CN1232536C - 人干细胞因子巨噬细胞集落刺激因子双功能蛋白及制备 - Google Patents
人干细胞因子巨噬细胞集落刺激因子双功能蛋白及制备 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。其目的是构建一种人干细胞因子-巨噬细胞集落刺激因子双功能蛋白基因，并在昆虫细胞中高效分泌表达和制备生物活性更高副作用更小的重组干细胞因子-巨噬细胞集落刺激因子双功能蛋白(rhSCF/M-CSF)。rhSCF/M-CSF基因为依次含有SCF完整的信号肽及1-165位氨基酸、15个氨基酸的连接肽和M-CSF的1-149位氨基酸编码序列及终止密码子的DNA片段。它可用于促进病人放疗、化疗及骨髓移植后造血与免疫功能重建和恢复，并增强其抗真菌感染的能力，也可用于干细胞的体外扩增研究。
Description
一、技术领域
本发明属于基因工程制各多肽药物技术领域。
二、背景技术
干细胞因子(SCF，也叫Kit配体，Steel因子，肥大细胞生长因子等)是一种多功能细胞因子，能促进早期造血干细胞的增殖而不诱导细胞的分化，能在多级造血水平与其他细胞因子协同作用促进造血干/祖细胞及各种造血细胞的存活和增殖。其受体广泛分布于不同系列、不同世代的造血细胞，包括红细胞系、单核粒巨细胞系及巨核细胞系等。SCF能增强这些细胞的增殖能力并激活这些细胞使之对相应的各种造血细胞因子的刺激更为敏感。临床主要应用于放疗，化疗和骨髓移植后造血和免疫功能的重建和恢复。另外SCF对骨髓干细胞体外扩增和干细胞移植及基因治疗中也有应用前景。SCF单独能促进肥大细胞的生长。并能活化成熟的肥大细胞，促进其脱颗粒，释放组胺。故在应用时大多数病人的注射部位会出现瘙痒风疹，少数病人还会发生变态样反应。这种副作用限制了SCF的临床应用[Broudy VC，Blood，1997；90：1345-1364]。
天然的SCF是一种糖蛋白，在正常生理条件下90％以上以单体形式存在，在局部高浓度时可以形成离子型同源二聚体，二聚体形式SCF活性比单体高10-20倍[Hsu YR，Journal of Biological Chemistry，1997；272(10)：6406-6415]、[秦浚川，中国发明专利，专利号ZL03112934.X]。
巨噬细胞集落刺激因子(M-CSF)是一种多功能的细胞因子，也是一种免疫调节剂。它能刺激骨髓干细胞分化成单核巨噬细胞并促进其增殖及成熟。它还能特异地促进单核巨噬细胞吞噬细菌、真菌和肿瘤细胞，并刺激巨噬细胞分泌G-CSF、GM-CSF、TNF、IL-6、IL-8等多种造血细胞因子[P，Praloran V.，Eur Cytokine Netw.1997；8(2)：125-136]。另外，它也是骨髓基质细胞的生长因子，参与造血微环境及骨髓重建。在临床上，M-CSF能有效提高骨髓移植及肿瘤病人放化疗后的免疫功能，显著提高这些病人感染致死性白色念珠菌和曲霉菌的存活率[Nemunaitis J，et al.Blood，1993，82(5)：1422-1427]。但长期和大剂量使用M-CSF，能抑制造血过程，并造成对多种器官的损伤。天然的M-CSF是一种通过链间二硫键共价连接的同源二聚体形式的糖蛋白。
SCF与M-CSF具有协同作用，尤其在无血清培养条件下，其协同作用更为明显。
利用M-CSF在天然条件下能通过Cys31-Cys31形成共价同源二聚体，且SCF与M-CSF具有协同作用这些特点，通过基因重组技术构建并在昆虫细胞中表达同源二聚体形式的干细胞因子-巨噬细胞集落刺激因子双功能蛋白(SCF/M-CSF)，以显著提高SCF与M-CSF的活性，克服在临床上因大剂量使用SCF及M-CSF时的引起的副作用。
三、发明内容
本发明专利需要解决的技术问题是设计出了能保证SCF与M-CSF各自天然活性不受影响的柔性连接肽，通过基因工程技术构建一种干细胞因子-巨噬细胞集落刺激因子双功能蛋白基因(rhSCF/M-CSF)，使之在Sf9细胞(Invitrogen)中高效分泌表达，并从培养液中分离纯化rhSCF/M-CSF，以达到可应用水平，成为一种性能比SCF和M-CSF单体蛋白更好的恢复造血和调节免疫功能的药物。
本发明的技术内容包括：1、重组人干细胞因子-巨噬细胞集落刺激因子双功能蛋白基因(rhSCF/M-CSF)的设计与构建。2、高效分泌表达的rhSCF/M-CSF重组苜蓿夜蛾核型多角体病毒AcNPV-SCF/M-CSF的构建。3、rhSCF/M-CSF在Sf9细胞中的分泌表达及其分离纯化和鉴定。
我们设计的rhSCF/M-CSF双功能蛋白基因是含有SCF的25个氨基酸的完整信号肽及1-165位氨基酸、15个氨基酸的连接肽和M-SCF的1-149位氨基酸编码序列及终止密码子的DNA片段。
ATG AAG AAG ACA CAA -61
Met Lys Lys Thr Gln -21
ACT TCG ATT CTC ACT TGC ATT TAT CTT CAG CTG CTC CTA TTT AAT CCT CTC GTC AAA ACT -1
