CN1053700C - 家蚕基因工程生产重组人巨噬细胞集落刺激因子 - Google Patents
家蚕基因工程生产重组人巨噬细胞集落刺激因子 Download PDFInfo
- Publication number
- CN1053700C CN1053700C CN94111226A CN94111226A CN1053700C CN 1053700 C CN1053700 C CN 1053700C CN 94111226 A CN94111226 A CN 94111226A CN 94111226 A CN94111226 A CN 94111226A CN 1053700 C CN1053700 C CN 1053700C
- Authority
- CN
- China
- Prior art keywords
- csf
- rhm
- gene
- silkworm
- hemolymph
- 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.)
- Expired - Fee Related
Links
- 241000255789 Bombyx mori Species 0.000 title claims abstract description 37
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 35
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 title claims description 4
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 title claims description 3
- 230000006798 recombination Effects 0.000 title claims description 3
- 238000005215 recombination Methods 0.000 title claims description 3
- 108020004705 Codon Proteins 0.000 claims abstract description 13
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 10
- 239000012634 fragment Substances 0.000 claims abstract description 7
- 108020005038 Terminator Codon Proteins 0.000 claims abstract description 5
- 210000000087 hemolymph Anatomy 0.000 claims description 24
- 241000700605 Viruses Species 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 13
- 238000000746 purification Methods 0.000 claims description 10
- 241000409811 Bombyx mori nucleopolyhedrovirus Species 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 239000003112 inhibitor Substances 0.000 claims description 4
- 230000003647 oxidation Effects 0.000 claims description 4
- 238000007254 oxidation reaction Methods 0.000 claims description 4
- 238000011218 seed culture Methods 0.000 claims description 4
- 230000003187 abdominal effect Effects 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 3
- 210000001015 abdomen Anatomy 0.000 claims description 2
- 230000008901 benefit Effects 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 239000002502 liposome Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000007669 thermal treatment Methods 0.000 claims description 2
- 102000007079 Peptide Fragments Human genes 0.000 claims 1
- 238000010276 construction Methods 0.000 claims 1
- 238000003780 insertion Methods 0.000 claims 1
- 230000037431 insertion Effects 0.000 claims 1
- 230000008521 reorganization Effects 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 239000013603 viral vector Substances 0.000 claims 1
