CN100336907C - 重组人血小板生成素/干细胞因子融合蛋白及其制备 - Google Patents
重组人血小板生成素/干细胞因子融合蛋白及其制备 Download PDFInfo
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Abstract
本发明属于生物技术中的基因工程制备多肽药物技术领域。其目的是构建一种血小板生成素/干细胞因子双功能融合蛋白基因，并在昆虫细胞中高效表达和制备生物活性更高、副作用更小的重组血小板生成素/干细胞因子双功能蛋白(rhTPO/SCF)。rhTPO/SCF基因为依次含有TPO的1-157位氨基酸编码序列、16个氨基酸的连接肽编码序列和SCF的1-145位氨基酸编码序列及终止密码子的DNA片段。从感染重组病毒的贴壁培养Sf9细胞中制备的rhTPO/SCF用于TF1细胞增殖实验，测定其比活为2.0×105units/mg；用于Mo7e细胞增殖实验，测定其比活为8.3×105units/mg。可用于治疗病人放疗、化疗及骨髓移植等病症引起的血小板低下。
Description
一.技术领域
本发明属于基因工程制备多肽药物技术领域。
二.技术背景
血小板是参与凝血作用的关键成分，造血干细胞首先经过逐步分化成为巨核系定向祖细胞，然后再分化为成熟的巨核细胞，最后裂解产生血小板。这一复杂的分化成熟过程受众多造血因子调控，同时也受造血干细胞与细胞外基质及周围的骨髓细胞之间的相互作用影响。目前，一致认为血小板生成素(Thrombopoietin，TPO)是调节巨核细胞生成和增殖的最重要的细胞因子，它与IL-3，SCF共同作用，促使造血干细胞分化为定向的巨核细胞，然后在TPO、IL-3、SCF、IL-6、IL-11等因子参与下发育成为成熟的巨核细胞[Rasko JE Inter.J.Biochem.Cell.Biol.1998，30：657]。1994年后Bartley等先后克隆得到TPO的cDNA基因，为治疗血小板缺乏症展示了希望[Bartley TD，et al.Cell，1994，77(7)：1117]。天然的人TPO全长332个氨基酸，其N端的153个氨基酸有完全的TPO受体结合活性和生物活性，而C端部分包含有多个糖基化位点，主要与成熟TPO的分泌有关，同时也与TPO在人体内的半衰期有关。rhTPO虽然能快速提高体内血小板的水平，但是血小板稳定的时间不长，效果不理想。
人干细胞因子(SCF)是促进造血干/祖细胞存活的细胞因子，可以抑制造血干细胞的凋亡[Domen J.，et al.J.Exp.Med.(2000)192：1707-1718]。SCF能够在多级造血水平上与包括TPO在内的其他细胞因子协同作用促进造血干/祖细胞及各种血细胞的增殖和存活。SCF虽然不能单独促进巨核细胞的生长或增殖，但是在TPO存在时，SCF有很强的协同作用，能有效的提高巨核细胞的增殖的速度和延长生存时间。
构建由不同造血细胞因子组成的融合蛋白可以改善使用单一细胞因子的不足。TPO/SCF融合蛋白除了能够提高巨核细胞的增殖的速度和延长生存时间外，其SCF基团还可以充分发挥其促进造血干细胞存活的功能，防止由于干细胞或巨核细胞的前体细胞供应不足而导致血小板升高后又迅速下降。
三.发明内容
本发明需要解决的问题是构建一种重组人血小板生成素/干细胞因子(rhTPO/SCF)双功能蛋白基因，并采用与谷胱甘肽硫转移酶基因融合表达的方式在Sf9细胞中高效表达并分离纯化重组蛋白，以达到可应用水平，使之成为一种性能比TPO更好的恢复血小板水平的药物。
本发明的技术内容包括：1.重组人血小板生成素/干细胞因子双功能融合蛋白基因的设计与构建。2.高效表达融合型GST-rhTPO/SCF的重组苜蓿夜蛾核型多角体病毒AcNPV-rhTPO/SCF的构建。3.rhTPO/SCF在Sf9细胞中的表达及分离纯化和鉴定。
1.天然的TPO全长332个氨基酸，天然的分泌型可溶性SCF为165个氨基酸。我们设计的rhTPO/SCF双功能蛋白基因是含有TPO的1-157位氨基酸、16个氨基酸的连接肽、SCF的1-145位氨基酸编码序列和终止密码的DNA片段。其DNA序列和氨基酸序列如下：
AGC CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC CTC AGT AAA CTA CTT CGT GAC TCC CAT 60
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His 20
GTC CTT CAC AGC AGA CTG AGC CAG TGC CCA GAG GTT CAC CCT TTG CCT ACA CCT GTC CTG 120
Val Leu His Ser Arg Leu Ser Asn Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu 40
CTG CCT GCT GTG GAC TTT AGC TTG GGA GAA TGG AAA ACC CAG ATG GAG GAG ACC AAG GCA 180
Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Asn Met Glu Glu Thr Lys Ala 60
CAG GAC ATT CTG GGA GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG GCA GCA CGG GGA CAA 240
Asn Asp Ile Leu Gly Aal Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Asn 80