Thr Trp Ile Leu Thr Cys Ile Tyr Leu Gln Leu Leu Leu Phe Asn Pro Leu Val Lvs Thr -1
GAA GGG ATC TGC AGG AAT CGT GTG ACT AAT AAT GTA AAA GAC GTC ACT AAA TTG GTG GCA 60
Glu Gly Ile Leu Arg Ash Arg Val Thr Asn Ash Val Lys Asp Val Thr Lys Leu Val Ala 20
AAT CTT CCA AAA GAC TAC ATG ATA ACC CTC AAA TAT GTC CCC GGG ATG GAT GTT TTG CCA 120
Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly Met Asp Val Leu Pro 40
AGT CAT TGT TGG ATA AGC GAG ATG GTA GTA CAA TTG TCA GAC AGC TTG ACT GAT CTT CTG 180
Ser His Cys Trp Ile Scr Glu Met Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu 60
GAC AAG TTT TCA AAT ATT TCT GAA GGC TTG AGT AAT TAT TCC ATC ATA GAC AAA CTT GTG 240
Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val 80
AAT ATA GTG GAT GAC CTT GTG GAG TGC GTG AAA GAA AAC TCA TCT AAG GAT CTA AAA AAA 300
Ash Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Ash Ser Ser Lys Asp Leu Lys Lys 100
TCA TTC AAG AGC CCA GAA CCC AGG CTC TTT ACT CCT GAA GAA TTC TTT AGA ATT TTT AAT 360
Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe Asn 120
AG
ATCC ATT GAT GCC TTC AAG GAC TTT GTA GTG GCA TCT GAA ACT AGT GAT TGT GTG GTT420
Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val 140
TCT TCA ACA TTA AGT CCT GAG AAA GAT TCC AGA GTC AGT GTC ACA AAA CCA TTT ATG TTA 480
Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu 160
CCC CCT GTT GCA GCC GGA GGA GGA GGA TCC GGA GGA GGA GGC TCC GGC GGC AGT ATC ACC 540
Pro Pro Val Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Ile Thr 180
GAG GAG GTG TCG GAG TAC TGT AGC CAC ATG ATT GGG AGT GGA CAC CTG CAG TCT CTG CAG 600
Glu Glu VaL Ser Glu Tyr Cys Ser His Met Ile Gly Ser Gly His Leu Gln Ser Leu Gln 200
CGG CTG ATT GAC AGT CAG ATG GAG ACC TCG TGC CAA ATT ACA TTT GAG TTT GTA GAC CAG 660
Leu Arg Ile Asp Ser Gln Met Glu Thr Ser Cys Gln Ile Thr Phe Glu Phe Val Asp Gln 220
GAA CAG TTG AAA GAT CCA GTG TGC TAC CTT AAG AAG GCA TTT CTC CTG GTA CAA GAC ATA 720
Glu Gln Leu Lys Asp Pro Val Cys Tyr Leu Lys Lys Ala Phe Leu Leu Val Gln Asp Ile 240
ATG GAG GAC ACC ATG CGC TTC AGA GAT AAC ACC CCC AAT GCC ATC GCC ATC GTG CAG CTG 780