- 230000003612 virological effect Effects 0.000 claims 1
- 150000001413 amino acids Chemical class 0.000 abstract description 9
- 229940079593 drug Drugs 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 4
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 4
- 229920001184 polypeptide Polymers 0.000 abstract description 2
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 2
- 230000009385 viral infection Effects 0.000 abstract description 2
- 210000004369 blood Anatomy 0.000 abstract 2
- 239000008280 blood Substances 0.000 abstract 2
- 210000002751 lymph Anatomy 0.000 abstract 2
- 241000233866 Fungi Species 0.000 abstract 1
- 206010028980 Neoplasm Diseases 0.000 abstract 1
- 210000000265 leukocyte Anatomy 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 11
- 238000000502 dialysis Methods 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 6
- 238000013016 damping Methods 0.000 description 6
- 239000012530 fluid Substances 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 239000012064 sodium phosphate buffer Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 230000002281 colonystimulating effect Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000011033 desalting Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 101100364969 Dictyostelium discoideum scai gene Proteins 0.000 description 2
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 2
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 208000003351 Melanosis Diseases 0.000 description 2
- 101100364971 Mus musculus Scai gene Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000004264 monolayer culture Methods 0.000 description 2
- 239000002516 radical scavenger Substances 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 239000012723 sample buffer Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 235000010576 Artemisia cina Nutrition 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 244000281762 Chenopodium ambrosioides Species 0.000 description 1
- 235000000509 Chenopodium ambrosioides Nutrition 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 206010017533 Fungal infection Diseases 0.000 description 1
- 102000012428 Hematopoietic Cell Growth Factors Human genes 0.000 description 1
- 108010022580 Hematopoietic Cell Growth Factors Proteins 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102000007330 LDL Lipoproteins Human genes 0.000 description 1
