CN1087747C - 用蚕生产重组人的血小板生成素制备药物的方法 - Google Patents
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Abstract
本发明属于生物技术制药工业中的基因工程生产多肽类药物的技术领域，利用家蚕幼虫、蛹作为生物反应器，通过基因工程重组技术获得带有人的血小板生成素基因的重组家蚕核型多角体病毒，利用重组病毒，在家蚕幼虫体内、蛹体内高效表达。表达产物经超声波处理后冷冻干燥后，以家蚕液冷干粉及蛹和蛾冷干粉为填充料配成口服胶囊药物的技术和方法，动物实验证明口服胶囊具有明确效果。首次实现用蚕生产口服血小板生成素口服药物发明。该方法原料易得，成本低，疗效明确，实施价值重大。
Description
本发明属于生物技术制药工业中的基因工程生产多肽类药物技术领域。
1958年Kelemen发现血小板增生症病人血清中存在促进血小板生成的体液调节因子，并称之为血小板生成素(Thromboprotein，TPO)，人的TPO基因全长约6.2kb，被5个内含子所分割，形成6个外显子(Sohma，1995)。转录的起始位点位于编码第一个氨基酸上游1949位核苷酸，终止密码子位于下游3745-3747位核苷酸，人的TPO主要用于因骨髓移植和肿瘤化疗及原因不明所引起的血小板减少症。目前，国内外尚无利用家蚕生产重组人的血小板生成素药物的报道和专利。
以家蚕作为生物反应器，利用家蚕核型多角体病毒表达系统，与从人血浆中合成cDNA进行重组，获得高效表达重组的TPO的重组病毒，表达后的产物经过分子筛纯化后，再经过高压液相分离，获得纯度为90％的TPO蛋白，经过小鼠及猕猴药效研究表明，用蚕生产的重组人的TPO具有明显的生血小板作用，有效率达90％以上。用蚕蛹接种TPO重组病毒后5-7天，收集表达产物经过纱布过滤，18000rpm离心后，再冷冻干燥装成胶囊，经口给药小鼠及猕猴，经过药效学研究表明，口服胶囊具有明确的疗效，有效率达87％，具有开发成新药的前景和广泛的应用价值。具体技术操作方法如下：
1.取人的肝组织100mg，在冰浩中研磨碎后，加入1ml的Trozol(GIBCO BRL公司产品)，充分混匀后在室温下放置10分钟，加入等体积的氯仿，混匀后在4℃条件下，12000rpm离心10分钟，取上清，加入等体积的异丙醇，混合均匀，4℃，12000rpm离心10分钟，弃上清，用70％乙醇洗沉淀，自然干燥后加入经DEPC处理过的灭菌双蒸水20μl。获得肝细胞的mRNA。
2.用GIBCO BRL的反转录试剂盒，取10μl的mRNA进行反转录，合成cDNA具体操作按说明书进行。
3.合成TPO基因编码区上下游的PCR引物，长分别为28bp和29bp，序列如下所示：引物15’AAAGCTTATGGAGCTGACTGGTGAGAAC3’；引物2为GGAATTCTTACCCTTCCTGAGACAGATTC-3’
4.取2μl反转录cDNA作为模板，加16mM 4dNTP 2μl，取引物12μl(1 O.D.引物加ddH2O 150μl)，引物22μl(1 O.D.引物加ddH2O 150μl)，加Taq酶5单位(宝灵曼公司产品)，以及其相应缓冲液及MgCl2(宝灵曼公司产品)，加入至100μl，最后加50μl石蜡油(分析纯)。
5.PCR的反应条件为预变性94℃，10分钟；变性温度94℃，45秒；延伸温度72℃，2秒；复性温度45℃，60秒；循环35次，最后72℃延伸10分钟。PCR产品经电泳鉴定，所扩增的片段长约为1100bp，与预计的完全一致，说明PCR扩增TPO基因成功。
6.将PCR产物，用1％的低融点胶(Sigma公司产品)分离，经电泳后，用手术刀电切下大小为1100bp的片段，回收于1.5ml离心管中，放置于65℃水浩10分钟，加等体积的饱和苯酚(pH8.0)，快速混匀，4℃，12000rpm离心10分钟后，取上清于一新的1.5ml离心管中，再用饱和苯酚提取一次，方法用上。取上清加等体积仿∶异戊醇(24∶1)，混合均匀，室温下放置10分钟，4℃12000rpm离心10分钟，取上清，加2倍体积冷乙醇，混匀，-20℃放置2小时。4℃，12000rpm离心10分钟，弃上清，用70％乙醇洗，自然干燥，加20μl TE溶解后，经电泳检查，片段已被正确回收，置-20℃备用。
7.将回收的片段与经SmaI酶切的pUC19(为GIBCO BRL公司产品)连接，连接条件为，取5μl外源片段，经酶切的pUC191μl，T4 DNA连接酶1单位，10×缓中液1μl，加水至10μl，20℃连接过夜(所用试剂均为宝灵曼公司产品)。
8.制备JM109(GIBCO BRL公司产品)感受态细胞(制备方见《分子克隆》科学出版社，1989，第一版)，转化连接产物，涂布于含氨卞青霉素、X-gal及IPTG的琼脂培养基上，37℃培养过夜，取白斑进小批量质粒提取(制备方见《分子克隆》科学出版社，1989，第一版)，用HindIII和EcoRI酶切鉴定克隆，筛选到正确的重组子。