CTG GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG CAA CTT TCT GGA CAG GTC CGT CTC CTC 300
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Asn Leu Ser Gly Asn Val Arg Leu Leu 100
CTT GGG GCC CTG CAG AGC CTC CTT GGA ACC CAG CTT CCT CCA CAG GGC AGG ACC ACA GCT 360
Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Asn Leu Pro Pro Asn Gly Arg Thr Thr Ala 120
CAC AAG GAT CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG CGT 420
His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg 140
TTC CTG ATG CTT GTA GGG GGG TCA ACA CTA TGT GTC AGG CGG GCC CCA CCC GGA GGA GGA 480
Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Gly Gly Gly 160
GGT TCC CCG GGC GGA TCC GGA GGA GGA GGC TCC GGC GGC GAA GGG ATC TGC AGG AAT CGT 540
Gly Ser Pro Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Gly Ile Cys Arg Asn Arg 180
GTG ACT AAT AAT GTA AAA GAC GTC ACT AAA TTG GTG GCA AAT CTT CCA AAA GAC TAC ATG 600
Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro Lys Asp Tyr Met 200
ATA ACC CTC AAA TAT GTC CCC GGG ATG GAT GTT TTG CCA AGT CAT TGT TGG ATA AGC GAG 660
Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu 220
ATG GTA GTA CAA TTG TCA GAC AGC TTG ACT GAT CTT CTG GAC AAG TTT TCA AAT ATT TCT 720
Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser 240
GAA GGC TTG AGT AAT TAT TCC ATC ATA GAC AAA CTT GTG AAT ATA GTG GAT GAC CTT GTG 780
Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val 260
GAG TGC GTG AAA GAA AAC TCA TCT AAG GAT CTA AAA AAA TCA TTC AAG AGC CCA GAA CCC 840
Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro 280
AGG CTC TTT ACT CCT GAA GAA TTC TTT AGA ATT TTT AAT AGA TCC ATT GAT GCC TTC AAG 900
Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys 300
GAC TTT GTA GTG GCA TCT GAA ACT AGT GAT TGT GTG GTT TCT TCA ACA TTA AGT TAG TAA 960
Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser 320
rhTPO/SCF基因的构建流程如图1所示，以pUC18-SCF为模板，用引物1，2经PCR法合成5’端带BamHI酶切位点和部分连接肽(GSGGGGSGG)的编码序列，3’端带两个连续终止密码子和HindIII酶切位点的含有SCF第1-145位氨基酸编码序列的基因片段，并将其克隆入pBluscript质粒载体的BamHI和HindIII位点之间，得到质粒pBluscript-3’SCF。然后以pUC18-TPO为模板，用引物3，4经PCR法，在pfu高温聚合酶的催化下，合成5’端带XbaI和BglII位点，3’端带部分连接肽(GGGGSPG)的编码序列，含有TPO的第1-157位氨基酸密码子的基因片段，该片段的末端为平端，经XbaI酶切后，克隆入pBluscript-3’SCF的XbaI和BamHI位点之间(pBluscript-3’SCF先经BamHI酶切，Klenow补平，再用XbaI酶切)，得到pBluscript-TS。在该重组质粒中，TPO的第1-157位氨基酸的编码序列与SCF的第1-145位氨基酸的编码序列通过16个氨基酸(GGGGSPGGSGGGGSGG)的连接肽编码区连接，从而获得TPO/SCF双功能蛋白基因，全长960bp。经测序证明，得到的基因序列与设计完全一致。(测序结果见附图2，3)。
引物1：5’CC
GGATCCGGAGGAGGAGGCTCCGGCGGCGAAGGGATCTGCA GGAATCGT 3’
引物2：5’GG
AAGCTTTTACTAACTTAATGTTGAAGAAAC 3’
引物3：5’CCTCTAGATCTAGCCCGGCTCCTCCTGCT 3’
