CN103074349A - 巢式一步法rt-pcr从单个b细胞扩增鼠源抗体重链和轻链可变区基因全序列的方法 - Google Patents
巢式一步法rt-pcr从单个b细胞扩增鼠源抗体重链和轻链可变区基因全序列的方法 Download PDFInfo
- Publication number
- CN103074349A CN103074349A CN2012104388163A CN201210438816A CN103074349A CN 103074349 A CN103074349 A CN 103074349A CN 2012104388163 A CN2012104388163 A CN 2012104388163A CN 201210438816 A CN201210438816 A CN 201210438816A CN 103074349 A CN103074349 A CN 103074349A
- Authority
- CN
- China
- Prior art keywords
- heavy chain
- variable region
- light chain
- primer
- linker
- 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.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 34
- 210000003719 b-lymphocyte Anatomy 0.000 title claims abstract description 22
- 238000003757 reverse transcription PCR Methods 0.000 title claims abstract description 16
- 241001529936 Murinae Species 0.000 title claims abstract description 7
- 230000003321 amplification Effects 0.000 claims abstract description 16
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 16
- 108091008146 restriction endonucleases Proteins 0.000 claims abstract description 12
- 101150117115 V gene Proteins 0.000 claims abstract description 8
- 239000013604 expression vector Substances 0.000 claims abstract description 6
- 238000011144 upstream manufacturing Methods 0.000 claims description 28
- 239000002299 complementary DNA Substances 0.000 claims description 6
- 238000013461 design Methods 0.000 claims description 6
- 230000000295 complement effect Effects 0.000 claims description 5
- 238000013467 fragmentation Methods 0.000 claims description 5
- 238000006062 fragmentation reaction Methods 0.000 claims description 5
- 108020004999 messenger RNA Proteins 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- 238000010839 reverse transcription Methods 0.000 claims description 3
- 230000002103 transcriptional effect Effects 0.000 claims description 3
- 108020004414 DNA Proteins 0.000 abstract description 42
- 239000012634 fragment Substances 0.000 abstract description 13
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000004907 flux Effects 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 108060003951 Immunoglobulin Proteins 0.000 abstract 1
- 230000002457 bidirectional effect Effects 0.000 abstract 1
- 102000018358 immunoglobulin Human genes 0.000 abstract 1
- 238000007857 nested PCR Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 102100034357 Casein kinase I isoform alpha Human genes 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 101710118321 Casein kinase I isoform alpha Proteins 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 229910052739 hydrogen Inorganic materials 0.000 description 5
- 229940125644 antibody drug Drugs 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 210000004408 hybridoma Anatomy 0.000 description 4
- 239000011535 reaction buffer Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 101150097493 D gene Proteins 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 2
- 101150111062 C gene Proteins 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 101150090724 3 gene Proteins 0.000 description 1
- 101150033839 4 gene Proteins 0.000 description 1
- 101100532034 Drosophila melanogaster RTase gene Proteins 0.000 description 1
- 101000994700 Homo sapiens Casein kinase I isoform alpha Proteins 0.000 description 1
- 102000012960 Immunoglobulin kappa-Chains Human genes 0.000 description 1
- 108010090227 Immunoglobulin kappa-Chains Proteins 0.000 description 1
- 108091036060 Linker DNA Proteins 0.000 description 1
- 101100107522 Mus musculus Slc1a5 gene Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229940125645 monoclonal antibody drug Drugs 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000002818 protein evolution Methods 0.000 description 1
- 230000003946 protein process Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims (3)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201210438816.3A CN103074349B (zh) | 2012-11-07 | 2012-11-07 | 巢式一步法rt-pcr从单个b细胞扩增鼠源抗体重链和轻链可变区基因全序列的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201210438816.3A CN103074349B (zh) | 2012-11-07 | 2012-11-07 | 巢式一步法rt-pcr从单个b细胞扩增鼠源抗体重链和轻链可变区基因全序列的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN103074349A true CN103074349A (zh) | 2013-05-01 |
CN103074349B CN103074349B (zh) | 2014-10-15 |
Family
ID=48151044
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201210438816.3A Active CN103074349B (zh) | 2012-11-07 | 2012-11-07 | 巢式一步法rt-pcr从单个b细胞扩增鼠源抗体重链和轻链可变区基因全序列的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN103074349B (zh) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015096063A1 (en) * | 2013-12-25 | 2015-07-02 | Coyote Bioscience Co., Ltd. | Methods and systems for nucleic acid amplification |
CN105121663A (zh) * | 2013-12-25 | 2015-12-02 | 卡尤迪生物科技(北京)有限公司 | 用于核酸扩增的方法和系统 |
CN106086009A (zh) * | 2016-06-17 | 2016-11-09 | 中国农业科学院哈尔滨兽医研究所 | 一种利用单个b细胞pcr技术生产全猪源单克隆抗体的方法 |
CN107779500A (zh) * | 2017-10-25 | 2018-03-09 | 上海药明生物技术有限公司 | 一种快速获取大鼠杂交瘤细胞单克隆抗体序列的测序方法及引物序列 |
CN108315342A (zh) * | 2018-02-12 | 2018-07-24 | 深圳市国创纳米抗体技术有限公司 | 一种利用流式细胞分选筛选纳米抗体的方法 |
CN108997493A (zh) * | 2018-08-15 | 2018-12-14 | 中国农业科学院兰州兽医研究所 | 一种全牛源o型口蹄疫病毒广谱中和抗体的制备方法 |
CN109972209A (zh) * | 2019-01-18 | 2019-07-05 | 南开大学 | 基于乳液pcr的一步法将单链抗体库重排为全长抗体库的方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050287538A1 (en) * | 2004-06-29 | 2005-12-29 | Wing-Tai Cheung | Frame-shifting PCR for germline immunoglobulin genes retrieval and antibody engineering |
WO2008104184A2 (en) * | 2007-03-01 | 2008-09-04 | Symphogen A/S | Method for cloning cognate antibodies |
CN101294308A (zh) * | 2007-10-22 | 2008-10-29 | 侯明 | 人源化重组噬菌体抗体库方法 |
CN101451134A (zh) * | 2007-11-29 | 2009-06-10 | 上海凯勃生物技术有限公司 | 从人微量b细胞中扩增人抗体重链及轻链基因片段的方法 |
-
2012
- 2012-11-07 CN CN201210438816.3A patent/CN103074349B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050287538A1 (en) * | 2004-06-29 | 2005-12-29 | Wing-Tai Cheung | Frame-shifting PCR for germline immunoglobulin genes retrieval and antibody engineering |
WO2008104184A2 (en) * | 2007-03-01 | 2008-09-04 | Symphogen A/S | Method for cloning cognate antibodies |
CN101622346A (zh) * | 2007-03-01 | 2010-01-06 | 西福根有限公司 | 克隆关联抗体的方法 |
CN101294308A (zh) * | 2007-10-22 | 2008-10-29 | 侯明 | 人源化重组噬菌体抗体库方法 |
CN101451134A (zh) * | 2007-11-29 | 2009-06-10 | 上海凯勃生物技术有限公司 | 从人微量b细胞中扩增人抗体重链及轻链基因片段的方法 |
Cited By (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10465239B2 (en) | 2013-12-25 | 2019-11-05 | Coyote Bioscience Co., Ltd. | Methods and systems for nucleic acid amplification |
WO2015096763A1 (en) * | 2013-12-25 | 2015-07-02 | Coyote Bioscience Co., Ltd. | Methods and systems for nucleic acid amplification |
CN105121663A (zh) * | 2013-12-25 | 2015-12-02 | 卡尤迪生物科技(北京)有限公司 | 用于核酸扩增的方法和系统 |
US9546389B2 (en) | 2013-12-25 | 2017-01-17 | Coyote Bioscience Co., Ltd. | Methods and systems for nucleic acid amplification |
WO2015096063A1 (en) * | 2013-12-25 | 2015-07-02 | Coyote Bioscience Co., Ltd. | Methods and systems for nucleic acid amplification |
CN112359099A (zh) * | 2013-12-25 | 2021-02-12 | 卡尤迪生物科技(北京)有限公司 | 用于核酸扩增的方法和系统 |
CN105121663B (zh) * | 2013-12-25 | 2020-11-13 | 卡尤迪生物科技(北京)有限公司 | 用于核酸扩增的方法和系统 |
CN106086009A (zh) * | 2016-06-17 | 2016-11-09 | 中国农业科学院哈尔滨兽医研究所 | 一种利用单个b细胞pcr技术生产全猪源单克隆抗体的方法 |
CN106086009B (zh) * | 2016-06-17 | 2020-03-17 | 中国农业科学院哈尔滨兽医研究所 | 一种利用单个b细胞pcr技术生产全猪源单克隆抗体的方法 |
CN107779500A (zh) * | 2017-10-25 | 2018-03-09 | 上海药明生物技术有限公司 | 一种快速获取大鼠杂交瘤细胞单克隆抗体序列的测序方法及引物序列 |
CN108315342A (zh) * | 2018-02-12 | 2018-07-24 | 深圳市国创纳米抗体技术有限公司 | 一种利用流式细胞分选筛选纳米抗体的方法 |
CN113004400A (zh) * | 2018-02-12 | 2021-06-22 | 深圳市国创纳米抗体技术有限公司 | 一种抗绿脓杆菌外毒素a的纳米抗体3h12 |
CN113004399A (zh) * | 2018-02-12 | 2021-06-22 | 深圳市国创纳米抗体技术有限公司 | 一种抗绿脓杆菌外毒素a的纳米抗体3c12 |
CN113061183A (zh) * | 2018-02-12 | 2021-07-02 | 深圳市国创纳米抗体技术有限公司 | 一种抗绿脓杆菌外毒素a的纳米抗体3c11 |
CN113061183B (zh) * | 2018-02-12 | 2022-06-21 | 深圳市国创纳米抗体技术有限公司 | 一种抗绿脓杆菌外毒素a的纳米抗体3c11 |
CN108997493B (zh) * | 2018-08-15 | 2019-07-09 | 中国农业科学院兰州兽医研究所 | 一种全牛源o型口蹄疫病毒广谱中和抗体的制备方法 |
US10766949B2 (en) | 2018-08-15 | 2020-09-08 | Lanzhour Veterinary Research Institute, Chinese Academy of Agricultural Sciences | Method for preparing whole bovine-derived broadly neutralizing antibody against serotype O foot-and-mouth disease virus |
CN108997493A (zh) * | 2018-08-15 | 2018-12-14 | 中国农业科学院兰州兽医研究所 | 一种全牛源o型口蹄疫病毒广谱中和抗体的制备方法 |
CN109972209A (zh) * | 2019-01-18 | 2019-07-05 | 南开大学 | 基于乳液pcr的一步法将单链抗体库重排为全长抗体库的方法 |
CN109972209B (zh) * | 2019-01-18 | 2022-05-03 | 南开大学 | 基于乳液pcr的一步法将单链抗体库重排为全长抗体库的方法 |
Also Published As
Publication number | Publication date |
---|---|
CN103074349B (zh) | 2014-10-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN103074349B (zh) | 巢式一步法rt-pcr从单个b细胞扩增鼠源抗体重链和轻链可变区基因全序列的方法 | |
US10912799B2 (en) | Compositions and methods for inhibition of lineage specific antigens | |
AU2023201162A1 (en) | Compositions and methods for immunooncology | |
CN109963944A (zh) | 体外敲除T细胞中靶基因的方法以及该方法中使用的crRNA | |
CA3118824A1 (en) | Anti-liv1 immune cell cancer therapy | |
CN101440130A (zh) | 一种抗人IL-13Rα2单克隆抗体的重链和轻链的可变区 | |
CN110372793B (zh) | Pd-l1的纳米抗体及其临床应用 | |
CN109705225B (zh) | 一种抗人caix抗原的嵌合抗原受体及其应用 | |
CN111018989B (zh) | 一种抗pd-l1单克隆抗体以及在制备抗癌药物方面的应用 | |
US20230340139A1 (en) | Immune cells having co-expressed shrnas and logic gate systems | |
AU2004316866B2 (en) | A kind of fusion protein, gene encoding it, expression method and use thereof | |
CN113583124B (zh) | 一种抗前胃泌素释放肽单克隆抗体及其制备方法 | |
CN105802975B (zh) | 靶向her2阳性肿瘤的细胞制备物及其用途 | |
CN101585880B (zh) | 一种抗人IL-13Rα2单克隆抗体的重链和轻链的可变区 | |
CN110950954A (zh) | 抗cd123人源化单链抗体及其应用 | |
CN104804090B (zh) | 一种抗b细胞生长刺激因子的纳米抗体和用途 | |
WO2015182749A1 (ja) | TCR cDNAの増幅方法 | |
CN110885378A (zh) | 一种小反刍兽疫重组融合蛋白、其制备方法及应用 | |
CN115125215B (zh) | 分泌猪IFN-λ4单克隆抗体的杂交瘤细胞株及其分泌的单克隆抗体与应用 | |
CN103524625B (zh) | 一种新型诱导抗体产生的方法 | |
CN102718846A (zh) | 活性增强的sec2突变蛋白及编码基因与制备和应用 | |
CN109988238B (zh) | Pd1单链抗体在大肠杆菌中的制备方法 | |
CN110790835A (zh) | 一种包虫重组蛋白及其制备方法和应用 | |
Zein et al. | Generation, characterization, and docking studies of DNA‐hydrolyzing recombinant Fab antibodies | |
CN117924487A (zh) | 一种由羊驼重链抗体可变区组成的特异性结合lag3蛋白的纳米抗体及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
ASS | Succession or assignment of patent right |
Owner name: BEIJING NORMAL UNIVERSITY Effective date: 20140423 |
|
C41 | Transfer of patent application or patent right or utility model | ||
TA01 | Transfer of patent application right |
Effective date of registration: 20140423 Address after: 3, building 2, building 288, No. 310052 Qiu Yi Road, Hangzhou, Zhejiang, Binjiang District Applicant after: Gmax Biopharm Co., Ltd. Applicant after: Beijing Normal University Address before: Hangzhou City, Zhejiang province Binjiang District 310052 Spring Road No. 1288, high-tech park building 2 floor Applicant before: Gmax Biopharm Co., Ltd. |
|
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CP03 | Change of name, title or address |
Address after: Hangzhou City, Zhejiang Province, 310052 Binjiang District Road No. 288, autumn overflow high tech Park Building No. 2 building 302 room 3 Co-patentee after: Beijing Normal University Patentee after: Hongyun Huaning (Hangzhou) Biological Medicine Co Ltd Address before: 3, building 2, building 288, No. 310052 Qiu Yi Road, Hangzhou, Zhejiang, Binjiang District Co-patentee before: Beijing Normal University Patentee before: Gmax Biopharm Co., Ltd. |
|
CP03 | Change of name, title or address |