CN110256571A - 重组人白蛋白-血红蛋白β亚基融合蛋白 - Google Patents
重组人白蛋白-血红蛋白β亚基融合蛋白 Download PDFInfo
- Publication number
- CN110256571A CN110256571A CN201811450566.9A CN201811450566A CN110256571A CN 110256571 A CN110256571 A CN 110256571A CN 201811450566 A CN201811450566 A CN 201811450566A CN 110256571 A CN110256571 A CN 110256571A
- Authority
- CN
- China
- Prior art keywords
- fusion protein
- hemoglobin
- recombinant human
- human albumin
- chain
- 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.)
- Pending
Links
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 47
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 47
- 102000001554 Hemoglobins Human genes 0.000 claims abstract description 22
- 108010054147 Hemoglobins Proteins 0.000 claims abstract description 22
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 19
- 229920001184 polypeptide Polymers 0.000 claims abstract description 12
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 12
- 102000009027 Albumins Human genes 0.000 claims abstract description 11
- 108010088751 Albumins Proteins 0.000 claims abstract description 11
- 210000002966 serum Anatomy 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 10
- 241000588724 Escherichia coli Species 0.000 claims abstract description 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 7
- 210000004369 blood Anatomy 0.000 claims abstract description 7
- 239000008280 blood Substances 0.000 claims abstract description 7
- 238000013467 fragmentation Methods 0.000 claims abstract description 7
- 238000006062 fragmentation reaction Methods 0.000 claims abstract description 7
- 239000013604 expression vector Substances 0.000 claims abstract description 4
- 230000009465 prokaryotic expression Effects 0.000 claims abstract description 4
- 239000000126 substance Substances 0.000 claims abstract description 3
- 238000006257 total synthesis reaction Methods 0.000 claims abstract description 3
- 230000014509 gene expression Effects 0.000 claims description 9
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 5
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 5
- 238000003257 protein preparation method Methods 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 238000010353 genetic engineering Methods 0.000 claims 1
- 210000002429 large intestine Anatomy 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 10
- 239000001301 oxygen Substances 0.000 abstract description 10
- 229910052760 oxygen Inorganic materials 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 5
- 239000000470 constituent Substances 0.000 abstract description 3
- 238000012408 PCR amplification Methods 0.000 abstract 1
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 abstract 1
- 238000003786 synthesis reaction Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 6