Met Glu Asp Thr Met Arg Phe Arg Asp Asn Thr Pro Asn Ala Ile Ala Ile Val Gln Leu 260
CAG GAA CTC TCT TTG AGG CTG AAG AGC TGC TTC ACC AAG GAT TAT GAA GAG CAT GAC AAG 840
Gln Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp Tyr Glu Glu His Asp Lys 280
GCC TGC GTC CGA ACT TTC TAT GAG ACA CCT CTC CAG TTG CTG GAG AAG GTC AAG AAT GTC 900
Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gln Leu Leu Glu Lys Val Lys Asn Val 300
TTT AAT GAA ACA AAG AAT CTC CTT GAC AAG GAC TGG AAT ATT TTC AGC AAG AAC TGC AAC 960
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AAC AGC TTT GCT GAA TGC TCC AGC CAA TAA TAG
Asn Ser Phe Ala Glu Cys Ser Ser Gln
1.1.SCF/M-CSF的构建：
A.以pUC18-SCF为模板，用引物1，2经PCR法合成5’端带Xba I酶切位点，3’端缺失终止密码子但带柔性连接肽(GGGGS)编码序列和BamH I位点的含有SCF25个氨基酸的完整信号肽及第1-165位氨基酸编码序列，并将其克隆入pUC18质粒载体(市售)中，得到pUC18-5’SCF质粒
引物1：5’GG
TCTAGATGAAGAAGACACAAACT-3’
引物2：5’CC
GGATCCTCCTCCTCCGGCTGCAACAGGGGGTAA-3’
B.以pUC18-M-CSF为模板，用引物3，4经PCR法合成5’端带BamH I酶切位点，柔性连接肽(GSGGGGSGGSIT)编码序列及3’端带有两个连续终止密码子和KpnI位点的含有M-CSF第1-149位氨基酸编码序列，得到pUC18-3’M-CSF质粒
引物3：5’-GG
GGATCCGGAGGAGGAGGCTCCGGCGGCAGTATCACCGAGGAGGTGTCG-3，
引物4：5’-GC
GGTACCGGGCTATTATTGGCTGGAGCATTC-3’
C.用Xba I和BamH I从pUC18-5’-SCF质粒中切下SCF基因片段，插入pUC18-3’M-CSF质粒的Xba I和BamH I之间，得到重组质粒pUC18-SCF/M-CSF。在该重组质粒中，SCF信号肽及N端1-165aa的编码序列与M-CSF N端1-149aa编码序列及两个终止密码子通过15个氨基酸(GGGGSGGGGSGGSIT)这段柔性连接肽连接在一起，从而获得SCF/M-CSF双功能蛋白基因，全长1068bp。
1.2.重组转移载体的构建及重组病毒的纯化：
A.用Xba I和Kpn I从pUC18-SCF/M-CSF质粒中切下带信号肽的SCF/M-CSF双功能蛋白基因片段，插入AcNPV转移载体pVL1392(Invitrogen)的Xba I与Kpn I位点之间，得到重组转移载体pVL1392-SCF/M-CSF。
B.将重组转移载体pVL1392-SCF/M-CSF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，经胞内同源重组和4轮有限稀疏法筛选并结合TF-1细胞MTT法测活筛选出高效分泌表达SCF/M-CSF的重组病毒AcNPV-SCF/M-CSF。
2.将重组SCF/M-CSF病毒以合适的感染复数(MOI)感染生长状态良好的Sf9细胞，感染后在无血清培养基Sf900或不加血清的TMN-FH中培养，表达72小时后收集培养液经M-CSF抗体柱亲和层析分离纯化得到SCF/M-CSF双功能蛋白。
本发明构建的rhSCF/M-CSF基因为将一个SCF单体基因(25个氨基酸信号肽及1-165位氨基酸)和一个M-CSF单体基因(1-149位氨基酸)用柔性的15肽(GGGGSGGGGSGGSIT)编码序列以头尾连接方式连接而形成的新型人干细胞因子-巨噬细胞集落刺激因子双功能蛋白基因。在昆虫细胞中表达的产物经TF-1细胞MTT法(SCF活性)及人骨髓巨噬细胞集落形成(CFU-M，M-CSF活性)测活分析，结果显示，在SCF活性检测中rhSCF/M-CSF比E.coli中表达的单体rhSCF(纯度＞98％)比活高5.3倍，而在促人骨髓巨噬细胞集落形成活性(M-CSF活性)检测中rhSCF/M-CSF比家蚕中表达的纯品rhM-CSF高出5倍。因此可以在临床应用时减少用量，降低其副作用。
