe ProATA ACA GAA AAA GGT TTT GTT GTC CCA GAT TTA TCA GAG CAT ATT AAA AAC CCTIle Thr Glu Lys Gly Phe Val Val Pro Asp Leu Ser Glu His Ile Lys Asn ProGGA TTC AAC TTA ATT ACA AAG GTT GTT ATA GAA AAG AAA TAAGly Phe Asn Leu Ile Thr Lys Val Val Ile Glu Lys Lys End
2.根据权利要求1所述的溶血栓药物重组葡激酶，其特征在于是通过工程菌株EcoliH15表达获得，该工程菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，其保藏号为CGMCC0432。
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| TR01 | Transfer of patent right |
Effective date of registration: 20070427 Address after: 134001 No. 258 water spring road, Jilin, Tonghua Patentee after: Tonghua jade gold pharmaceutical Limited by Share Ltd. Address before: 200031 No. 320, Yueyang Road, Shanghai Patentee before: SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES |
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| C56 | Change in the name or address of the patentee |
Owner name: JILIN YUJIN PHARMACEUTICAL CO., LTD. Free format text: FORMER NAME: TONGHUA YUJIN PHARMACEUTICAL CO., LTD. Owner name: TONGHUA ZHONGXIN YUJIN PHARMACEUTICAL CO., LTD. Free format text: FORMER NAME: JILIN YUJIN PHARMACEUTICAL CO., LTD. |
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| CP01 | Change in the name or title of a patent holder |
Address after: 134001 No. 258 water spring road, Jilin, Tonghua Patentee after: Tonghua Xinyu Pharmaceutical Co.,Ltd. Address before: 134001 No. 258 water spring road, Jilin, Tonghua Patentee before: Jilin jade gold pharmaceutical Limited by Share Ltd. Address after: 134001 No. 258 water spring road, Jilin, Tonghua Patentee after: Jilin jade gold pharmaceutical Limited by Share Ltd. Address before: 134001 No. 258 water spring road, Jilin, Tonghua Patentee before: Tonghua jade gold pharmaceutical Limited by Share Ltd. |
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| CP01 | Change in the name or title of a patent holder |
Address after: 134001 No. 258 water spring road, Jilin, Tonghua Patentee after: Tonghua Kangyuan Yujin Pharmaceutical Co.,Ltd. Address before: 134001 No. 258 water spring road, Jilin, Tonghua Patentee before: Tonghua Xinyu Pharmaceutical Co.,Ltd. |
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| CP01 | Change in the name or title of a patent holder | ||
| CX01 | Expiry of patent term |
Granted publication date: 20040414 |
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| CX01 | Expiry of patent term |