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 6
- 230000001939 inductive effect Effects 0.000 description 5
- 210000003000 inclusion body Anatomy 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003633 blood substitute Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000002525 ultrasonication Methods 0.000 description 2
- 206010067484 Adverse reaction Diseases 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
- 241001465754 Metazoa Species 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 108700020412 bacteria Inh Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
Abstract
重组人白蛋白‑血红蛋白β亚基融合蛋白，涉及一种构成血红蛋氧载体的组成成分，属于生物医药技术领域，是由人血清白蛋白和人血红蛋白β亚基组成的融合蛋白。人血清白蛋白和人血红蛋白β亚基之间由连接肽连接而成；其制备方法：通过PCR扩增的方法或用化学全合成方法合成所述的编码融合蛋白多肽链氨基酸序列的脱氧核苷酸序列（DNA片段）；将该DNA片段克隆至原核表达载体中；将表达载体转化至大肠杆菌细胞中表达并对所表达的融合蛋白进行分离和纯化；所述的融合蛋白亚基有较大的分子量大和携带很多负电荷这两个因素在构建血红蛋白氧载体时(α2β2四聚体)，能降低血红蛋白氧载体对人体的副作用。
Description
技术领域
本发明涉及一种构成血红蛋白氧载体的组成成分：重组人白蛋白-血红蛋白β亚基融合蛋白，属于生物制药技术领域。
背景技术
输血属于临床基本治疗手段。由于医疗技术的进步导致医疗用血增加等原因，许多城市出现了血荒现象。因此，具有携氧功能的血液代用品研究和开发具有重大的经济价值和社会效益。血红蛋白氧载体是目前最有希望成功的血液代用品。血红蛋白氧载体是利用人或动物的血红蛋白做原料经修饰后能替代红细胞输送氧气的一类产品。人的血红蛋白是由两个α亚基和两个β亚基组成的四聚体。游离的血红蛋白四聚体可以降解成αβ二聚体以及单体，有许多副作用。为了降低副作用，目前多采用化学修饰的办法，如交联，聚合和偶联等，加大血红蛋白分子量。化学修饰的产物此外，用生物技术，如基因重组的方法，也可以达到这个目的。
发明内容
本发明的目的在于克服现有技术中的不足之处，提供一种构成血红蛋氧载体的组成成分：重组人白蛋白-血红蛋白β亚基融合蛋白。
为了实现本发明的目的，我们采用如下的技术方案予以实施：
重组人白蛋白-血红蛋白β亚基融合蛋白，其特征在于：
所述的融合蛋白是由人血清白蛋白和人血红蛋白β亚基组成的；
所述的融合蛋白是由人血清白蛋白和人血红蛋白由连接肽连接而成；
所述的融合蛋白的N-末端由人血清白蛋白和C-末端的人血红蛋白β结构域组成；
所述的融合蛋白多肽链氨基酸序列：
MDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL GSGGGGSKL VHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH
进一步，所述的连接肽的改变相应改变融合蛋白多肽链氨基酸序列和长度，但所表达的融合蛋白结构和功能不变。
重组人白蛋白-血红蛋白β亚基融合蛋白的制备方法，其特征在于如下几点：
一、用RT-PCR扩增的方法获得所述的融合蛋白多肽链DNA片段或用化学全合成方法合成所述的融合蛋白DNA片段；
二、将步骤一所述的能编码融合蛋白多肽链氨基酸序列的DNA片段克隆至原核表达载体中；
三、将步骤二所述的表达载体转化至大肠杆菌表达体系的细胞中；
四、在大肠杆菌细胞中诱导表达重组人白蛋白-血红蛋白β亚基融合蛋白；
五、对步骤四所表达的重组人白蛋白-血红蛋白β亚基融合蛋白进行分离和纯化。
有益效果
本发明所述的重组人白蛋白-血红蛋白β亚基融合蛋白的制备方法，用于生产血红蛋白氧载体的组成成分，目的是解决血红蛋白四聚体解离后的亚基分子量变小引起不良反应的难题。
本发明利用基因重组技术，将人血清白蛋白和人血红蛋白通过连接肽构建成融合蛋白。融合蛋白的人血清白蛋白部分（66kD）约是血红蛋白部分（16kD）的分子量的四倍。同时，人血清白蛋白在生理条件下有大量的负电荷，因此，所生产出的修饰后的血红蛋白会也会携带这些负电荷。分子量大和携带很多负电荷这两个因素可以降低对人体的副作用。
附图说明
图1为IPTG诱导人白蛋白-血红蛋白β亚基融合蛋白的表达效果。
为没有诱导的样品，2和3为IPTG诱导的样品，4和5分别为为用IPTG诱导的样品的沉淀和上清液，M为标准分子量蛋白。
图2为纯化后的人白蛋白-血红蛋白β亚基融合蛋白。
其中1为纯化的人白蛋白-血红蛋白β亚基融合蛋白，M为标准分子量蛋白。
具体实施方式
实施例：
一、用化学全合成的方法合成编码融合蛋白多肽链的DNA序列：
ATGGACGCCCACAAATCGGAAGTCGCTCATCGCTTTAAGGACCTGGGTGAAGAAAACTTTAAGGCTCTGGTGC TGATCGCATTCGCACAGTATCTGCAGCAATGCCCGTTTGAAGATCATGTCAAACTGGTGAACGAAGTTACCGAATTT GCAAAAACGTGCGTCGCGGATGAATCAGCCGAAAATTGTGACAAGTCGCTGCACACCCTGTTTGGCGACAAACTGTG CACCGTGGCCACGCTGCGTGAAACGTACGGTGAAATGGCAGATTGCTGTGCTAAACAGGAACCGGAACGCAACGAAT GCTTTCTGCAACATAAAGATGACAACCCGAATCTGCCGCGTCTGGTGCGCCCGGAAGTTGATGTCATGTGTACCGCG TTTCATGACAATGAAGAAACGTTCCTGAAAAAGTATCTGTACGAAATTGCCCGTCGCCACCCGTATTTTTACGCACC