- 108010007622 LDL Lipoproteins Proteins 0.000 description 1
- 240000000249 Morus alba Species 0.000 description 1
- 235000008708 Morus alba Nutrition 0.000 description 1
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 1
- 241000256259 Noctuidae Species 0.000 description 1
- 108700022034 Opsonin Proteins Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102000001938 Plasminogen Activators Human genes 0.000 description 1
- 108010001014 Plasminogen Activators Proteins 0.000 description 1
- 102000018594 Tumour necrosis factor Human genes 0.000 description 1
- 108050007852 Tumour necrosis factor Proteins 0.000 description 1
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000001857 anti-mycotic effect Effects 0.000 description 1
- 230000000118 anti-neoplastic effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 239000002543 antimycotic Substances 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 235000021405 artificial diet Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000005059 dormancy Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000009585 enzyme analysis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 208000024386 fungal infectious disease Diseases 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002607 hemopoietic effect Effects 0.000 description 1
- 231100000652 hormesis Toxicity 0.000 description 1
- -1 il-1 Proteins 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 238000003819 low-pressure liquid chromatography Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229940127126 plasminogen activator Drugs 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于生物技术制药工业中的基因工程生产多肽药物的技术领域，其目的是利用我国资源十分丰富的家蚕为原料，从家蚕幼虫中的血淋巴中大量表达rhM-CSF，表达水平可达250毫克/升，rhM-CSF基因为含有完整的天然信号肽和基因第1-149位氨基酸密码子及终止密码的DNA片段。并从家蚕血淋巴中分离纯化出比活大于107单位/毫克，纯度98%的rhM-CSF精品，用于生产抗真菌，抗病毒感染，抗肿瘤及升白血球等用途的药物。
Description
本发明属于生物技术制药工业中的基因工程生产多肽药物技术领域。
人巨噬细胞集落刺激因子(hM-CSF)是一种重要的造血细胞生长因子。它对巨噬细胞的分化、增殖及成熟后的功能具有特异的刺激作用。同时它也是一种免疫效应剂，能促进和诱导效应细胞分泌干扰素，肿瘤坏死因子、白介素-1、粒细胞集落刺激因子(G-CSF)、前列腺素及纤溶酶原激活剂等，有效地增强巨噬细胞在炎症反应中的抗炎症作用。临床上主要应用于促进造血功能的恢复。如放疗化疗后的白细胞减少或骨髓移植造成的骨髓发育不良；增强机体抗感染、抗肿瘤的防御能力，对治疗某些致死性真菌病和某些病毒病特别有效；此外，还能选择性地降低血清中的低密度脂蛋白，用于抗动脉硬化。国外基因工程生产的rhM-CSF已进入III期临床。1985年美国Cetus公司首先分离到hM-CSF的短链cDNA基因(Science(1985)230：291)，并在COS细胞中表达出有活性的rhM-CSF，但表达水平仅为2×103单位/毫升，随后不同实验室相继从大肠杆菌、酵母或昆虫细胞中表达获得rhM-CSF。但表达水平一般在10毫克/升。1988年Cetus公司采用大规模高密度培养的草地贪夜蛾(S.F)细胞，使rhM-CSF在培养细胞中的表达量达到40毫克/升。(Biotechnology(1988)6：1406)。但成本偏高。不利于投入生产。
