CN1322011C - 链亲和素/粒细胞-巨噬细胞集落刺激因子融合蛋白 - Google Patents
链亲和素/粒细胞-巨噬细胞集落刺激因子融合蛋白 Download PDFInfo
- Publication number
- CN1322011C CN1322011C CNB2004100525364A CN200410052536A CN1322011C CN 1322011 C CN1322011 C CN 1322011C CN B2004100525364 A CNB2004100525364 A CN B2004100525364A CN 200410052536 A CN200410052536 A CN 200410052536A CN 1322011 C CN1322011 C CN 1322011C
- Authority
- CN
- China
- Prior art keywords
- csf
- 6his
- fusion rotein
- cell
- fusion protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102000037865 fusion proteins Human genes 0.000 title abstract description 11
- 108020001507 fusion proteins Proteins 0.000 title abstract description 11
- 239000003242 anti bacterial agent Substances 0.000 title 1
- 229940088710 antibiotic agent Drugs 0.000 title 1
- 230000004936 stimulating effect Effects 0.000 title 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 claims abstract description 35
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims abstract description 35
- 230000004927 fusion Effects 0.000 claims abstract description 35
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 8
- 230000014509 gene expression Effects 0.000 claims abstract description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 6
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims abstract description 4
- 108010090804 Streptavidin Proteins 0.000 claims description 22
- 239000004471 Glycine Substances 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 230000006798 recombination Effects 0.000 claims 1
- 238000005215 recombination Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 14
- 239000013612 plasmid Substances 0.000 abstract description 10
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 abstract description 8
- 239000002299 complementary DNA Substances 0.000 abstract description 8
- 206010028980 Neoplasm Diseases 0.000 abstract description 7
- 230000001745 anti-biotin effect Effects 0.000 abstract description 7
- 238000000034 method Methods 0.000 abstract description 7
- 238000004153 renaturation Methods 0.000 abstract description 5
- 239000000427 antigen Substances 0.000 abstract description 3
- 102000036639 antigens Human genes 0.000 abstract description 3
- 108091007433 antigens Proteins 0.000 abstract description 3
- 238000012986 modification Methods 0.000 abstract description 3
- 230000004048 modification Effects 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 3