构建的重组病毒按本发明所述方法感染单层Sf9细胞，收集细胞上清液，上清液中累积的重组蛋白表达量约为1mg/L。
按本发明提供的分离纯化方法可以从Sf9细胞培养液中获得的SCF/M-CSF比活为1.0×106units/mg。非还原条件SDS-PAGE后经考马斯亮蓝染色检测，rhSCF/M-CSF呈分子量约84KD的一条带，而还原条件下SDS-PAGE后经考马斯亮蓝染色检测，rhSCF/M-CSF呈分子量约42KD的一条带。纯度约为90％。
本发明所述人干细胞因子巨噬细胞集落刺激因子双功能蛋白可用于促进病人放疗、化疗及骨髓移植后造血与免疫功能重建和恢复，并增强其抗真菌感染的能力，也可用于干细胞的体外扩增研究。
四.附图说明
图1rhSCF/M-CS基因的构建流程图
图2rhSCF/M-CSF基因的测序结果图(5’端测序结果)
图3rhSCF/M-CSF基因的测序结果图(3’端测序结果)
图4rhSCF/M-CSF与rhSCF单体的活性比较图
图5rhSCF/M-CSF SDS-PAGE及Westen-blot图谱
五.具体实施方式
1.1.以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pUC18质粒载体，得到pUC18-5’SCF质粒，以pUC18-M-CSF为模板，用引物3，4经PCR法合成M-CSF基因片段，并将其克隆入pUC18质粒载体得到pUC18-3’M-CSF质粒，将SCF基因片段克隆到pUC18-3’M-CSF得pUC18-SCF/M-CSF。
1.2.将pUC18-SCF/M-CSF酶切得SCF/M-CSF基因片段，并插入AcNPV转移载体的Xba I与Kpn I位点之间，得到重组转移载体pVL1392-M-CSF/SCF。将pVL1392-SCF/M-CSF与AcNPV DNA共转染Sf9，经筛选并结合TF-1细胞测活选出不含野生型病毒的重组病毒AcNPV-SCF/M-CSF。
2.取倍增时间为21小时，活细胞占90％，密度为1×106细胞/mLSf9细胞，按感染复数(MOI)＝10感染重组病毒1小时后，倾去病毒液，换成无血清的TMN-FH培养基。27℃培养72小时后，用TF-1细胞测培养液重组蛋白SCF/M-CSF，得到SCF/M-CSF表达上清液的活性为1000units/mL。低速离心收集的细胞冻融后细胞抽提液中未分泌的重组蛋白活性仅为50units/6×106cells，说明重组蛋白分泌完全。表达量约为1mg/L。将40mL细胞培养上清液对1×PBS(pH7.4)透析12小时，以1mL/min流速上样至0.5mL的M-CSF单抗抗体柱亲和层析柱，层析柱预先用1×PBS平衡。上样后，先用1×PBS洗至基线，再用含0.8％Triton X-100的1×PBS洗涤，再次用1×PBS洗至基线后，用洗脱液(pH 8.0，50mM Tris-Cl，0.5Mol/L NaCl)以2mL/min的流速洗脱，收集洗脱峰15mL。洗脱液对蒸馏水透析并冻干，得到SCF/M-CSF双功能蛋白精品。测定蛋白含量和活性。结果显示，在SCF活性检测中rhSCF/M-CSF比活为1.0×106units/mg，比E.coli中表达的单体rhSCF(纯度＞98％)比活高6；在促人骨髓巨噬细胞集落形成活性(M-CSF活性)检测中rhSCF/M-CSF比活为7×106CFU/mg，比家蚕中表达的rhM-CSF纯品高出5倍。非还原条件下SDS-PAGE后经考马斯亮蓝染色检测，rhSCF/M-CSF呈分子量约84KD的一条带，而还原条件下SDS-PAGE后经考马斯亮蓝染色检测，rhSCF/M-CSF呈分子量约42KD的一条带。纯度约为90％。免疫印迹(Westernblot)分析结果与其一致(见附图5)。
人干细胞因子巨噬细胞集落刺激因子双功能蛋白基因序列表.txt
人干细胞因子巨噬细胞集落刺激因子双功能蛋白基因序列表
<110>南京大学
<120>人干细胞因子巨噬细胞集落刺激因子双功能蛋白及制备
<210>1
<211>1068
<212>DNA
<213>人(Homo sapiens)
<400>1
atgaagaaga cacaaacttg gattctcact tgcatttatc ttcagctgct cctatttaat 60
cctctcgtca aaactgaagg gatctgcagg aatcgtgtga ctaataatgt aaaagacgtc 120
actaaattgg tggcaaatct tccaaaagac tacatgataa ccctcaaata tgtccccggg 180
atggatgttt tgccaagtca ttgttggata agcgagatgg tagtacaatt gtcagacagc 240
ttgactgatc ttctggacaa gttttcaaat atttctgaag gcttgagtaa ttattccatc 300
atagacaaac ttgtgaatat agtggatgac cttgtggagt gcgtgaaaga aaactcatct 360