引物4：5’CGCCCGGGGAACCTCCTCCTCCGGGTGGGGCCCGCCTGAC 3’
1.pBluscript-TS经HindIII酶切，Klenow补平，BglII酶切后得到TPO/SCF基因片段，并克隆于杆状病毒载体pAcSecG2T(PharMingen International)的BamHI和SmaI位点之间得到pAcSecG2T-TS。将上述重组转移载体DNA与野生型苜蓿夜蛾核型多角体病毒DNA用脂质体介导法共转染草地夜蛾细胞Sf9，经细胞内同源重组和4轮有限稀释法纯化并结合重组蛋白的体外活性测定，筛选出高效表达GST-rhTPO/SCF的重组病毒AcNPV-rhTPO/SCF。
2.将重组病毒以合适的感染复数(MOI)感染生长状态良好的Sf9细胞，感染后48小时收集细胞，悬浮于1×PBS溶液，超声破碎细胞，离心后，取上清液用于Glutathione Sephrose4B柱亲和层析(Amersham Pharmacia Biotech Inc)，分离纯化得到GST-TPO/SCF，最后经凝血酶切割和再次Glutathione Sephrose4B柱亲和层析即可获得重组人血小板生成素/干细胞因子双功能蛋白精品(图4)。
本发明构建的rhTPO/SCF双功能基因将一段截短的TPO基因(1-157位氨基酸)和一段截短的SCF单体基因(1-145位氨基酸)用一段16肽(GGGGSPGGSGGGGSGG)编码序列以头尾连接方式连接形成新型人血小板生成素/干细胞因子双功能融合蛋白基因。该基因在昆虫细胞中表达的产物在保持TPO活性的同时，由于同时具有TPO受体和SCF受体的结合能力，能够有效的促进造血干细胞的存活，刺激造血干细胞向巨核细胞的分化和增殖，改善单独使用TPO时，血小板升高太快和血小板水平恢复后稳定时间短的问题。
构建的重组病毒按本发明所述方法感染单层Sf9细胞，收集细胞，细胞内累积的重组蛋白表达量约为0.5mg/L。
按本发明提供的分离纯化方法从Sf9细胞中获得的rhTPO/SCF，经SDS-PAGE和考马斯亮蓝染色检测，呈分子量约35KD的一条带，纯度大于90％。用于TF1细胞增殖实验，测定其比活为2.0×105units/mg；用于Mo7e细胞增殖实验，测定其比活为8.3×105units/mg。
四.附图说明
附图1人血小板生成素/干细胞因子基因的构建流程图
附图2rhTPO/SCF基因的测序结果(3’端测序结果)
附图3rhTPO/SCF基因的测序结果(5’端测序结果)
附图4SDSPAGE电泳图谱
a)Glutathione Sephrose4B亲和柱层析洗脱峰
b)Glutathione Sephrose4B亲和柱层析洗脱峰经凝血酶切刻后的产物
c)谷胱甘肽硫转移酶
d)rhTPO/SCF
M.分子量标准蛋白
五.具体实施方式
1.pUC18-SCF为模板，用引物1，2经PCR法合成SCF基因片段并将其克隆入pBluscript质粒载体，得到pBluscript-3’SCF质粒，然后以pUC18-TPO为模板，用引物3，4经PCR法合成TPO基因片段并将其克隆入pBluscript-3’SCF质粒载体，得到pBluscript-TS质粒。经测序证明，结果与设计序列完全一致。(流程见附图1)
2.将pBluscript-TS质粒酶切，得到TPO/SCF基因片段，并插入AcNPV转移载体pAcSecG2T，得到重组转移载体pAcSecG2T-TS。将pAcSecG2T-TS与AcNPV DNA共转染Sf9，经筛选并结合测活选出不含野生型病毒的重组病毒AcNPV-rhTPO/SCF。
3.取倍增时间为21小时，活细胞占90％，密度为6×105细胞/mL的单层Sf9细胞，按感染复数(MOI)＝10感染重组病毒1小时后，倾去病毒液，换成新鲜的培养基。27℃培养48小时后，离心收集细胞，悬浮于1×PBS，超声破碎细胞后，12,000离心20分钟。收集离心后的上清液以1mL/min流速上样至2mL的Glutathione Sephrose4B层析柱，层析柱预先用1×PBS平衡。上样后，先用1×PBS洗至基线，而后用洗脱液(pH 8.0，50mM Tris-Cl，10mM还原型谷胱肽)以1mL/min的流速洗脱，收集洗脱峰10mL。洗脱液对蒸馏水透析并冻干，冻干后，溶于300μl×PBS，再加入5单位的凝血酶，22℃酶切16小时，再以1mL/min流速上样至2mL的Glutathione Sephrose4B层析柱，收集穿过峰，对蒸馏水透析后冻干，得到TPO/SCF双功能蛋白精品。还原条件SDS-PAGE后经考马斯亮蓝染色检测，rhTPO/SCF呈分子量约35KD的一条带，纯度大于90％。免疫印迹(Westenblotting)分析结果与其一致。
序列表.txt
重组人血小板生成素/干细胞因子基因序列表
<110>申请人的姓名或名称：南京大学
<120>发明名称：重组人血小板生成素/干细胞因子双功能融合蛋白
<160>序列表中序列的个数：1
<210>序列标识符：1
<211>序列的长度：960
<212>序列的类型：DNA
<213>生物体：人(Homo sapiens)
<400>核苷酸序列和氨基酸序列：
AGC CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC CTC AGT AAA CTA CTT CGT GAC TCC CAT 60
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His 20
1 6 11 16
GTC CTT CAC AGC AGA CTG AGC CAG TGC CCA GAG GTT CAC CCT TTG CCT ACA CCT GTC CTG 120
Val Leu His Ser Arg Leu Ser Asn Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu 40