- 239000013613 expression plasmid Substances 0.000 abstract description 2
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 2
- 208000035473 Communicable disease Diseases 0.000 abstract 1
- 229960002685 biotin Drugs 0.000 abstract 1
- 235000020958 biotin Nutrition 0.000 abstract 1
- 239000011616 biotin Substances 0.000 abstract 1
- 230000009977 dual effect Effects 0.000 abstract 1
- 230000008105 immune reaction Effects 0.000 abstract 1
- 230000005847 immunogenicity Effects 0.000 abstract 1
- 238000002407 reforming Methods 0.000 abstract 1
- 210000004881 tumor cell Anatomy 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 9
- 238000013467 fragmentation Methods 0.000 description 7
- 238000006062 fragmentation reaction Methods 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 229930003756 Vitamin B7 Natural products 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 239000011735 vitamin B7 Substances 0.000 description 6
- 235000011912 vitamin B7 Nutrition 0.000 description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 5
- 210000001015 abdomen Anatomy 0.000 description 5
- 239000004202 carbamide Substances 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000001588 bifunctional effect Effects 0.000 description 4
- 229940030156 cell vaccine Drugs 0.000 description 4
- 230000009849 deactivation Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 210000003000 inclusion body Anatomy 0.000 description 4
- 230000000968 intestinal effect Effects 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000011740 C57BL/6 mouse Methods 0.000 description 3
- 241001655322 Streptomycetales Species 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 210000004493 neutrocyte Anatomy 0.000 description 3
- 239000002516 radical scavenger Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000012064 sodium phosphate buffer Substances 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- WWVANQJRLPIHNS-BKPPORCPSA-N 2-iminobiotin Chemical compound N1C(=N)N[C@H]2[C@H](CCCCC(=O)O)SC[C@H]21 WWVANQJRLPIHNS-BKPPORCPSA-N 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 108091081024 Start codon Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000006287 biotinylation Effects 0.000 description 2
