CN1232039A - 一种基因工程双特异抗体及其应用 - Google Patents
一种基因工程双特异抗体及其应用 Download PDFInfo
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Abstract
本发明公开了一种由抗红细胞和抗乙肝表面抗原组成的基因工程双特异抗体及其应用,是利用基因工程重组技术通过缩短单链抗体连接肽的方法将抗红细胞和抗乙肝表面抗原改造成杂合抗体基因,组装于同一表达载体中,构成抗红细胞和抗乙肝表面抗原双特异抗体;可做为乙肝表面抗原检测试剂,检测血中乙肝表面抗原。由抗红细胞和抗乙肝表面抗原组成的双特异抗体,由于基因工程产品,可直接从细菌培养上清中获取,降低了产品的制备成本;在检测过程中不需要使用特殊设备,不受时间和地理位置的影响,具有操作简便、快捷的优点。
Description
本发明公开了一种基因工程双特异抗体及其应用，特别涉及一种由抗红细胞和抗乙肝表面抗原组成的基因工程双特异抗体及其应用。
目前，公知的双特异抗体主要有1993年英国剑桥大学的Winter教授通过缩短单链抗体连接肽的方法制备的由抗肿瘤细胞和抗免疫细胞表面标志CD3组成的双特异抗体，称为双特异Diabody，其在临床上主要作用于抗肿瘤治疗。由抗红细胞和抗乙肝表面抗原组成的基因工程双特异抗体及其应用尚无记载与应用。
本发明的目的在于提供一种由抗红细胞和抗乙肝表面抗原组成的基因工程双特异抗体及其应用。
本发明的目的是这样实现的：
A．双特异抗体基因：
本发明利用基因工程重组技术通过缩短单链抗体连接肽的方法将抗红细胞和抗乙肝表面抗原改造成杂合抗体基因，组装于PCOMb3H表达载体中(载体PCOMb3H的参考文献为CarlosF.BarbasⅢ andJurgen Wagner Synthetic Human Antibodies:Selecting andEvolving Functional Proteins METHODS:A Companion toMethods in Enzymology 1995；8:94-103)，构成抗红细胞和抗乙肝表面抗原双特异抗体；双特异抗体基因序列为
GCCGAGCTCA CCCAGTCTCC ATCCTTCCTG TCTGCATCTA
TAGGAGACAG CGTCACCATC ACTTGCCGGA CCAGTCAGGA
CATTGATAAT TATTTAGCCT GGTATCAGCA AAAACCAGGA
AAAGCCCCTA AGCTCCTGGT CTATTCTGCA TCCGCCTTGC
AAGGTGGGGT CCCATCAAGG TTCAGCGGCA GTGGATCTAG
GACAGATTTC ACTCTCACAA TCAACAGCCT GCAGCCTGAA
GATTCTGCAA CTTATTACTG TCAACAGCTT GGAAGTTACC
CACTCACTTT CGGCGGAGGG ACCAAGGTGG AGATCAAACG
ATCTAGAGGT GGTGGATCCG AGGTGCAGCT GCTCGAGCTT
GCGGAGTCTG GGGGAGGCTT AGTGAAGCCT GGAGGGTCCC
TGAAACTCTC CTGTACAACC TCTGGATTCG CTTTCAGTAG
TTATGACATG TCTTGGGTTC GCCAGACTCC GGAGAAGAGG
CTGGAGTGGG TCGCATACAT TAGTAGTGCT GGTGGTAGTA
TTTATTATTC AGACACTGTG AAGGACCGCT TCACCATCTC
CAGAGACAAT GTCAAAAACA CCCTGTACCT GCAAATGAAC
AGTCTGAATT CTGAGGACAC AGCCATATAT TACTGTACAA
GACACGGGCT CGGCTCCTAC TATGTTATGG ACTACTGGGG
TCAAGGAACC TCAGTCACCG TCTCCTCAAC TAGTGAACAA
AAACTCATCT CAGAAGAAGA TCTGGCTAGC CATCACCATC
ACCATCACTA AGTCGACAGG AGGAATTTAA AATGAAAAAG
ACAGCTATCG CGATTGCAGT GGCACTGGCT GGTTTCGCTA
CCGTGGCCCA GGCGGCCGAG CTCCAGATGA CCCAGTCTCC
AGCCTCCCTA TCTGCATCTG TGGGAGAAAC TGTCACCATC
ACATGTCGAG CAAGTGAGAA TATTTACAGT TATTTAGCAT
GGTATCAGCA GAAACAGGGA AAATCTCCTC AGCTCCTGGT
CTATAATGCA AAAACCTTAG CAGAAGGTGT GCCATCAAGG
TTCAGTGGCA GTGGATCAGG CACACAGTTT TCTCTGAAGA
TCAACAGCCT GCAGCCTGAA GATTTTGGGA GTTATTACTG
TCAACATCAT TATGGTACTC GTCTCACGTT CGGTGCTGGG
ACCAAGCTGG AGCTGAAACG GTCTAGAGGT GGTGGATCCG
AGGTGCAGGT GCTCGAGGAG TCTGGGGGAG GCTTGGTACA
GGCTGGGGGG TCCCTGAGAC TCTCCTGTGC AATCTCTGGA
TTCACCTTTA GTGACTATGC CGTGAGTTGG GTCCGCCAGG
CTCCTGGGAA GGGGCTGGAG TGGGTCTCAA CCATCAGTGG
CAGTGCTGGA AACACATACT ACGCAGACTC CGTGAAGGGC
CGATTGATCA TCTCCAGAGA CACTTCCAGG AACATGCTAT
TTCTGCAAAT GAACAGCCTG AGAGCCGAGG ACACGGCCGT
ATATTACTGT GCGAAAGTCA AGATGGTTCG GGGTGGCTAC
TGGTTCTACG GTATGGACGT CTGGGGCCAA GGGACCGCGG
TCAGAGTCTC CTCA；
B．应用：
将双特异抗体基因转化大肠杆菌并分泌表达，离心收集细菌培养上清，上清中即含有抗红细胞和抗乙肝表面抗原双特异抗体，可做为乙肝表面抗原检测试剂，检测血中乙肝表面抗原。
采用抗红细胞和抗乙肝表面抗原双特异基因制备而成的双特异抗体，由于基因工程产品，可直接从细菌培养上清中获取，降低了产品的制备成本；在检测过程中不需要使用特殊设备，只需采集未梢血，在室温放置1～2分钟便可完成检测，不受时间和地理位置的影响，具有操作简便、快捷的优点。
使用双特异抗体检测血中乙肝表面抗原的方法是在玻片上滴加双特异抗体试剂50ml，采集未梢血(指血或耳垂血)20ml与试剂混匀，在室温放置1～2分钟，将玻片摇动观察凝集情况，判定结果，血球凝集者为乙肝表面抗原阳性。
Claims (2)
1．一种基因工程双特异抗体及其应用，利用基因工程重组技术通过缩短单链抗体连接肽的方法将抗红细胞和抗乙肝表面抗原改造成杂合抗体基因，组装于PCOMb3H表达载体中，构成抗红细胞和抗乙肝表面抗原双特异抗体基因；其特征在于：双特异抗体基因序列为
GCCGAGCTCA CCCAGTCTCC ATCCTTCCTG TCTGCATCTA
TAGGAGACAG CGTCACCATC ACTTGCCGGA CCAGTCAGGA
CATTGATAAT TATTTAGCCT GGTATCAGCA AAAACCAGGA
AAAGCCCCTA AGCTCCTGGT CTATTCTGCA TCCGCCTTGC
AAGGTGGGGT CCCATCAAGG TTCAGCGGCA GTGGATCTAG
GACAGATTTC ACTCTCACAA TCAACAGCCT GCAGCCTGAA
GATTCTGCAA CTTATTACTG TCAACAGCTT GGAAGTTACC
CACTCACTTT CGGCGGAGGG ACCAAGGTGG AGATCAAACG
ATCTAGAGGT GGTGGATCCG AGGTGCAGCT GCTCGAGCTT
GCGGAGTCTG GGGGAGGCTT AGTGAAGCCT GGAGGGTCCC
TGAAACTCTC CTGTACAACC TCTGGATTCG CTTTCAGTAG
TTATGACATG TCTTGGGTTC GCCAGACTCC GGAGAAGAGG
CTGGAGTGGG TCGCATACAT TAGTAGTGCT GGTGGTAGTA
TTTATTATTC AGACACTGTG AAGGACCGCT TCACCATCTC
CAGAGACAAT GTCAAAAACA CCCTGTACCT GCAAATGAAC
AGTCTGAATT CTGAGGACAC AGCCATATAT TACTGTACAA
GACACGGGCT CGGCTCCTAC TATGTTATGG ACTACTGGGG
TCAAGGAACC TCAGTCACCG TCTCCTCAAC TAGTGAACAA
AAACTCATCT CAGAAGAAGA TCTGGCTAGC CATCACCATC
ACCATCACTA AGTCGACAGG AGGAATTTAA AATGAAAAAG
ACAGCTATCG CGATTGCAGT GGCACTGGCT GGTTTCGCTA
CCGTGGCCCA GGCGGCCGAG CTCCAGATGA CCCAGTCTCC
AGCCTCCCTA TCTGCATCTG TGGGAGAAAC TGTCACCATC
ACATGTCGAG CAAGTGAGAA TATTTACAGT TATTTAGCAT
GGTATCAGCA GAAACAGGGA AAATCTCCTC AGCTCCTGGT
CTATAATGCA AAAACCTTAG CAGAAGGTGT GCCATCAAGG
TTCAGTGGCA GTGGATCAGG CACACAGTTT TCTCTGAAGA
TCAACAGCCT GCAGCCTGAA GATTTTGGGA GTTATTACTG
TCAACATCAT TATGGTACTC GTCTCACGTT CGGTGCTGGG
ACCAAGCTGG AGCTGAAACG GTCTAGAGGT GGTGGATCCG
AGGTGCAGCT GCTCGAGGAG TCTGGGGGAG GCTTGGTACA
GCCTGGGGGG TCCCTGAGAC TCTCCTGTGC AATCTCTGGA
TTCACCTTTA GTGACTATGC CGTGAGTTGG GTCCGCCAGG
CTCCTGGGAA GGGGCTGGAG TGGGTCTCAA CCATCAGTGG
CAGTGCTGGA AACACATACT ACGCAGACTC CGTGAAGGGC
CGATTGATCA TCTCCAGAGA CACTTCCAGG AACATGCTAT
TTCTGCAAAT GAACAGCCTG AGAGCCGAGG ACACGGCCGT
ATATTACTGT GCGAAAGTCA AGATGGTTCG GGGTGGCTAC
TGGTTCTACG GTATGGACGT CTGGGGCCAA GGGACCGCGG
TCAGAGTCTC CTCA；
2．一种基因工程双特异抗体及其应用，其特征在于：双特异抗体可做为乙肝表面抗原检测试剂，检测血中乙肝表面抗原。
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101668771A (zh) * | 2007-03-12 | 2010-03-10 | 艾斯巴技术股份公司 | 单链抗体的基于序列的工程改造和最优化 |
| CN103068846A (zh) * | 2010-08-24 | 2013-04-24 | 弗·哈夫曼-拉罗切有限公司 | 包含二硫键稳定性Fv片段的双特异性抗体 |
| US9688758B2 (en) | 2012-02-10 | 2017-06-27 | Genentech, Inc. | Single-chain antibodies and other heteromultimers |
| US9890204B2 (en) | 2009-04-07 | 2018-02-13 | Hoffmann-La Roche Inc. | Trivalent, bispecific antibodies |
| US9994646B2 (en) | 2009-09-16 | 2018-06-12 | Genentech, Inc. | Coiled coil and/or tether containing protein complexes and uses thereof |
| US10106612B2 (en) | 2012-06-27 | 2018-10-23 | Hoffmann-La Roche Inc. | Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof |
| US10106600B2 (en) | 2010-03-26 | 2018-10-23 | Roche Glycart Ag | Bispecific antibodies |
| US10323099B2 (en) | 2013-10-11 | 2019-06-18 | Hoffmann-La Roche Inc. | Multispecific domain exchanged common variable light chain antibodies |
| US10611825B2 (en) | 2011-02-28 | 2020-04-07 | Hoffmann La-Roche Inc. | Monovalent antigen binding proteins |
| US10633457B2 (en) | 2014-12-03 | 2020-04-28 | Hoffmann-La Roche Inc. | Multispecific antibodies |
| US10640555B2 (en) | 2009-06-16 | 2020-05-05 | Hoffmann-La Roche Inc. | Bispecific antigen binding proteins |
| US10793621B2 (en) | 2011-02-28 | 2020-10-06 | Hoffmann-La Roche Inc. | Nucleic acid encoding dual Fc antigen binding proteins |
| US10927163B2 (en) | 2007-12-21 | 2021-02-23 | Hoffmann-La Roche, Inc. | Bivalent, bispecific antibodies |
| US11421022B2 (en) | 2012-06-27 | 2022-08-23 | Hoffmann-La Roche Inc. | Method for making antibody Fc-region conjugates comprising at least one binding entity that specifically binds to a target and uses thereof |
| US11618790B2 (en) | 2010-12-23 | 2023-04-04 | Hoffmann-La Roche Inc. | Polypeptide-polynucleotide-complex and its use in targeted effector moiety delivery |
-
1999
- 1999-04-02 CN CN 99103517 patent/CN1232039A/zh active Pending
Cited By (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101668771B (zh) * | 2007-03-12 | 2013-08-21 | 艾斯巴技术,爱尔康生物医药研究装置有限责任公司 | 单链抗体的基于序列的工程改造和最优化 |
| CN101668771A (zh) * | 2007-03-12 | 2010-03-10 | 艾斯巴技术股份公司 | 单链抗体的基于序列的工程改造和最优化 |
| US10927163B2 (en) | 2007-12-21 | 2021-02-23 | Hoffmann-La Roche, Inc. | Bivalent, bispecific antibodies |
| US11993642B2 (en) | 2009-04-07 | 2024-05-28 | Hoffmann-La Roche Inc. | Trivalent, bispecific antibodies |
| US9890204B2 (en) | 2009-04-07 | 2018-02-13 | Hoffmann-La Roche Inc. | Trivalent, bispecific antibodies |
| US10640555B2 (en) | 2009-06-16 | 2020-05-05 | Hoffmann-La Roche Inc. | Bispecific antigen binding proteins |
| US11673945B2 (en) | 2009-06-16 | 2023-06-13 | Hoffmann-La Roche Inc. | Bispecific antigen binding proteins |
| US9994646B2 (en) | 2009-09-16 | 2018-06-12 | Genentech, Inc. | Coiled coil and/or tether containing protein complexes and uses thereof |
| US10106600B2 (en) | 2010-03-26 | 2018-10-23 | Roche Glycart Ag | Bispecific antibodies |
| CN103068846A (zh) * | 2010-08-24 | 2013-04-24 | 弗·哈夫曼-拉罗切有限公司 | 包含二硫键稳定性Fv片段的双特异性抗体 |
| US9879095B2 (en) | 2010-08-24 | 2018-01-30 | Hoffman-La Roche Inc. | Bispecific antibodies comprising a disulfide stabilized-Fv fragment |
| US11618790B2 (en) | 2010-12-23 | 2023-04-04 | Hoffmann-La Roche Inc. | Polypeptide-polynucleotide-complex and its use in targeted effector moiety delivery |
| US10611825B2 (en) | 2011-02-28 | 2020-04-07 | Hoffmann La-Roche Inc. | Monovalent antigen binding proteins |
| US10793621B2 (en) | 2011-02-28 | 2020-10-06 | Hoffmann-La Roche Inc. | Nucleic acid encoding dual Fc antigen binding proteins |
| US9688758B2 (en) | 2012-02-10 | 2017-06-27 | Genentech, Inc. | Single-chain antibodies and other heteromultimers |
| US11407836B2 (en) | 2012-06-27 | 2022-08-09 | Hoffmann-La Roche Inc. | Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof |
| US11421022B2 (en) | 2012-06-27 | 2022-08-23 | Hoffmann-La Roche Inc. | Method for making antibody Fc-region conjugates comprising at least one binding entity that specifically binds to a target and uses thereof |
| US10106612B2 (en) | 2012-06-27 | 2018-10-23 | Hoffmann-La Roche Inc. | Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof |
| US10323099B2 (en) | 2013-10-11 | 2019-06-18 | Hoffmann-La Roche Inc. | Multispecific domain exchanged common variable light chain antibodies |
| US10633457B2 (en) | 2014-12-03 | 2020-04-28 | Hoffmann-La Roche Inc. | Multispecific antibodies |
| US11999801B2 (en) | 2014-12-03 | 2024-06-04 | Hoffman-La Roche Inc. | Multispecific antibodies |
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