21 26 31 36
CTG CCT GCT GTG GAC TTT AGC TTG GGA GAA TGG AAA ACC CAG ATG GAG GAG ACC AAG GCA 180
Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Asn Met Glu Glu Thr Lys Ala 60
41 46 51 56
CAG GAC ATT CTG GGA GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG GCA GCA CGG GGA CAA 240
Asn Asp Ile Leu Gly Aal Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Asn 80
61 66 71 76
CTG GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG CAA CTT TCT GGA CAG GTC CGT CTC CTC 300
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Asn Leu Ser Gly Asn Val Arg Leu Leu 100
81 86 91 96
CTT GGG GCC CTG CAG AGC CTC CTT GGA ACC CAG CTT CCT CCA CAG GGC AGG ACC ACA GCT 360
Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Asn Leu Pro Pro Asn Gly Arg Thr Thr Ala 120
101 106 111 116
CAC AAG GAT CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG CGT 420
His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg 140
121 126 131 136
TTC CTG ATG CTT GTA GGG GGG TCA ACA CTA TGT GTC AGG CGG GCC CCA CCC GGA GGA GGA 480
Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Gly Gly Gly 160
141 146 151 156
GGT TCC CCG GGC GGA TCC GGA GGA GGA GGC TCC GGC GGC GAA GGG ATC TGC AGG AAT CGT 540
Gly Ser Pro Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Gly Ile Cys Arg Asn Arg 180
161 166 171 176
GTG ACT AAT AAT GTA AAA GAC GTC ACT AAA TTG GTG GCA AAT CTT CCA AAA GAC TAC ATG 600
Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro Lys Asp Tyr Met 200
181 186 191 196
ATA ACC CTC AAA TAT GTC CCC GGG ATG GAT GTT TTG CCA AGT CAT TGT TGG ATA AGC GAG 660
Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu 220
201 206 211 216
ATG GTA GTA CAA TTG TCA GAC AGC TTG ACT GAT CTT CTG GAC AAG TTT TCA AAT ATT TCT 720
Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser 240
221 226 231 236
GAA GGC TTG AGT AAT TAT TCC ATC ATA GAC AAA CTT GTG AAT ATA GTG GAT GAC CTT GTG 780
Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val 260
241 246 251 256
GAG TGC GTG AAA GAA AAC TCA TCT AAG GAT CTA AAA AAA TCA TTC AAG AGC CCA GAA CCC 840
Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro 280
261 266 271 276
AGG CTC TTT ACT CCT GAA GAA TTC TTT AGA ATT TTT AAT AGA TCC ATT GAT GCC TTC AAG 900
Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys 300
281 286 291 296
GAC TTT GTA GTG GCA TCT GAA ACT AGT GAT TGT GTG GTT TCT TCA ACA TTA AGT TAG TAA 960
Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser 320
301 306 311 316
氨基酸序列为：
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His