GGAACTGCTGTTTTTCGCTAAGCGTTATAAAGCGGCCTTCACCGAATGCTGTCAGGCAGCTGATAAGGCGGCCTGCC TGCTGCCGAAACTGGATGAACTGCGTGACGAAGGCAAAGCAAGCTCTGCTAAGCAGCGCCTGAAATGTGCGTCTCTG CAAAAGTTTGGTGAACGTGCCTTCAAAGCCTGGGCAGTTGCTCGTCTGTCTCAGCGCTTTCCGAAAGCGGAATTTGC CGAAGTGAGCAAGCTGGTTACCGATCTGACGAAAGTGCATACCGAATGCTGTCACGGCGATCTGCTGGAATGCGCGG ATGACCGCGCAGACCTGGCTAAGTACATCTGTGAAAACCAGGATTCGATCAGTTCCAAACTGAAGGAATGCTGTGAA AAGCCGCTGCTGGAAAAAAGCCATTGCATTGCAGAAGTTGAAAACGATGAAATGCCGGCTGACCTGCCGAGCCTGGC AGCTGATTTTGTTGAATCTAAGGACGTCTGTAAAAATTATGCGGAAGCCAAAGACGTGTTTCTGGGCATGTTCCTGT ATGAATACGCCCGTCGCCATCCGGATTATAGCGTGGTTCTGCTGCTGCGTCTGGCAAAGACCTACGAAACCACGCTG GAAAAATGCTGTGCAGCAGCAGACCCGCACGAATGCTACGCAAAGGTCTTTGATGAATTTAAACCGCTGGTTGAAGA ACCGCAGAACCTGATCAAACAAAATTGTGAACTGTTTGAACAGCTGGGTGAATATAAATTCCAAAACGCGCTGCTGG TTCGCTACACCAAAAAGGTCCCGCAGGTGAGTACCCCGACCCTGGTGGAAGTGAGCCGTAATCTGGGCAAAGTGGGT TCAAAGTGCTGTAAACATCCGGAAGCGAAACGCATGCCGTGCGCCGAAGATTATCTGTCCGTCGTGCTGAATCAACT GTGTGTGCTGCACGAAAAGACCCCGGTCTCAGATCGTGTGACCAAATGCTGTACGGAATCGCTGGTTAACCGTCGCC CGTGCTTTAGCGCGCTGGAAGTGGATGAAACGTACGTTCCGAAAGAATTTAATGCGGAAACCTTTACGTTCCATGCC GATATTTGTACCCTGAGCGAAAAAGAACGCCAGATCAAAAAGCAAACGGCGCTGGTTGAACTGGTCAAACACAAGCC GAAAGCAACCAAGGAACAGCTGAAAGCTGTTATGGATGACTTTGCTGCGTTCGTCGAAAAGTGCTGTAAAGCCGATG ACAAAGAAACGTGTTTCGCTGAAGAAGGTAAAAAGCTGGTTGCGGCATCACAGGCGGCACTGGGTCTG GGATCCGGC GGTGGCGGTAGCAAGCTTGTTCATCTGACCCCGGAAGAAAAGTCGGCGGTTACGGCTCTGTGGGGCAAGGTTAATGTTGACGAAGTGGGCGGCGAAGCTCTGGGCCGTCTGCTGGTTGTGTATCCGTGGACCCAGCGCTTTTTCGAATCTTTTGGTGATCTGTCTACGCCGGACGCAGTCATGGGCAACCCGAAAGTGAAGGCTCATGGCAAAAAGGTTCTGGGTGCGTTTAGTGATGGCCTGGCCCACCTGGACAATCTGAAAGGTACCTTCGCGACGCTGAGCGAACTGCATTGCGATAAGCTGCACGTTGACCCGGAAAACTTCCGTCTGCTGGGTAATGTCCTGGTGTGTGTTCTGGCACATCACTTTGGCAAAGAATTTACCCCGCCGGTCCAAGCTGCGTATCAGAAGGTTGTTGCGGGTGTGGCGAATGCCCTGGCTCATAAGTATCACTAA
所述的DNA序列可以编码以下氨基酸序列的蛋白质：氨基酸1- 586是人血清白蛋白序列（下划线）；587-595是连接肽(斜体)；最后596-742是人血红蛋白β亚基：
MDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYF YAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKA EFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLP SLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPL VEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVL NQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVK HKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL GSGGGGSKLVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH
二、将上述DNA序列克隆至原核表达载体中。
三、将所述的表达载体转化至大肠杆菌细胞中；
四、通过IPTG诱导表达的方法鉴定大肠杆菌细胞中含有所述的融合蛋白多肽链的细胞:
A、 IPTG诱导后，收集大肠杆菌细胞进行诱导表达分析。SDS-PAGE电泳分析法证明，IPTG诱导表达重组人白蛋白-血红蛋白β亚基融合蛋白；
B、纯化：将1.6 L诱导表达的培养菌液低温离心6000g， 10min，菌体沉淀重悬与20 mllysis buffer (20 mMTris-HCl containing 1 mM PMSF and bacteria proteaseinhibitor cocktail, pH 8.0)，超声破碎(功率 400W，工作4sec，间歇 8sec，共 20min)；
2.将超声破碎的细胞裂解液 4℃，10000g离心20min，收集沉淀；