9.大批量制备重组子质粒DNA，用BamHI和EcoRI酶切，切出TPO基因，与pBacPAK8载体(Clontech公司产品)，连接方法同第7步。用EcoRI、BamHI、HindIII等酶进行酶切方法鉴定重组子，获得重组转移载体，称为pBacTPO，并鉴定正确的插入方法，确定外源基因被正确地置于多角体基因启动子的控制之下。并大批量制备重组子的质粒DNA(方法见《分子克隆》科学出版社，1989，第1版)，用于共转染。
10.取2μg重组子pBacTPO与线性化家蚕核型多角体病毒基因组DNA(本实验保存株)，与Dospor混合，共转染家蚕BmN细胞(操作方法见Summers and Smith(1986)A Manual ofMethods for Baculovirus Vector and Insect cell culture procedures；Texas A &M University，USA)。
11.通过TPO ELISA试剂盒(宝灵曼公司产品，操作方法按说明书)测定TPO表达活性筛选带有TPO基因并能高效表达的重组杆状病毒，我们把这种重组病毒称之为pBMTPO，该重组病毒已于1998年1月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC NO 0338。
12.用重组病毒pBMTPO接种家蚕幼虫和家蚕蛹，经TPO ELISA测定，表达量达500μg/蚕，1mg/蛹，生物活性与天然相似，表达产物大小为40kD。
13.用大量家蚕蛹(化蛹后第2天)接种重组病毒，感染7-8天后收集蚕蛹，经TPO ELISA活性检测，证明TPO得到高效表达，平均表达量达800μg/蛹。
14.表达好的蛹在无菌条件下经过低温匀浆后，匀浆转速每分钟300转，用纱布过滤，滤去不溶物，过滤后的溶液经超声波处理后，经过1800rpm离心30分钟，取上清，放置于冷冻干燥器中，冷冻干燥后，冷冻成粉后，在无菌条件下装成胶囊。每粒胶囊容量为200mg，活性含量为500μg TPO。胶囊称为TPO胶囊。
15.取四周龄Balb/c小鼠，♀♂各100只，体重均在16-18g左右，注射抗血小板抗体(购自浙江医科大学平喘药教研室)每天每只注射0.01μg，连续注射三天，使小鼠血小板下降至每ml50单位以下，经口给药TPO胶囊，给药量为每kg 500μg，每天给药一次，连续给药7-10天，每天测定血小板变化(具体操作参照卫生部《新药研究指南》)。结果表明所给药物具有明确的升血小板作用。
16.用4-6岁猕猴，体重在4-6kg，共30只，分成5组，每组6只，♀♂各半，按对照组，阳性对照组，高、中、低给药剂量组分组，高剂量给药量为每kg 1000μg，中剂量为每kg 200μg，低剂量为每kg给药50μg，每天经口给药一次，每天检测血小板变化，结果表明，口服TPO胶囊有明确的升血小板功能和作用(具体操作参照卫生部《新药研究指南》。药物有效达90％。开发口服TPO药物具有广泛的市场和重大经济价值。
参考文献Vignon I，et al.，Molecular clonng and characterization of MPL，the human homology of the v-
mpl oncogene：identification of a member of the hematopoietic growth factor receptor
superfamily，Proc Natl Acad Sci USA，1992，89：5640-44Methia N，et al.，Oligodeoxynucleotides antisense to the protooncogene c-mpl specifically
inhibit in virto megakaryocutopoiesis，Blood，1993，82：1395-1401de Sauvage FJ，et al.，Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl
ligand，Nature，1994，369：533-538Kaushansky K，et al.，Promotion of megakaryocyte progenitor expansion and differentiation by
the c-Mpl ligand Thrombopoietin，Nature，1994，369：568-571Lok S，et al.，Cloning and expression of murine thrombopoietin cDNA and stimulation of
platelet production in viro，Nature，1994，369：565-568Wendling F，et al.，c-Mpl ligand is a humoral regulator of megakaryocytopoiesis，Nature，