1 6 11 16
Val Leu His Ser Arg Leu Ser Asn Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu
21 26 31 36
Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Asn Met Glu Glu Thr Lys Ala
41 46 51 56
Asn Asp Ile Leu Gly Aal Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Asn
61 66 71 76
Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Asn Leu Ser Gly Asn Val Arg Leu Leu
81 86 91 96
Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Asn Leu Pro Pro Asn Gly Arg Thr Thr Ala
101 106 111 116
His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg
121 126 131 136
Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Gly Gly Gly
141 146 151 156
Gly Ser Pro Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Gly Ile Cys Arg Asn Arg
161 166 171 176
Val Thr Asn Asn Val Lys Asp Val Thr Lys Leu Val Ala Asn Leu Pro Lys Asp Tyr Met
181 186 191 196
Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu
201 206 211 216
Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser
221 226 231 236
Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val
241 246 251 256
Glu Cys Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro
261 266 271 276
Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys
281 286 291 296
Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu Ser
301 306 311 316
Claims (4)
1.一种重组人血小板生成素/干细胞因子双功能融合蛋白基因，其特征是设计并构建一个人血小板生成素的1-157位氨基酸的编码区、16个氨基酸GGGGSPGGSGGGGSGG的连接肽编码区、可溶性人干细胞因子的1-145位氨基酸编码区和终止密码子依次首尾相连接的DNA片段，重组人血小板生成素/干细胞因子融合蛋白基因的核苷酸序列为：
agcccggctc ctcctgcttg tgacctccga gtcctcagta aactacttcg tgactcccat 60
gtccttcaca gcagactgag ccagtgccca gaggttcacc ctttgcctac acctgtcctg 120
ctgcctgctg tggactttag cttgggagaa tggaaaaccc agatggagga gaccaaggca 180
caggacattc tgggagcagt gacccttctg ctggagggag tgatggcagc acggggacaa 240
ctgggaccca cttgcctctc atccctcctg gggcaacttt ctggacaggt ccgtctcctc 300
cttggggccc tgcagagcct ccttggaacc cagcttcctc cacagggcag gaccacagct 360
cacaaggatc ccaatgccat cttcctgagc ttccaacacc tgctccgagg aaaggtgcgt 420
ttcctgatgc ttgtaggggg gtcaacacta tgtgtcaggc gggccccacc c
ggaggagga480
ggttccccgg gcggatccgg aggaggaggc tccggcggcgaagggatctg caggaatcgt 540
gtgactaata atgtaaaaga cgtcactaaa ttggtggcaa atcttccaaa agactacatg 600
ataaccctca aatatgtccc cgggatggat gttttgccaa gtcattgttg gataagcgag 660
atggtagtac aattgtcaga cagcttgact gatcttctgg acaagttttc aaatatttct 720
gaaggcttga gtaattattc catcatagac aaacttgtga atatagtgga tgaccttgtg 780
gagtgcgtga aagaaaactc atctaaggat ctaaaaaaat cattcaagag cccagaaccc 840
aggctcttta ctcctgaaga attctttaga atttttaata gatccattga tgccttcaag 900