- 238000007413 biotinylation Methods 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 210000003714 granulocyte Anatomy 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 235000014304 histidine Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- JJGWLCLUQNFDIS-GTSONSFRSA-M sodium;1-[6-[5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]hexanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCCCNC(=O)CCCC[C@H]1[C@H]2NC(=O)N[C@H]2CS1 JJGWLCLUQNFDIS-GTSONSFRSA-M 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000186983 Streptomyces avidinii Species 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 210000003995 blood forming stem cell Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000003677 hemocyte Anatomy 0.000 description 1
- 229940000351 hemocyte Drugs 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 150000002411 histidines Chemical class 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明为一种基因工程技术生产的链亲和素/粒细胞-巨噬细胞集落刺激因子融合蛋白(SA/GM-CSF融合蛋白)。通过对SA和GM-CSF的cDNA进行改造,如引入甘氨酸/丝氨酸丰富的17肽作为两者间链接接头等措施,获得了SA和GM-CSF融合基因,构建了SA/GM-CSF表达质粒,将其转化大肠杆菌并得到了原核高效表达;SA/GM-CSF融合蛋白经纯化复性后具有SA和GM-CSF的双重活性,即既对生物素有高亲合性,又具有相应的GM-CSF的生物活性。因此,这些SA/GM-CSF融合蛋白可用于已生物素化的细胞、组织或各类抗原的修饰以增强其免疫原性并调节机体的免疫反应,从而预防和治疗肿瘤和传染病。
Description
一、技术领域
本发明属基因工程领域,特别是涉及一种利用基因工程技术生产的融合蛋白)。
二、背景技术
GM-CSF有如下功能:维持和促进造血干细胞以及某些成熟的血细胞(如中性粒细胞、巨噬细胞、单核细胞和噬酸性粒细胞)的生长;激活中性粒细胞、巨噬细胞和噬酸性粒细胞对病原体及肿瘤的杀伤活性,增强树突状细胞的抗原提呈功能,从而提高机体对特异性肿瘤抗原和某些胞内病原体(如病毒、寄生虫、分枝杆菌)抗原的识别能力;并可作为中性粒细胞和巨噬细胞的趋化因子。因此,GM-CSF能使机体有效地清除肿瘤细胞、病毒、细菌或真菌感染的细胞,在疾病的预防和治疗中扮演了重要的角色。
链亲和素(SA)是由抗生物素链霉菌产生的非糖基化同源四聚体蛋白,故一个链亲和素蛋白可结合四个生物素。它能与生物素紧密非共价结合(Kd=10-15M),其结合力是抗原一抗体间作用力的一千至一百万倍。由于链亲和素可与生物素快速且几乎不可逆的强力结合,以及生物素较容易参入各种生物分子(如蛋白质,核酸和脂多糖)中,即生物素化,故链亲和素一生物素间的强力作用早已用于生物医学的许多领域多领域(Sano,T.and Cantor,C.R.(2000)Streptavidin-containing chimericproteins:design and production.Methods Enzymol.326,305-311.)。
三、发明内容
SA/GM-CSF融合蛋白的发明过程如下:为了获得含有SA/GM-CSF融合基因的质粒,我们对GM-CSF或SA cDNA进行了克隆和末端改造,如除去其终止密码子和引入甘氨酸/丝氨酸丰富的17肽作为接头,构建了GMCSF-L-SA-pET24和SA-L-GM-CSF-pET24重组质粒;将这两个重组质粒转化大肠杆菌BL21(DE3),获得相应的工程菌大肠杆菌GM-CSF-L-SA/pET24和SA-L-GM-CSF/pET24,并对GMCSF-L-SA和SA-L-GM-CSF融合基因实现了表达,表达效率为20-30%;融合蛋白在细菌内主要以包涵体形式存在;分离纯化蛋白并复性处理后,获得了双功能融合蛋白。
本发明SA-L-GM-CSF和GM-CSF-L-SA双功能融合蛋白,其中SA能强力结合生物素,而GM-CSF的生物学活性为ED50=0.1~0.5ng/ml。
这些SA/GM-CSF双功能融合蛋白可通过SA与生物素的强力结合将GM-CSF锚定在生物素化的肿瘤细胞表面,且能在r射线灭活肿瘤细胞表面稳定存在,并仍保持GM-CSF的活性。
动物试验表明,经SA/GM-CSF双功能融合蛋白修饰的肿瘤疫苗具有预防和治疗肿瘤的效用。
四、附图说明
图1是质粒GM-CSF-L-SA-pET24和SA-L-GM-CSF-pET24的示意图。
图2是GM-CSF-L-SA-6His和6His-SA-L-GM-CSF融合蛋白在大肠杆菌中的高效表达。
图3是GM-CSF-L-SA-6His和6His-SA-L-GM-CSF融合蛋白锚定在生物素化的B16.F10肿瘤细胞表面。
图4是锚定在生物素化且γ射线灭活肿瘤细胞表面的GM-CSF-L-SA-6His融合蛋白的稳定性测定。
图5是锚定在生物素化的肿瘤细胞表面GM-CSF-L-SA-6His融合蛋白的GM-CSF生物活性的测定。
图6是GM-CSF-L-SA-6His修饰的B6.F10肿瘤细胞致瘤性研究。
图7是GM-CSF-L-SA-6His修饰的B6.F10肿瘤细胞疫苗的预防肿瘤性研究。