1994，369：571-574Bartley T，et al.，Identification and cloning of a megakaryocyte growth and development factor
that is a ligand for the cytokine receptor Mpl，Cell，1994，77：1117-1124Hoffman RC，et al.，Peptide，disulfide and glycosylation mapping of recombinant human
thrombopoietin from Serl to Arg246，Biochemistry，1996，35(47)：14849-14861Kuter DJ，et al.，Thrombopoietin：biology，clinical applications role in the donor setting，J Clin
Apheresis，1996，11(3)：149-159Barr PJ，Mammalian subtilisins：the long-sought dibasic processing endoproteases，Cell，
1991，6691)：1-3Wada T，et al.，Characterization of the truncated thrombopoietin variants，Biochem Biophys Res
Commun，1995，213(3)：1091-1098Hunt P，et al.，The physiologic role and therapeutic potential of the Mpl-ligand in
thrombopoiesis Stem Cells，1995，13(6)：579-587Sambrook J，et al.，Molecular Cloning：A Laboratory Mamal，New York：Cold Spring Harbor
laboratory Press，1989Summers MD.Smith GE.Amanual of methods for baculovirus vectors and insect cell culture
procedures，1987，Texas：Texas Agricultural Experiment Station and Texas A & M
University College Station，Bulletin No.1555Jiang X，et al.，Translational initiation region plays an important role in the expression of human
thrombopoietin in Escherichia coli，Biochem Mol Biol Int，1996，39(6)：1109-1113
Claims (2)
1.一种用蚕幼虫和蛹及蛾生产重组人的血小板生成素(TPO)口服药物或注射剂型的方法，其特征在于应用家蚕幼虫、蛹、蛾为生物反应器，通过基因工程重组技术获得带有人的血小板生成素基因的重组家蚕核型多角体病毒pBMEPO CGMCC NO 0338，利用重组病毒感染家蚕幼虫、蛹及蛾，接种家蚕幼虫和家蚕蛹表达产生人TPO，在体内高效表达TPO，表达产物经纯化去病毒及超声波处理，经冷冻干燥制成纯品，成为针剂或以家蚕幼虫血液、蛹、蛾为填充料制备成口服胶囊药物。
2.如权利要求1所述的方法，其特征在于从人的肝细胞中通过反转录合成TPO的cDNA基因，长为1100bp，5’端带有BamHI位点，3’端带有EcoRI位点，被克隆进入pBacPAK8载体中，获得重组转移载体pBacTPO；与线性化家蚕核型多角体病毒基因组DNA进行重组，
通过筛选获得高效表达TPO的重组家蚕核型多角体病毒，表达产物分子量为40kD。
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1185485A (zh) * | 1996-12-16 | 1998-06-24 | 中国人民解放军军事医学科学院放射医学研究所 | 血小板生成素在原核细胞中的表达及纯化方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1185485A (zh) * | 1996-12-16 | 1998-06-24 | 中国人民解放军军事医学科学院放射医学研究所 | 血小板生成素在原核细胞中的表达及纯化方法 |
Non-Patent Citations (1)
| Title |
|---|
| FEBS LETR 353(1) 1994.10.1 SOHMA Y等,MOLECALAR CLONING AND CHROMOSONAL LOCALIZATION OF HTE HUMAN THROMBOPOIEF IN GENE * |
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