gactttgtag tggcatctga aactagtgat tgtgtggttt cttcaacatt aagttagtaa 960。
2.权利要求1所提供的基因构建的一种能在昆虫Sf9中高效表达谷胱甘肽硫转移酶-人血小板生成素/干细胞因子融合蛋白的重组苜蓿夜蛾核型多角体病毒AcNPV-rhTPO/SCF，其特征是将人血小板生成素/干细胞因子融合蛋白基因克隆于杆状病毒转移载体pAcSecG2T的BamHI和SmaI位点之间，得到重组转移载体pAcSecG2T-rhTPO/SCF，其中rhTPO/SCF基因上游融合了带凝血酶切割位点的谷胱甘肽硫转移酶基因片段，整个融合基因处于AcNPV的多角体蛋白启动子控制之下；将重组转移载体DNA与野生型AcNPV DNA共转染Sf9细胞，经4轮有限稀释法纯化并结合重组蛋白的体外活性测定，筛选出重组病毒AcNPV-rhTPO/SCF。
3.一种重组人血小板生成素/干细胞因子融合蛋白的制备方法，其特征是用权利要求2所构建的重组病毒感染贴壁培养的Sf9细胞，27℃表达48小时，细胞内表达的血小板生成素/干细胞因子融合蛋白具有TF1细胞和Mo7e细胞增殖的生物活性；收集表达细胞，悬浮于1×PBS溶液，超声破碎细胞，经离心后，取上清液用于Glutathione Sephrose4B柱亲和层析，最后经凝血酶切割和再次的Glutathione Sephrose4B柱亲和层析即可获得纯度大于90％的重组人血小板生成素/干细胞因子融合蛋白，SDS-PAGE分析后经考马斯亮蓝染色检测，rhTPO/SCF呈分子量35KD的一条带，用于TF1细胞增殖实验，测定其比活为2.0×105units/mg；用于Mo7e细胞增殖实验，测定其比活为8.3×105units/mg。
4.根据权利要求3所述融合蛋白在制备治疗放疗、化疗及骨髓移植病症引起的血小板低下症的药物中的应用。
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| WO2015047062A1 (en) * | 2013-09-27 | 2015-04-02 | Uab Profarma | Fused proteins of granulocyte colony-stimulating factor with other partners of growth factor, preferably with stem cell factor, and method of preparation thereof |
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| CN101863982A (zh) * | 2009-04-17 | 2010-10-20 | 哈药集团生物工程有限公司 | 一种用于升高血小板的融合蛋白及其制备方法 |
| JP6584956B2 (ja) | 2012-12-21 | 2019-10-02 | アステラス インスティテュート フォー リジェネレイティブ メディシン | 多能性幹細胞から血小板を生産するための方法およびその組成物 |
| CN104181290B (zh) * | 2014-08-08 | 2017-07-25 | 北京泰德制药股份有限公司 | 一种评价促血小板生成素受体激动剂体外活性的分析方法 |
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| WO2003039232A2 (en) * | 2001-10-26 | 2003-05-15 | Large Scale Biology Corporation | Endothelial cell derived hemotopoietic growth factor |
| CN1122716C (zh) * | 1994-03-07 | 2003-10-01 | 依默耐克斯有限公司 | 体外细胞培养和移植试剂盒 |
| CN1556114A (zh) * | 2004-01-08 | 2004-12-22 | �Ϻ���ͨ��ѧ | 人干细胞因子巨噬细胞集落刺激因子双功能蛋白及制备 |
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| CN1122716C (zh) * | 1994-03-07 | 2003-10-01 | 依默耐克斯有限公司 | 体外细胞培养和移植试剂盒 |
| WO2003039232A2 (en) * | 2001-10-26 | 2003-05-15 | Large Scale Biology Corporation | Endothelial cell derived hemotopoietic growth factor |
| CN1556114A (zh) * | 2004-01-08 | 2004-12-22 | �Ϻ���ͨ��ѧ | 人干细胞因子巨噬细胞集落刺激因子双功能蛋白及制备 |
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| WO2015047062A1 (en) * | 2013-09-27 | 2015-04-02 | Uab Profarma | Fused proteins of granulocyte colony-stimulating factor with other partners of growth factor, preferably with stem cell factor, and method of preparation thereof |
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