aaggatctaa aaaaatcatt caagagccca gaacccaggc tctttactcc tgaagaattc 420
tttagaattt ttaatagatc cattgatgcc ttcaaggact ttgtagtggc atctgaaact 480
agtgattgtg tggtttcttc aacattaagt cctgagaaag attccagagt cagtgtcaca 540
aaaccattta tgttaccccc tgttgcagcc ggaggaggag gatccggagg aggaggctcc 600
ggcggcagta tcaccgagga ggtgtcggag tactgtagcc acatgattgg gagtggacac 660
ctgcagtctc tgcagcggct gattgacagt cagatggaga cctcgtgcca aattacattt 720
gagtttgtag accaggaaca gttgaaagat ccagtgtgct accttaagaa ggcatttctc 780
ctggtacaag acataatgga ggacaccatg cgcttcagag ataacacccc caatgccatc 840
gccatcgtgc agctgcagga actctctttg aggctgaaga gctgcttcac caaggattat 900
gaagagcatg acaaggcctg cgtccgaact ttctatgaga cacctctcca gttgctggag 960
aaggtcaaga atgtctttaa tgaaacaaag aatctccttg acaaggactg gaatattttc 1020
agcaagaact gcaacaacag ctttgctgaa tgctccagcc aataatag
人干细胞因子巨噬细胞集落刺激因子双功能蛋白基因DNA序列
Met Lys Lys Thr Gln Thr Trp Ile Leu Thr Cys Ile Tyr Leu Gln Leu Leu Leu Phe Asn
5 10 15 20
Pro Leu Val Lys Thr Glu Gly Ile Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val
25 30 35 40
Thr Lys Leu Val Ala Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr Val Pro Gly
45 50 55 60
Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu Met Val Val Gln Leu Ser Asp Ser
65 70 75 80
Leu Thr Asp Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Cly Leu Ser Asn Tyr Ser Ile
85 90 95 100
Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser
105 110 115 120
Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe
125 130 135 140
Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser Glu Thr
145 150 155 160
Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr
165 170 175 180
Lys Pro Phe Met Leu Pro Pro Val Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
185 190 195 200
Gly Cly Ser Ile Thr Glu Glu VaL Ser Glu Tyr Cys Ser His Met Ile Gly Ser Gly His
205 210 215 220
Leu Gln Ser Leu Gln Leu Arg Ile Asp Ser Gln Met Glu Thr Ser Cys Gln Ile Thr Phe
225 230 235 240
Glu Phe Val Asp Gln Glu Gln Leu Lys Asp Pro Val Cys Tyr Leu Lys Lys Ala Phe Leu
245 250 255 260
Leu Val Gln Asp Ile Met Glu Asp Thr Met Arg Phe Arg Asp Asn Thr Pro Asn Ala Ile
265 270 275 280
