Anti-p185<erbB2> human-mouse chimeric antibody ChAb26, mammary gland specific expression vectors, transgenic FVB mouse, and preparation method of transgenic FVB mouse

A p185erbb2, human-mouse chimeric antibody technology, applied in the field of bioengineering, can solve the problems of high treatment costs and high production costs for tumor patients, and achieve the effects of avoiding anti-antibody reactions, low cost, and easy scale.

Inactive Publication Date: 2016-06-08
GUANGXI MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Its production cost is extremely high, resulting in high cost of treatment for tumor patients. The preparation of anti-p18

Method used

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  • Anti-p185&lt;erbB2&gt; human-mouse chimeric antibody ChAb26, mammary gland specific expression vectors, transgenic FVB mouse, and preparation method of transgenic FVB mouse
  • Anti-p185&lt;erbB2&gt; human-mouse chimeric antibody ChAb26, mammary gland specific expression vectors, transgenic FVB mouse, and preparation method of transgenic FVB mouse
  • Anti-p185&lt;erbB2&gt; human-mouse chimeric antibody ChAb26, mammary gland specific expression vectors, transgenic FVB mouse, and preparation method of transgenic FVB mouse

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Anti-p185 erbB2 Construction of mammary gland-specific expression vector of human-mouse chimeric antibody ChAb26 transgenic mouse

[0072] 1. Obtaining the target gene

[0073] anti-p185 erbB2 Human-mouse chimeric antibody ChAb26 heavy chain H and light chain gene L pUC57 / pBCN-H-F2A-L-ployA-EGFP-Neo plasmid as a template ( figure 1 ), under the action of LATaqDNA polymerase, respectively H-F / H-R (SEQ.ID.NO.1 / SEQ.ID.NO.2) and L-F / L-R (SEQ.ID.NO.4 / SEQ.ID.NO. 5) Amplify anti-p185 for primers erbB2 Human-mouse chimeric antibody ChAb26 heavy chain gene H (SEQ.ID.NO.3) and light chain gene L (SEQ.ID.NO.6). After identification by 1% agarose gel electrophoresis, the specific bands of the heavy chain gene H and the light chain gene L were consistent with the theoretical values, and the sizes were 2113bp and 799bp ( figure 2 ).

[0074] The amplification primer of table 1 object gene (the underlined part is the sequence of restriction endonuclease XhoI)

[0075...

Embodiment 2

[0101] Example 2 Anti-p185 erbB2 Preparation of human-mouse chimeric antibody ChAb26 transgenic FVB mice

[0102] 1. Anti-p185 erbB2 Linearization of human-mouse chimeric antibody mammary gland-specific expression vector pBC1-H / pBC1-L

[0103] The anti-p185 constructed in Example 1 was digested with SalI and NotI endonucleases erbB2 The human-mouse chimeric antibody ChAb26 mammary gland-specific expression plasmid pBC1-H / pBC1-L was linearized, and the results were as follows Figure 23 and Figure 24 shown. Mammary gland-specific expression plasmids pBC1-H (23735bp) or pBC1-L (22421bp) were linearized into pBC1-H-linear fragments (17861bp, SEQ.ID. NO.11) or pBC1-L-linear fragment (16547bp, SEQ.ID.NO.12).

[0104] 2. Preparation of transgenic mice by microco-injection

[0105] With the assistance of Saiye (Suzhou) Biotechnology Co., Ltd., the linearized mammary gland-specific expression plasmids pBC1-H-linear and pBC1-L-linear were diluted to an appropriate concentration...

Embodiment 3

[0124] Example 3 Anti-p185 erbB2 Acquisition and identification of human-mouse chimeric antibody ChAb26

[0125] 1. Anti-p185 erbB2 Acquisition of human-mouse chimeric antibody ChAb26

[0126] 10 days after the pregnant mice gave birth, use the mouse milker (WAT2006) to collect milk from the transgenic mother mice (lactation period); then transfer the milk collected in the collection tubes to sterilized Ep tubes, 4°C, 4000×g Centrifuge for 20 minutes; after centrifugation, the milk is divided into three layers, the upper layer is milky white milk fat, the lower layer is insoluble matter, and the middle layer is clear liquid. Use a 1ml disposable syringe to pierce the Ep tube at the lower edge of the middle layer, and gently suck out the clear liquid in the middle layer, which is anti-p185 erbB2 Human mouse chimeric antibody ChAb26 solution.

[0127] 2. Western blot detection of the expression of chimeric antibody ChAb26 in the milk of transgenic double-positive mice

[01...

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Abstract

The invention discloses an anti-p185<erbB2> human-mouse chimeric antibody ChAb26. The anti-p185<erbB2> human-mouse chimeric antibody ChAb26 is mainly composed of a heavy chain gene H and a light chain gene L, and the heavy chain gene H and the light chain gene L are respectively connected to a mammary gland specific expression plasmid pBC1 in order to obtain p185<erbB2> human-mouse chimeric antibody ChAb26 mammary gland specific expression vectors pBC1-H and pBC1-L. The pBC1-H and pBC1-L are linearized, the linearized pBC1-H and pBC1-L are introduced to FVB mouse zygotes to produce a transgenic FVB mouse, and the anti-p185<erbB2> human-mouse chimeric antibody ChAb26 can be obtained through mammary gland specific expression with the transgenic FVB mouse as a mammary gland bioreactor. The antibody reduces the immunogenicity of the v monoclonal antibody, and a medicinal recombinant protein produced through the transgenic FVB mouse has the advantages of high output, low cost, high activity, easy large-scale formation, shortening of the manufacturing period of new medicines, and huge market potential.

Description

technical field [0001] The technology of the invention belongs to the field of bioengineering, in particular to an anti-p185 erbB2 Human-mouse chimeric antibody ChAb26, mammary gland-specific expression vector, transgenic FVB mouse and preparation method thereof. Background technique [0002] Shih et al. (1981) first discovered a new proto-oncogene in rat embryonic neuroblasts and named it the neu gene. Subsequently, the neu proto-oncogene and the proto-oncogene erbB were found to be highly homologous through nucleic acid sequence analysis and chromosomal profile analysis. Therefore, the neu proto-oncogene was considered as a related gene of the erbB proto-oncogene and was named erbB2 / HER2. Coussens et al. found that the human proto-oncogene erbB2 is located in the 2 region 1 band (17q21) of the long arm of human chromosome 17, and its encoded product is a receptor tyrosine kinase (RTK, receptortyrosinekinase) with a molecular weight of 185kDa, so it is also known as p185...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/85C12N15/66A01K67/027
CPCA01K67/0278A01K2227/105A01K2267/01C07K16/18C07K2317/24C12N15/66C12N15/8509
Inventor 李力梁振鑫
Owner GUANGXI MEDICAL UNIVERSITY
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