3.使用包涵体洗涤液 (20mM Tris，1mM EDTA，2M 尿素，1M NaCl，1％Triton X-100，pH8.0)洗涤包涵体3次；
4.用溶解缓冲液(20 mMTris，5mM DTT，8M尿素，pH8.0)，按一定比例溶解包涵体，4℃，放置过夜；室温，15000rpm离心 15 min；
5. 将包涵体溶解液滴加到 20mM Tris，pH8.0 的缓冲液，逐步成倍梯度稀释，缓慢搅拌；至尿素浓度达到0.5 M时，将蛋白溶液装入透析袋于4℃在20 mM PBS， pH7.4中透析过夜。
6. 进行 10% SDS-PAGE 分析。
纯化出来的重组人白蛋白-血红蛋白β亚基融合蛋白纯度可达80%以上。
Claims (4)
1.重组人白蛋白-血红蛋白β亚基融合蛋白，其特征在于：所述的重组人白蛋白-血红蛋白β亚基融合蛋白是由人血清白蛋白和人血红蛋白β亚基组成的融合蛋白；所述的融合蛋白是由人血清白蛋白和人血红蛋白之间由连接肽连接而成；所述的融合蛋白的N-末端由人血清白蛋白结构域和C-末端的人血红蛋白β结构域组成；所述的融合蛋白多肽链氨基酸序列：
MDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSKLVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH。
2.重组人白蛋白-血红蛋白β亚基融合蛋白的制备方法，其特征在于：a、用基因工程技术或用化学全合成方法获得编码所述的融合蛋白多肽链的DNA片段；b、将编码步骤一所述的融合蛋白多肽链的DNA片段克隆至原核表达载体中；c、将步骤二所述的表达载体转化至大肠杆菌中；d、所述的融合蛋白在大肠杆菌表达。
3.根据权利要求1所述的重组人白蛋白-血红蛋白β亚基融合蛋白，其特征在于: 所述的重组人白蛋白-血红蛋白β亚基融合蛋白多肽链序列及结构和功能；所述的连接肽序列的改变相应改变融合蛋白多肽链氨基酸序列和长度，但整个融合蛋白结构和功能不变。
4.根据权利要求2所述的重组人白蛋白-血红蛋白β亚基融合蛋白的制备方法，其特征在于：用基因工程技术构建的表达重组人白蛋白-血红蛋白β亚基融合蛋白的载体在大肠杆菌中表达生产。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811450566.9A CN110256571A (zh) | 2018-11-30 | 2018-11-30 | 重组人白蛋白-血红蛋白β亚基融合蛋白 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811450566.9A CN110256571A (zh) | 2018-11-30 | 2018-11-30 | 重组人白蛋白-血红蛋白β亚基融合蛋白 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110256571A true CN110256571A (zh) | 2019-09-20 |
Family
ID=67911886
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811450566.9A Pending CN110256571A (zh) | 2018-11-30 | 2018-11-30 | 重组人白蛋白-血红蛋白β亚基融合蛋白 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110256571A (zh) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020155995A1 (en) * | 2000-10-16 | 2002-10-24 | Proteopharma Aps | Function of a haptoglobin-haemoglobin receptor and the uses thereof |
| US20050244931A1 (en) * | 2001-04-12 | 2005-11-03 | Human Genome Sciences, Inc. | Albumin fusion proteins |
| US20070154993A1 (en) * | 2004-09-15 | 2007-07-05 | Olson John S | Enhancing Recombinant Hemoglobin Production By Co-Expression With Alpha Hemoglobin Stabilizing Protein |
| CN101402688A (zh) * | 2008-11-18 | 2009-04-08 | 中国人民解放军军事医学科学院生物工程研究所 | 一种融合蛋白及其编码基因与应用 |
| CN101429245A (zh) * | 2007-11-06 | 2009-05-13 | 尼普洛株式会社 | 利用毕赤酵母制备重组人血红蛋白 |
| CN102711795A (zh) * | 2009-12-08 | 2012-10-03 | 泰华制药工业有限公司 | 用于治疗可卡因滥用的BChE白蛋白融合体 |
| CN104163867A (zh) * | 2013-05-20 | 2014-11-26 | 王子元 | 一种多负电荷血红蛋白α亚基 |
| US20160168228A1 (en) * | 2014-12-10 | 2016-06-16 | S-Aima Holding Company, Llc | Generation of hemoglobin-based oxygen carriers using elastin-like polypeptides |
| CN106715468A (zh) * | 2014-07-02 | 2017-05-24 | 环喜有限公司 | 用于癌症靶向治疗的包含基于重组血红蛋白蛋白质或亚基的治疗剂的药物组合物 |
-
2018
- 2018-11-30 CN CN201811450566.9A patent/CN110256571A/zh active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020155995A1 (en) * | 2000-10-16 | 2002-10-24 | Proteopharma Aps | Function of a haptoglobin-haemoglobin receptor and the uses thereof |