Ala Ile Val Gln Leu Gln Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp Tyr
285 290 295 300
Glu Glu His Asp Lys Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gln Leu Leu Glu
305 310 315 320
Lys Val Lys Asn Val Phe Asn Glu Thr Lys Asn Leu Leu Asp Lys Asp Trp Asn Ile Phe
325 330 335 340
Ser Lys Asn Cys Asn Asn Ser Phe Ala Glu Cys Ser Ser G1n
345 350
人干细胞因子巨噬细胞集落刺激因子双功能蛋白氨基酸序列
Claims (3)
1.一种干细胞因子-巨噬细胞集落刺激因子双功能蛋白基因(rhSCF/M-CSF)，其核苷酸序列为：
atgaagaaga cacaaacttg gattctcact tgcatttatc ttcagctgct cctatttaat
cctctcgtca aaactgaagg gatctgcagg aatcgtgtga ctaataatgt aaaagacgtc
actaaattgg tggcaaatct tccaaaagac tacatgataa ccctcaaata tgtccccggg
atggatgttt tgccaagtca ttgttggata agcgagatgg tagtacaatt gtcagacagc
ttgactgatc ttctggacaa gttttcaaat atttctgaag gcttgagtaa ttattccatc
atagacaaac ttgtgaatat agtggatgac cttgtggagt gcgtgaaaga aaactcatct
aaggatctaa aaaaatcatt caagagccca gaacccaggc tctttactcc tgaagaattc
tttagaattt ttaatagatc cattgatgcc ttcaaggact ttgtagtggc atctgaaact
agtgattgtg tggtttcttc aacattaagt cctgagaaag attccagagt cagtgtcaca
aaaccattta tgttaccccc tgttgcagcc 8gaggaggag gatccggagg aggaggctcc
ggcggcagta tcaccgagga ggtgtcggag tactgtagcc acatgattgg gagtggacac
ctgcagtctc tgcagcggct gattgacagt cagatggaga cctcgtgcca aattacattt
gagtttgtag accaggaaca gttgaaagat ccagtgtgct accttaagaa ggcatttctc
ctggtacaag acataatgga ggacaccatg cgcttcagag ataacacccc caatgccatc
gccatcgtgc agctgcagga actctctttg aggctgaaga gctgcttcac caaggattat
gaagagcatg acaaggcctg cgtccgaact ttctatgaga cacctctcca gttgctggag
aaggtcaaga atgtctttaa tgaaacaaag aatctccttg acaaggactg gaatattttc
agcaagaact gcaacaacag ctttgctgaa tgctccagcc aataatag 。
2.一种构建含有权利要求1所述基因rhSCF/M-CSF的重组苜蓿夜蛾核型多角体病毒(AcNPV-rhSCF/M-CSF)的方法，其特征是将rhSCF/M-CSF基因克隆于杆状病毒转移载体pVL1392的BamH I和Sma I位点之间，得到重组转移载体pVL1392rhSCF/M-CSF，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pVL1392rhSCF/M-CSF载体DNA与野生型AcNPV DNA共转染Sf9细胞，经过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-rhSCF/M-CSF。
3.根据权利要求1所述rhSCF/M-CSF基因所编码蛋白的制备方法，其特征是用权利要求2所构建的重组病毒(AcNPV-rhSCF/M-CSF)转染贴壁的Sf9细胞，27℃在无血清培养基中表达72小时，收集细胞表达液，经M-CSF单抗抗体柱亲和层析分离纯化得到SCF/M-CSF双功能蛋白。
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