本发明的目的是利用我国资源来源十分广泛便宜的家蚕为原料，代替大肠杆菌或酵母发酵，或昆虫SF培养细胞，从家蚕幼虫的血淋巴中大量表达rhM-CSF，表达水平可达250毫克/升，并创造一条简便可行的工艺，从家蚕血淋巴中分离纯化出比活大于107单位/毫克，纯度98％的rhM-CSF精品。使整个工艺成本大为降低，成为一条更适于大规模生产可供临床应用的rhM-CSF生产工艺。
本发明的技术内容包括：1.家蚕基因工程表达载体----带hM-CSF(1-149aa)的重组家蚕核型多角体病毒BmNPV-M-CSF的构建；2.重组病毒对家蚕幼虫的感染及rhM-CSF在家蚕血淋巴中的高效表达和分泌；3.从家蚕血淋巴中分离纯化rhM-CSF的精品。
1.天然的hM-CSF具有长中短三种型式，它们的cDNA基因具有相同的信号肽(32个氨基酸)和N端第1-149位氨基酸密码子，据此，我们设计的rhM-CSF基因为含有完整的天然信号肽(32个氨基酸包括起始密码ATG)和基因第1-149位氨基酸密码子及终止密码的DNA片段。具体合成过程与重组病毒的构建过程如下：hM-CSF信号肽质粒含有hM-CSF信号肽32个氨基酸及基因第1-6位氨基酸密码子，经化学合成后克隆于pUC18的EcoRI、PstI位点中，由中科院上海生物工程研究中心合成提供，另以hM-CSF的长链cDNA基因片段为模板，用PCR法合成5’端带EcoRI位点3’端带终止密码及Hind III位点的含hM-CSF基因第3-149位密码子的基因片段，克隆于M13mp18之EcoRI、Hind III位点中。利用hM-CSF基因第6密码子中的ScaI位点，将信号肽与基因片段拼接成5’端有EcoRI位点，含信号肽32个密码子、基因第1-149位密码子及终止密码，3’端有Hind III位点的分泌型短基因，克隆于pUC18的EcoRI、Hind III位点中。经序列分析证明序列正确，ScaI位点恢复。上述质粒经扩增纯化后用EcoRI、Hind III双酶解切出带信号肽及N端1-149位密码子的rhM-CSF基因片段，用klenow酶补平后插入至家蚕转移质粒pBE284(S.Maeda提供)之补平的XbaI位点中限制酶分析筛选出重组方向正确----即多角体基因启动子与基因编码方向一致的表达转移质粒pBE-M-CSF。扩增纯化后与纯化的野生型家蚕核型多角体病毒Bm NPV(C6株)的基因组DNA混合，经脂质体(Lipofectin)共转染培养的单层家蚕细胞Bm N.27℃，培养7天后在琼脂板上用原位杂交筛选出阳性重组病毒空斑。再经4次以上96孔板有限稀释法纯化病毒。并结合测定集落刺激活性选出高水平表达rhM-CSF的不含野生型病毒的纯净重组病毒BmNPV-M-CSF。
2.重组病毒感染家蚕幼虫及表达rhM-CSF的家蚕血淋巴的收获：纯化后的重组病毒以感染指数(MOI)＝0.1空斑形成单位(PFU)/细胞感染单层培养的BmN细胞，27℃培养4-5天后，4000转/分离心弃细胞收集上清液作为毒种。毒种液滴度为107CFU/ml。用天然桑叶或人工饲料饲养的家蚕，5龄起眠后第1天，以10微升(105PFU)/虫毒种腹部注射或穿刺感染重组病毒。继续饲养4-5天后，剪腹足加一倍体积抗氧剂保护液(1mMDTT，10mM抗坏血酸)防止血淋巴黑化，离心8,000-10,000转/分，4℃，20分钟或低温压滤收集血淋巴。血淋巴可在-20℃存放。测定血淋巴的集落刺激活性可达2-3×106集落刺激单位(CFU)/ml。
3.从家蚕血淋巴中分离纯化rhM-CSF：上述方法收集的家蚕血淋巴，在70℃水浴保温15分钟，8,000转/分离心除去热处理沉淀的杂蛋白。上清液对pH6.0，0.05M NaAc缓冲液透析，上DFAE 52柱，柱子预先用样品缓冲液平衡，先用0.05M NaAc，0.05M NaCl，pH6.0缓冲液充分洗涤，然后用pH6.0，0.05M NaAc，0.2M NaCl缓冲液洗脱rhM-CSF。收集洗脱液对pH6.5，3mM磷酸钠缓冲液透析，上预先平衡的羟基磷灰石柱子。先用10mM，pH6.5磷酸钠缓冲液充分洗涤，再用50mM，pH6.5，磷酸钠缓冲液洗脱rhM-CSF。收集洗脱液透析浓缩后，于低压液相层析系统上经SP柱离子交换层析。用pH4.5，0.02M NaAc缓冲液盐浓度梯度0-0.5M NaCl进行梯度洗脱，梯度时间30分钟，流速1ml/分，rhM-CSF洗脱峰位于20-25分钟区段。收集该区段rhM-CSF洗脱液，透析脱盐后冻干保存。
本发明构建的重组家蚕核型多角体病毒BmNPV-M-CSF，其中带完整信号肽的hM-CSF基因(1-149)位于BmNPV的强大的多角体基因启动子控制下，多角体基因被hM-CSF基因所取代。在家蚕的培养细胞、幼虫或蛹体内，随着重组病毒的感染和复制，rhM-CSF能高效表达并分泌至细胞外。表达产物经免疫印迹法(western-blotting)检定，产生的rhM-CSF为糖基化程度不同分子量分别为18、20、22KD三种免疫特异性及生物活性相似的蛋白。精品经N末端分析N端十个氨基酸序列为Glu-Glu-Val-Ser-Glu-Tyr-Ser-His-Met，表明rhM-CSF的天然信号肽在表达分泌过程中已被正确地切除。
采用本发明构建的重组病毒按本发明所述方法感染家蚕幼虫和收集血淋巴，家蚕血淋巴中表达积累的rhM-CSF可达3×106CFU/ml，按精品比活1.2×107CFU/mg折算，表达量为250mg/L。实现了rhM-CSF的高效表达。
按本发明的技术路线，热处理可简便有效地灭活家蚕血淋巴中存在的大量酪氨酸氧化酶，防止了黑化并除去一部分易热变性的杂蛋白。按本发明提供的分离纯化方法可以从家蚕感染重组病毒后采集的血淋巴中获得纯化程度基本符合基因工程制药要求的rhM-CSF精品。rhM-CSF精品比活达1.2×107CFU/mg以上。在高压液相层析(HPLC)系统TSK2000SW分子筛柱上分析呈一个主峰，纯度＞98％。用SDS-PAGE银染法检测，无明显的杂蛋白电泳区带。rhM-CSF精品临床可用于生产抗真菌、抗病毒感染、抗肿瘤及升白血球等药物。