图8是GM-CSF-L-SA-6His修饰的B6.F10细胞疫苗的治疗肿瘤性研究。
五、具体实施方式
通过下述实施例对本发明的技术特征作详细描述。
材料与方法:
一、细胞株、菌株与质粒:XS106细胞株(GM-CSF依赖细胞)和B16.F10(鼠黑色素瘤细胞株);菌株Streptomyces avidinii(抗生物素链霉菌,ATCC),DH5a和BL21(DE3);原核表达质粒pET24akanar,Novagen)。
二、主要生化试剂及材料:DNeasy组织试剂盒和质粒DNA的制备试剂盒(Qiagen),寡核苷酸的合成(Sigma),Trizol,SuperScriptII逆转录酶,Platinum Pfx DNA聚合酶和T4 DNA连接酶(Invitrogen);GM-CSF标准品(R&D Systems),琼脂糖和SDS-PAGE(Biorad);2-Iminobiotin(Sigma)和Ni-NTA(Qiagen)填料;Sulfo-NHS-LC-Biotin(Pierce)和抗GM-CSF单克隆抗体(BD Biosciences Pharmingen)。
三、DNA片段的连接、转化及转化子筛选,限制性内切酶分析,SDS-聚丙烯酰胺凝胶点泳等常规方法均参照文献(Sambrook J,et al.Molecular Cloning-A Laboratory Manual,Cold Spring HarborLaboratory Press,New York,2nd edition,1989)或厂家提供的产品说明书;DNA序列分析在UTSW at Dallas的DNA测序服务中心完成。
实施例1.成熟链亲和素(SA)cDNA的制备。
用DNeasy组织试剂盒从抗生物素链霉菌抽提出细菌的基因组DNA,然后用其作为模板,通过Platinum pfx DNA聚合酶进行PCR制备成熟链亲和素cDNA。
实施例2.成熟GM-CSF cDNA的制备。
用Trizol抽提起PHA-活化的外周血单个核细胞的总RNA,并以其作为模板,进行RT-PCR制备成熟GM-CSF cDNA。
实施例3.GM-CSF-L-SA-pET24和SA-L-GM-CSF-pET24重组质粒的构建。
用PCR制备GM-CSF-L-SA-6His和6His-SA-L-GM-CSF-pET24融合基因,并进行核苷酸序列测定;然后将其分别克隆于pET24a,获得GM-CSF-L-SA-pET24和SA-L-GM-CSF-pET24重组质粒(图1),并对其进行限制性内切酶分析鉴定。
实施例4.GM-CSF-L-SA-6His和6His-SA-L-GM-CSF融合基因在大肠杆菌中的高效表达。
将GM-CSF-L-SA-pET24和SA-L-GM-CSF-pET24重组质粒分别转化大肠杆菌BL21(DE3),获得相应的工程菌GM-CSF-L-SA/pET24和SA-L-GM-CSF/pET24;并对GM-CSF-L-SA-pET24和6His-SA-L-GM-CSF融合基因实现了表达,表达效率为20-30%,表达产物主要以包涵体的形式存在(图2)。
实施例5.GM-CSF-L-SA-6His6和His-SA-L-GM-CSF融合蛋白的制备。
1.制备包涵体。5克菌体悬于100ml 1xPBS中,冰浴中超声(电流270mA),30秒×10次(每次间隔30秒);裂解液于4℃离心10分钟(8000g),将沉淀悬浮于100ml 1xPBS(内含4mol/L尿素,0.5%Triton X-100,20mmol/L EDTA)中进行漂洗,随后离心(1000g×10分钟)收集沉淀;漂洗两次后,溶于100mmol/L磷酸钠缓冲液,8mol/L尿素(pH 8.0)中,15000g×15分钟得上清。
2.Ni-NTA柱层析:将制备的包涵体溶解液上样于Ni-NTA柱(2.6×5cm),用平衡液(100mmol/L磷酸钠缓冲液,8mol/L尿素,pH8.0)进行冲洗至样品A280恢复至基线;然后用洗脱液(100mmol/L磷酸钠缓冲液,8mol/L尿素,pH4.5)进行洗脱,收集融合蛋白质峰(SDS-PAGE鉴定)。
3.透析复性:将Ni-NTA柱层析纯化获得的GM-CSF-L-SA-6His和6His-SA-L-GM-CSF融合蛋白调至OD280=0.2,在大于20倍体积的透析液(100mmol/L NaHCO3,1.0mol/L尿素,lmmol/L还原型谷胱苷肽,0.2mmol/L氧化型谷胱苷肽,pH 9.0)中4℃透析复性12小时;然后在50mmol/L NaHCO3,500mmol/L NaCl,pH 11中继续透析6小时;离心除去不溶物,收集上清。
4.2-Iminobiotin亲和柱层析:用平衡液(50mmol/L NaHCO3,500mmol/L NaCl,pH 11)洗脱5个柱体积后(柱体积0.5×2cm),将收集的已复性融合蛋白质液上亲和层析柱,并用平衡液洗脱至样品A280恢复至基线;然后,用50mmol/L NaAc,pH4.0洗脱,分管收集各洗脱峰,并用10x平衡液(500mmol/LNaHCO3,5mol/L NaCl,pH 11)将收集的融合蛋白质液调至pH8.0,并过滤除菌分装,储存于-20℃备用。
实施例6.GM-CSF-L-SA-6His和6His-SA-L-GM-CSF融合蛋白中GM-CSF活性的测定。
利用XS106细胞株3H掺入法对GM-CSF的活性进行测定,测得的GM-CSF活性为ED50=0.1~0.5ng/ml。
实施例7.将GM-CSF-L-SA-6His和6His-SA-L-GM-CSF融合蛋白锚定在生物素化的B16.F10表面。
将107B16.F10细胞悬浮在1ml 1xPBS中,加入0.5mg Sulfo-NHS-LC-Biotin并混匀后,室温下作用30分钟;用1xPBS洗涤细胞3次后,每106B16.F10细胞加入200ng GM-CSF-L-SA-6His或6His-SA-L-GM-CSF融合蛋白,冰上作用30分钟;1xPBS洗涤细胞1次后,用抗GM-CSF单克隆抗体及荧光标记的二抗经流式细胞仪对锚定在生物素化的B16.F10表面上的GM-CSF-L-SA-6His或6His-SA-L-GM-CSF融合蛋白进行检测(图3),并对已锚定的GM-CSF-L-SA-6His融合蛋白的稳定性进行分析(图4)。
实施例8.对锚定在生物素化的B16.F10表面上的GM-CSF-L-SA-6His融合蛋白进行GM-CSF生物活性的测定。
首先将表面已锚定了GM-CSF-L-SA-6His或6His-SA-L-GM-CSF融合蛋白的106B16.F10经超声破碎,离心收集其不溶的膜性成分;然后将其悬浮于100μl完全培养基中,用XS106细胞株3H掺入法对其中的GM-CSF进行生物活性测定(图5)。
实施例9.GM-CSF-L-SA-6His修饰的B6.F10肿瘤细胞致瘤性研究。
分别将105GFP(绿色荧光蛋白)-L-SA-6His(阴性对照)和GM-CSF-L-SA-6His修饰的、活力好的B6.F10肿瘤细胞接种于C57BL/6小鼠左肋腹部的皮下,然后观察肿瘤的生长情况和小鼠在45天内的成活情况(图6)。
实施例10.GM-CSF-L-SA-6His修饰的B6.F10肿瘤细胞疫苗的预防肿瘤性研究。
分别将106GFP-L-SA-6His和GM-CSF-L-SA-6His修饰的、γ射线灭活(20000rad)的B6.F10肿瘤细胞接种于C57BL/6小鼠左肋腹部的皮内,14天后加强一次;7天后,将105未经任何处理的B6.F10肿瘤细胞接种于小鼠右肋腹部的皮下,然后观察肿瘤的生长情况和小鼠在40天内的成活情况(图7)。
实施例10.GM-CSF-L-SA-6His修饰的B6.F10细胞疫苗的治疗肿瘤性研究。
将105未经任何处理的B6.F10肿瘤细胞接种于小鼠右肋腹部的皮下;4,11和18天后,分别将106GFP-L-SA-6His或GM-CSF-L-SA-6His修饰的、γ射线灭活(20000rad)的B6.F10肿瘤细胞接种于C57BL/6小鼠左肋腹部的皮内,然后观察肿瘤的生长情况和小鼠在40天内的成活情况(图8)。
链亲和素/粒细胞-巨噬细胞集落刺激因子融合蛋白
<110>高基民
<120>链亲和素/粒细胞-巨噬细胞集落刺激因子融合蛋白
<160>2
<210>1
<211>930bp
<212>DNA
<213>人工DNA
<220>
<221>GMCSF-L-SA-6His
<222>(1-381bp)
<223>该DNA片段为编码小鼠成熟GM-CSF的cDNA,其中5’端含有ATG,作为大肠杆菌翻译GMCSF-L-SA-6His融合蛋白的起始密码,3’端含有一个EcoRI的酶切位点。
<220>
<221>GMCSF-L-SA-6His
<222>(382-426bp)
<223>该DNA片段(45bp)编码富含甘氨酸和丝氨酸的链接肽(L),此15肽非常灵活,有助于融合蛋白中各单元蛋白质分子的独立折叠,从而保存各自的生物活性,最终提高融合蛋白的双重活性。此外,其3’端含有一个BamHI的酶切位点。
<220>
<221>GMCSF-L-SA-6His
<222>(427-930bp)
<223>DNA片段(504bp)编码链霉菌成熟的全长抗生物素(SA),其3’端含有编码一个XhoI位点,6个组氨酸和终止码的序列(904-930bp)。
<400>1
1 ATGGCACCCACCCGCTCACCCATCACTGTCACCCGGCCTTGGAAGCATGTAGAGGCCATC 60
MetAlaProThrArgSerProIleThrValThrArgProTrpLysHisValGluAlaIle
61 AAAGAAGCCCTGAACCTCCTGGATGACATGCCTGTCACATTGAATGAAGAGGTAGAAGTC 120
LysGluAlaLeuAsnLeuLeuAspAspMetProValThrLeuAsnGluGluValGluVal
121 GTCTCTAACGAGTTCTCCTTCAAGAAGCTAACATGTGTGCAGACCCGCCTGAAGATATTC 180
ValSerAsnGluPheSerPheLysLysLeuThrCysValGlnThrArgLeuLysIlePhe
181 GAGCAGGGTCTACGGGGCAATTTCACCAAACTCAAGGGCGCCTTGAACATGACAGCCAGC 240
GluGlnGlyLeuArgGlyAsnPheThrLysLeuLysGlyAlaLeuAsnMetThrAlaSer
241 TACTACCAGACATACTGCCCCCCAACTCCGGAAACGGACTGTGAAACACAAGTTACCACC 300
TyrTyrGlnThrTyrCysProProThrProGluThrAspCysGluThrGlnValThrThr
301 TATGCGGATTTCATAGACAGCCTTAAAACCTTTCTGACTGATATCCCCTTTGAATGCAAA 360
TyrAlaAspPheIleAspSerLeuLysThrPheLeuThrAspIleProPheGluCysLys
361 AAACCAGTCCAAAAAGAATTCTCGAGCGGGGGCAGCGGGGGCGGCAGCAGCGGCGGGGGC 420
LysProValGlnLysGluPheSerSerGlyGlySerGlyGlyGlyGlySerGlyGlyGly
421 GGATCCGACCCCTCCAAGGACTCGAAGGCCCAGGTCTCGGCCGCCGAGGCCGGCATCACC 480