| US20050244931A1 (en) * | 2001-04-12 | 2005-11-03 | Human Genome Sciences, Inc. | Albumin fusion proteins |
| US20070154993A1 (en) * | 2004-09-15 | 2007-07-05 | Olson John S | Enhancing Recombinant Hemoglobin Production By Co-Expression With Alpha Hemoglobin Stabilizing Protein |
| CN101429245A (zh) * | 2007-11-06 | 2009-05-13 | 尼普洛株式会社 | 利用毕赤酵母制备重组人血红蛋白 |
| CN101402688A (zh) * | 2008-11-18 | 2009-04-08 | 中国人民解放军军事医学科学院生物工程研究所 | 一种融合蛋白及其编码基因与应用 |
| CN102711795A (zh) * | 2009-12-08 | 2012-10-03 | 泰华制药工业有限公司 | 用于治疗可卡因滥用的BChE白蛋白融合体 |
| CN104163867A (zh) * | 2013-05-20 | 2014-11-26 | 王子元 | 一种多负电荷血红蛋白α亚基 |
| CN106715468A (zh) * | 2014-07-02 | 2017-05-24 | 环喜有限公司 | 用于癌症靶向治疗的包含基于重组血红蛋白蛋白质或亚基的治疗剂的药物组合物 |
| US20160168228A1 (en) * | 2014-12-10 | 2016-06-16 | S-Aima Holding Company, Llc | Generation of hemoglobin-based oxygen carriers using elastin-like polypeptides |
Non-Patent Citations (1)
| Title |
|---|
| 纪昌伟: "重组血红蛋白原核双表达载体的构建与表达", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Peña et al. | Granulysin, a new human cytolytic granule-associated protein with possible involvement in cell-mediated cytotoxicity | |
| CN110845603A (zh) | 人胶原蛋白17型多肽、其生产方法和用途 | |
| JPS60115528A (ja) | ヒトインタ―ロイキン―２蛋白質を含有する抗腫瘍用または免疫機能低下疾患治療用組成物 | |
| CN110267674A (zh) | 包含瘦素的融合蛋白及其生产和使用方法 | |
| Wang et al. | A new cytolysin from the sea anemone, Heteractis magnifica: isolation, cDNA cloning and functional expression | |
| CN110903383A (zh) | 一种重组人源i型胶原蛋白、编码基因、工程菌及其应用 | |
| CN101698682A (zh) | 一种基于抗菌肽的双功能融合蛋白及其制备方法和用途 | |
| CN105622761B (zh) | 一种凝集素蛋白寡聚复合体的构建方法及其应用 | |
| CN112980865A (zh) | 一种重组类人胶原蛋白工程菌的构建方法 | |
| CN112500495A (zh) | 一种elp-ⅲ型胶原蛋白的纯化方法及应用 | |
| CN110964096B (zh) | 一种重组人源c反应蛋白的制备方法 | |
| Cai et al. | Expression, purification and antibacterial activity of NK-lysin mature peptides from the channel catfish (Ictalurus punctatus) | |
| Guo et al. | Production of soluble bioactive mouse leukemia inhibitory factor from Escherichia coli using MBP tag | |
| CN103641901B (zh) | 一种抗菌肽 | |
| CN108003233B (zh) | 长牡蛎含DM9结构域蛋白CgDM9CP-2、制备方法及应用 | |
| CN110256571A (zh) | 重组人白蛋白-血红蛋白β亚基融合蛋白 | |
| CN110066330B (zh) | 一种仿刺参葡聚糖结合蛋白及其制备方法和应用 | |
| Chen et al. | Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli | |
| CN116925240A (zh) | 一种重组胶原蛋白及其表达方法和应用 | |
| Peng et al. | Preparation and characterization of monomers to tetramers of a collagen-like domain from Streptococcus pyogenes | |
| CN107226846A (zh) | 新型透明质酸结合肽及透皮吸收与皮下靶向释放制剂 | |
| Fröderberg et al. | Complementation of bacterial SecE by a chloroplastic homologue | |
| CN109776653B (zh) | 一种人血清白蛋白黏附肽及其应用 | |
| CN104263747B (zh) | 一种长效蛋白质药物表达复性修饰的双标签载体及方法 | |
| JPH02273193A (ja) | 組換えインターロイキン―２融合蛋白質類 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190920 |