下面是本发明的实施例：1.rhM-CSF在不同家蚕幼虫中的表达。以5龄家蚕幼虫，品种苏5×苏6，每批5,000条为材料，以M.O.I＝105PFU/虫的剂量注射感染重组病毒，第4天剪去一对腹足，加一倍体积抗氧剂溶液(1mMDTT，10mM抗坏血酸)，离心8,000转/分，4℃，20分种，收集血淋巴。第5天家蚕幼虫开始大批死亡。将收集的血淋巴存放于-20℃。血淋巴测定集落刺激活性，1993年春蚕二批的平均表达水平为2×106CFU/ml血淋巴原液。93年秋蚕二批平均表达水平为3×106CFU/ml血淋巴原液，约合250微克/毫升。
单层培养的家蚕细胞BmN，在接近汇合时，以MOI＝10PFU/细胞剂量感染重组病毒BmNPV-M-CSF，感染后27℃继续培养，第6天培养液中rhM-CSF的表达量达10-14×104CFU/ml(3×106细胞)。
2.取家蚕血淋巴表达液100ml，加抗氧剂液稀释1倍后，70℃保温15分钟，8,000转/分离心20分钟，弃杂蛋白沉淀取上清液。对pH6.0，0.05M NaAc缓冲液透析后，上DEAE52柱(16mm×200mm)，柱子预先用样品缓冲液平衡。先用三倍床体积之pH6.0，0.05M，NaAc，洗涤，然后用pH6.0，0.05M NaAc。0.2M NaCl缓冲液洗出rhM-CSF，收集洗出液。柱子用pH6.0，0.05M NaAc，0.5M NaCl缓冲液洗除杂蛋白，再生平衡后再使用。rhM-CSF洗出液对pH6.5，3mM磷酸钠缓冲液透析后，上预先平衡的羟基磷灰石柱。先用三倍床体积pH6.5，10mm磷酸钠缓冲液洗涤，而后用pH6.5，50mM磷酸钠缓冲液洗出rhM-CSF组分。留存的杂蛋白可用0.5M磷酸缓冲液洗净后再生平衡待用。收集的rhM-CSF洗出液经透析脱盐浓缩后，于Waters 650E蛋白纯化系统上经SP离子交换柱层析(FPLC)。选择梯度缓冲液为pH4.5，0.02M NaAc，0-0.5M Nacl，梯度时间30分钟，流速1ml/分。收集20-25分钟区段rhM-CSF洗出峰。上述rhM-CSF洗出液经透析脱盐后，冷冻干燥分装成rhM-CSF精品。分别测定蛋白质含量和集落刺激活性，算出精品比活为1.2×107CFU/mg。HPLC测定纯度＞98％。整个分离纯化过程纯化倍数为200倍以上。活性得率大于30％。
按本发明所述方法每一万条秋蚕可得血淋巴表达液约6升，可提取rhM-CSF精品约500毫克。
Claims (2)
1.一种家蚕基因工程生产人巨噬细胞集落刺激因子(rhM-CSF)的方法，其所用的重组病毒载体的构建特征是，将化学法合成的hM-CSF信号肽片段与PCR法合成的人M-CSF基因片段，经体外重组拼接成含人M-CSF信号肽32个密码子和基因第1-149位密码子及终止密码、两端有适当的限制酶切位点的分泌型、rhM-CSF基因片段，通过平头连接插入家蚕转移质粒pBE284中补平的XbaI位点，选出重组方向正确即多角体基因启动子与rhM-CSF基因编码方向一致的表达转移质粒pBE-M-CSF，扩增纯化后再与纯净的野生型家蚕核型多角体病毒(BmN-PV)C6株的基因组DNA混合，经脂质体共转染培养的单层家蚕细胞BmN，再经多次病毒的纯化筛选出高水平表达rhM-CSF的纯净重组病毒BmNPV-M-CSF。
2.根据权利要求1所述的方法，从感染重组病毒的家蚕血淋巴中表达和分离纯化rhM-CSF的特征是，用105PFU/虫的量将重组病毒10微升毒种腹部注射或穿刺感染5天龄家蚕幼虫，剪腹足加抗氧剂保护后，离心收集血淋巴，然后经热处理→DEAE52柱→羟基磷灰石柱→SP-FPLC逐步纯化，获得比活大于1.2×107集落刺激单位(CFU)/毫克，纯度经HPLC分析达98％以上的rhM-CSF精品，活性得率为30％以上。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94111226A CN1053700C (zh) | 1994-01-24 | 1994-01-24 | 家蚕基因工程生产重组人巨噬细胞集落刺激因子 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN94111226A CN1053700C (zh) | 1994-01-24 | 1994-01-24 | 家蚕基因工程生产重组人巨噬细胞集落刺激因子 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1096541A CN1096541A (zh) | 1994-12-21 |
| CN1053700C true CN1053700C (zh) | 2000-06-21 |
Family
ID=5035117
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN94111226A Expired - Fee Related CN1053700C (zh) | 1994-01-24 | 1994-01-24 | 家蚕基因工程生产重组人巨噬细胞集落刺激因子 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1053700C (zh) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1064405C (zh) * | 1996-04-08 | 2001-04-11 | 中国农业科学院上海家畜寄生虫病研究所 | 利用家蚕系统生产重组日本血吸虫谷胱甘肽－转移酶 |