GlySerAspProSerLysAspSerLysAlaGlnValSerAlaAlaGluAlaGlylleThr
481 GGCACCTGGTACAACCAGCTCGGCTCGACCTTCATCGTGACCGCGGGCGCCGACGGCGCC 540
GlyThrTrpTyrAsnGlnLeuGlySerThrPheIleValThrAlaGlyAlaAspGlyAla
541 CTGACCGGAACTACGAGTCGGCCGTCGGCAACGCCGAGAGCCGCTACGTCCTGACCGGT 600
LeuThrGlyThrTyrGluSerAlaValGlyAsnAlaGluSerArgTyrValLeuThrGly
601 CGTTACGACAGCGCCCCGGCCACCGACGGCAGCGGCACCGCCCTCGGTTGGACGGTGGCC 660
ArgTyrAspSerAlaProAlaThrAspGlySerGlyThrAlaLeuGlyTrpThrValAla
661 TGGAAGAATAACTACCGCAACGCCCACTCCGCGACCACGTGGAGCGGCCAGTACGTCGGC 720
TrpLysAsnAsnTyrArgAsnAlaHisSerAlaThrThrTrpSerGlyGlnTyrValGly
721 GGCGCCGAGGCGAGGATCAACACCCAGTGGCTGCTGCCTCCGGCACCACCGAGGCCAAC 780
GlyAlaGluAlaArgIleAsnThrGlnTrpLeuLeuThrSerGlyThrThrGluAlaAsn
781 GCCTGGAAGTCCACGCTGGTCGGCCACGACACCTTCACCAAGGTGAAGCCGTCCGCCGCC 840
AlaTrpLysSerThrLeuValGlyHisAspThrPheThrLysValLysProSerAlaAla
841 TCCATCGACGCGGCGAAGAAGGCCGGCGTCAACAACGGCAACCCGCTCGACGCCGTTCAG 900
SerIleAspAlaAlaLysLysAlaGlyValAsnAsnGlyAsnProLeuAspAlaValGln
901 CAGCTCGAGCACCACCACCACCACCACTGA 930
GlnLeuGluHisHisHisHisHisHis *
<210>2
<211>891bp
<212>DNA
<213>人工DNA
<220>
<221>6His-SA-L-GMCSF
<222>(1-459bp)
<223>该DNA片段为编码链霉菌抗生物素核心部分(与成熟的全长抗生物素相比,在N-端缺失了13个氨基酸)的cDNA,其中5’端含有ATG起始密码子和6个组氨酸密码子。
<220>
<221>6His-SA-L-GMCSF
<222>(460-513bp)
<223>该DNA片段编码L链接肽(460-504bp),并且其3’端含有BamHI和EcoRI的酶切位点。
<220>
<221>6His-SA-L-GMCSF
<222>(514-891bp)
<223>该DNA片段编码小鼠成熟GM-CSF,其5’端含有ATG,从而编码一额外的甲硫氨酸。
<400>2
1 ATGCATCATCACCATCACCATGAGGCCGGCATCACCGGCACCTGGTACAACCAGCTCGGC 60
MetHisHisHisHisHisHisGluAlaGlyIleThrGlyThrTrpTyrAsnGlnLeuGly
61 TCGACCTTCATCGTGACCGCGGGCGCCGACGGCGCCCTGACCGGAACCTACGAGTCGGCC 120
SerThrPheIleValThrAlaGlyAlaAspGlyAlaLeuThrGlyThrTyrGluSerAla
121 GTCGGCAACGCCGAGAGCCGCTACGTCCTGACCGGTCGTTACGACAGCGCCCCGGCCACC 180
ValGlyAsnAlaGluSerArgTyrValLeuThrGlyArgTyrAspSerAlaProAlaThr
181 GACGGCAGCGGCACCGCCCTCGGTTGGACGGTGGCCTGGAAGAATAACTACCGCAACGCC 240
AspGlySerGlyThrAlaLeuGlyTrpThrValAlaTrpLysAsnAsnTyrArgAsnAla
241 CACTCCGCGACCACGTGGAGCGGCCAGTACGTCGGCGGCGCCGAGGCGAGGATCAACACC 300
HisSerAlaThrThrTrpSerGlyGlnTyrvalGlyGlyAlaGluAlaArgIleAsnThr
301 CAGTGGCTGCTGACCTCCGGCACCACCGAGGCCAACGCCTGGAAGTCCACGCTGGTCGGC 360
GlnTrpLeuLeuThrSerGlyThrThrGluAlaAsnAlaTrpLysSerThrLeuValGly
361 CACGACACCTTCACCAAGGTGAAGCCGTCCGCCGCCTCCATCGACGCGGCGAAGAAGGCC 420
HisAspThrPheThrLysValLysProSerAlaAlaSerIleAspAlaAlaLysLysAla
421 GGCGTCAACAACGGCAACCCGCTCGACGCCGTTCAGCAGTCGAGCGGGGGCAGCGGGGGC 480
GlyValAsnAsnGlyAsnProLeuAspAlaValGlnGlnSerSerGlyGlySerGlyGly
481 GGAGGCAGCGGCGGGGGCGGATCCGCCGAATTCATGGCACCCACCCGCTCACCCATCACT 540
GlyGlySerGlyGlyGlyGlySerAlaGluPheMetAlaProThrArgSerProIleThr
541 GTCACCCGGCCTTGGAAGCATGTAGAGGCCATCAAAGAAGCCCTGAACCTCCTGGATGAC 600
ValThrArgProTrpLysHisValGluAlaIleLysGluAlaLeuAsnLLeuLeuAspAsp
601 ATGCCTGTCACATTGAATGAAGAGGAAGAAGTCGTCTCTAACGAGTTCTCCTTCAAGAAG 660
MetProvalThrLeuAsnGluGluvalGluvalvalSerAsnGluPheSerPheLysLys
661 CTAACATGTGTGCAGACCCGCCTGAAGATATTCGAGCAGGGTCTACGGGGCAATTTCACC 720
LeuThrCysValGlnThrArgLeuLysIlePheGluGlnGlyLeuArgGlyAsnPheThr
721 AAACTCAAGGGCGCCTTGAACATGACAGCCAGCTACTACCAGACATACTGCCCCCCAACT 780
LysLeuLysGlyAlaLeuAsnMetThrAlaSerTyrTyrGlnThrTyrCysProProThr
781 CCGGAAACGGACTGTGAAACACAAGTTACCACCTATGCGGATTTCATAGACAGCCTTAAA 840
ProGluThrAspCysGluThrGlnValThrThrTyrAlaAspPheIleAspSerLeuLys
841 ACCTTTCTGACTGATATCCCCTTTGAATGCAAAAACCAGTCCAAAAATGA 891
ThrPheLeuThrAspIleProPheGluCysLysLysProValGlnLys *
Claims (3)
1.一种融合蛋白,其特征在于将链亲和素的基因与粒细胞-巨噬细胞集落刺激因子基因经过基因重组、表达而产生的融合蛋白,即链亲和素/粒细胞-巨噬细胞集落刺激因子融合蛋白。
2.根据权利要求1所述的SA/GM-CSF融合蛋白,其特征在于SA序列位于融合蛋白的N端或C端。
3.根据权利要求1或2所述的SA/GM-CSF融合蛋白,其特征在于SA与GM-CSF之间的接头L是甘氨酸/丝氨酸丰富的17肽EFSSGGSGGGGSGGGGS,接头的核苷酸序列为GAA TTC TCGAGC GGG GGC AGC GGG GGC GGA GGC AGC GGC GGG GGC GGATCC。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB2004100525364A CN1322011C (zh) | 2004-12-07 | 2004-12-07 | 链亲和素/粒细胞-巨噬细胞集落刺激因子融合蛋白 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB2004100525364A CN1322011C (zh) | 2004-12-07 | 2004-12-07 | 链亲和素/粒细胞-巨噬细胞集落刺激因子融合蛋白 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1651464A CN1651464A (zh) | 2005-08-10 |
CN1322011C true CN1322011C (zh) | 2007-06-20 |
Family
ID=34868740
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNB2004100525364A Active CN1322011C (zh) | 2004-12-07 | 2004-12-07 | 链亲和素/粒细胞-巨噬细胞集落刺激因子融合蛋白 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN1322011C (zh) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101121752B (zh) * | 2007-06-29 | 2011-02-09 | 上海市计划生育科学研究所 | 以链亲和素为载体的短肽融合表达热诱导重组质粒及其制备方法 |
CN101372513B (zh) * | 2008-10-20 | 2012-05-02 | 中国人民解放军第二军医大学 | Sg融合蛋白 |
CN101863984B (zh) * | 2010-05-27 | 2013-06-05 | 温州医学院 | GM-CSF/TNF-α膜表面修饰的前列腺癌治疗性疫苗 |
CN107759695A (zh) * | 2016-08-18 | 2018-03-06 | 温州医科大学 | 纯化同时复性大规模制备SA‑hGM‑CSF双功能融合蛋白 |
CN107773537A (zh) * | 2016-08-18 | 2018-03-09 | 温州医科大学 | 大规模制备可长期稳定保存的SA‑hGM‑CSF双功能融合蛋白冻干制剂的方法 |
CN116510022B (zh) * | 2022-11-23 | 2023-12-05 | 武汉滨会生物科技股份有限公司 | 一种用于抗肿瘤的组合物、重组质粒组合物及其应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1221754A (zh) * | 1997-12-30 | 1999-07-07 | 中国人民解放军军事医学科学院放射医学研究所殷小刚 | 血小板生成素/粒、巨噬细胞集落刺激因子融合蛋白及用途 |
CN1225368A (zh) * | 1999-02-05 | 1999-08-11 | 中国科学院上海生物化学研究所 | 白细胞介素-2/粒细胞-巨噬细胞集落剌激因子融合蛋白 |
-
2004
- 2004-12-07 CN CNB2004100525364A patent/CN1322011C/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1221754A (zh) * | 1997-12-30 | 1999-07-07 | 中国人民解放军军事医学科学院放射医学研究所殷小刚 | 血小板生成素/粒、巨噬细胞集落刺激因子融合蛋白及用途 |
CN1225368A (zh) * | 1999-02-05 | 1999-08-11 | 中国科学院上海生物化学研究所 | 白细胞介素-2/粒细胞-巨噬细胞集落剌激因子融合蛋白 |
Also Published As
Publication number | Publication date |
---|---|
CN1651464A (zh) | 2005-08-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101319247B (zh) | Il-2选择性激动剂和拮抗剂 | |
WO2001051510A2 (en) | G-csf conjugates | |
JP6174710B2 (ja) | ケモカイン−サイトカイン融合タンパク質およびその利用 | |
ZA200400159B (en) | G-CSF Conjugates. | |
KR20040111357A (ko) | 미생물 감염증을 치료하기 위한 조성물 및 방법 | |
CA2451353A1 (en) | Methods and pharmaceutical compositions for immune deception, particularly useful in the treatment of cancer | |
CN1322011C (zh) | 链亲和素/粒细胞-巨噬细胞集落刺激因子融合蛋白 | |
JP2005523681A5 (zh) | ||
CN108864292B (zh) | 一种干扰素重组融合蛋白及其应用 | |
JP2001517422A (ja) | 単離したおよび組換えによる抗菌ペプチドトロンボシジン−1(tc−1)およびトロンボシジン−2(tc−2)またはその変異体 | |
JPH0615477B2 (ja) | 感染防禦剤 | |
WO2013075553A1 (zh) | 癌靶向超抗原融合蛋白、其制备方法和用途 | |
CN101148478A (zh) | 链亲和素-肿瘤坏死因子α融合蛋白 | |
CN115947868A (zh) | Cxcl-bpi融合蛋白及其用途 | |
CN104292341A (zh) | 一种凝血八因子融合蛋白及其制备方法和用途 | |
CN114805603A (zh) | 一种ctb/ltb多肽与vp8的融合蛋白及轮状病毒疫苗 | |
CN1163513C (zh) | 一种高效生产BPI-Fc重组蛋白的方法 | |
JPWO2012020842A1 (ja) | 腫瘍ワクチン効果を有する複合体及びその用途 | |
CN101531720A (zh) | 链亲和素/绿色荧光融合蛋白 | |
CN101942020A (zh) | 链亲和素/白细胞介素7融合蛋白 | |
WO2012088739A1 (zh) | 结核分歧杆菌抗原的融合蛋白的制备及其应用 | |
CN108048475B (zh) | 缢蛏i型溶菌酶-2基因、编码蛋白及重组缢蛏i型溶菌酶-2基因工程菌的构建方法 | |
CN107281487B (zh) | 一种兽用疫苗佐剂 | |
CN100513569C (zh) | 重组幽门螺杆菌热休克蛋白60 | |
CN101148477A (zh) | 链亲和素-白细胞介素2融合蛋白 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
ASS | Succession or assignment of patent right |
Owner name: WENZHOU MEDICAL COLLEGE Free format text: FORMER OWNER: GAO JIMIN Effective date: 20090410 |
|
C41 | Transfer of patent application or patent right or utility model | ||
TR01 | Transfer of patent right |
Effective date of registration: 20090410 Address after: Dasan Zhejiang city of Wenzhou Province Higher Education Park Patentee after: Wenzhou Medical College Address before: Laboratory of biological medicine No. 13 Gang Road Guangzhou city Guangdong province Chigang pomegranate Patentee before: Gao Jimin |