| CN1110563C (zh) * | 1997-11-05 | 2003-06-04 | 浙江农业大学 | 家蚕生产基因工程生白细胞药物的方法 |
| EP1222301B2 (en) * | 1999-10-19 | 2008-05-07 | Minos Biosystems Limited | Protein production system |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1062001A (zh) * | 1991-11-12 | 1992-06-17 | 北京大学 | 利用家蚕高效表达天花粉蛋白及其他有用蛋白的方法 |
-
1994
- 1994-01-24 CN CN94111226A patent/CN1053700C/zh not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1062001A (zh) * | 1991-11-12 | 1992-06-17 | 北京大学 | 利用家蚕高效表达天花粉蛋白及其他有用蛋白的方法 |
Non-Patent Citations (1)
| Title |
|---|
| 生物工程进展VOL.14,NO.3 1993.1.1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1096541A (zh) | 1994-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1317244C (en) | Production of biologically active forms of csf using a baculovirus (acnpv)-insect cell expression system | |
| DE69834179T2 (de) | Rekombinantes pockenvirus das das lösliche chemokine-bindende protein kodierende gen a41l nicht exprimieren kann. | |
| CN85109132A (zh) | 克隆编码具有人粒性巨噬细胞和嗜伊红细胞生长因子活性多肽的cDNA | |
| CA2186423C (en) | Protein which induces interferon-gamma production by immunocompetent cell | |
| CN1053700C (zh) | 家蚕基因工程生产重组人巨噬细胞集落刺激因子 | |
| KR930000273B1 (ko) | 약물의 유전공학적 제조방법 | |
| CN101629186B (zh) | 以家蚕作为宿主生产重组蝎毒素蛋白的方法 | |
| Dee et al. | Expression and secretion of a functional scorpion insecticidal toxin in cultured mouse cells | |
| CN1966709B (zh) | 一种重组新疆家蚕抗菌肽制备方法及其获得的新疆家蚕抗菌肽与应用 | |
| CN102492714A (zh) | 一种重组中华田园犬α干扰素的制备方法 | |
| CN106916803A (zh) | 一种双齿围沙蚕纤溶蛋白酶及其制备方法和用途 | |
| CN103613668A (zh) | 犬、猫长效融合干扰素及其制备方法与应用 | |
| CN1170927C (zh) | 用蚕表达人表皮生长因子生产药物的方法 | |
| EP0672684A1 (en) | Novel megakaryocyte colony stimulating factor and production thereof | |
| Zhang et al. | Can 29 kDa rhGM-CSF expressed by silkworm pupae bioreactor bring into effect as active cytokine through orally administration? | |
| CN101948518B (zh) | 用蜂王浆制备功能蛋白RJCPs的方法与应用 | |
| CN1321690A (zh) | 一种新型双功能水蛭素及其制备方法和应用 | |
| CN101481692A (zh) | 无血清制备重组人类凝血因子ix | |
| CN1110563C (zh) | 家蚕生产基因工程生白细胞药物的方法 | |
| JPH05227967A (ja) | カイコからの有用タンパク質の製造方法 | |
| CN1087746C (zh) | 用蚕表达重组人的促红细胞生成素制备药物的方法 | |
| US5681936A (en) | Method of purification of recombinant human interleukin-5 | |
| CN1277921C (zh) | 利用基因工程技术在家蚕体内生产重组人乳铁蛋白的方法 | |
| CN1064405C (zh) | 利用家蚕系统生产重组日本血吸虫谷胱甘肽－转移酶 | |
| CN1057564C (zh) | 能生产活性重组藻胆蛋白的微生物及其制备方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| C10 | Entry into substantive examination | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |