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Method for producing monoclonal antibodies by using transgenic animal mammary gland bioreactor

A monoclonal antibody, mammary gland technology, applied in biochemical equipment and methods, botanical equipment and methods, anti-animal/human immunoglobulin, etc. , complex production process and other problems

Inactive Publication Date: 2015-08-26
NANJING JENOMED BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] To sum up, various systems in the prior art cannot produce or separate and purify a sufficient amount of active human monoclonal antibodies, and the total market demand for these therapeutic monoclonal antibody drugs usually reaches 250-1000 kg, Or the produced active human monoclonal antibody products are not uniform, and the production process is complex and expensive

Method used

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  • Method for producing monoclonal antibodies by using transgenic animal mammary gland bioreactor
  • Method for producing monoclonal antibodies by using transgenic animal mammary gland bioreactor
  • Method for producing monoclonal antibodies by using transgenic animal mammary gland bioreactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0182] Example 1. Construction and preparation of random expression cassettes

[0183] Experimental materials, methods and results:

[0184] Experimental materials: chemical reagents were purchased from Sigma-Aldrich; endonucleases and other molecular biology reagents were purchased from NEB and Promega; media were purchased from Gibco.

[0185] Construction of random expression cassettes:

[0186] Plasmid pUC19 (Promega) was cut with SacI and BamHI endonucleases and ligated to an insulator fragment from the upstream region of the chicken β-globin gene (Proc Natl Acad Sci USA 94:575-80, 1997). The insulator fragment is a cis primer containing SacI, SacII, NotI, SalI sites and part of the DNA sequence of the upstream region of the chicken β-globin gene such as SEQ ID NO.: 3 (5'GAC ACT GAG CTC CAC CGC GGA CTGCGG CCG CTC GTC GAC GGG ACA GCC CCC CCC CAA AGC CCC CAG 3'), and the reverse primer containing BamHI, XhoI site and part of the chicken β-globin gene upstream region DNA s...

Embodiment 2

[0191] Example 2. Construction of the β-casein gene knock-in donor vector integrated in exon 1 of the β-casein gene:

[0192] Use cis primers containing SalI, NotI, AscI sites and part of the 5' end DNA sequence of goat β-casein gene such as SEQ ID NO.: 9 (5'ACG CGT GTC GAC GCG GCC GCG GCG CGC CAA TCCCTC AAC AAA TCC CCA 3') and TALEN cleavage site located in the middle reaches of exon 1 of goat β-casein promoter containing FseI, HindIII site and part of the reverse primer of the 5' end DNA sequence of goat β-casein gene such as SEQ ID NO .: 10 (5'AGG TCA GGG CCG GCC AAG CTT ATT TTC TCT ACA TAGAAC 3'), the genomic DNA isolated from goat blood was used as a template for PCR amplification to obtain a 2kb amplified product.

[0193]Using gene synthesis technology, synthesize a single-chain monoclonal antibody gene sequence containing FseI at the 5' end and an AgeI site at the 3' end, and connect it to the above-mentioned 2Kbβ-casein gene sequence at the 5' end through the FseI sit...

Embodiment 3

[0197] Example 3. TALEN vector construction:

[0198] According to the method described in (Nat Biotechnol 29,699-700,2011), construct TALEN carrier ( image 3 ).

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Abstract

The present invention relates to a method for large-scale production of recombinant monoclonal antibodies by using a transgenic animal mammary gland biological platform, and specifically provides an antibody element construct or expression cassette for specific expression of the mammary gland, wherein nuclear transplantation is performed after the construct or expression cassette is transformed into mammalian cells so as to make the transgenic animal mammary gland express and secrete the exogenous recombinant antibody protein, such that the monoclonal antibodies with characteristics of easy separation and easy purification are prepared. According to the present invention, the method is simple and efficient, and the produced recombinant monoclonal antibodies can be widely used in diagnosis, prevention, and treatment of cancers and other serious diseases research, and immune mechanism research, especially in the field of malignant tumor treatment or autoimmune disease treatment.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a method for large-scale production of recombinant monoclonal antibodies using a transgenic animal mammary gland biological platform. Background technique [0002] Biopharmaceutical technology has evolved very rapidly over the past forty years. Biopharmaceuticals have the advantages of good curative effect, small side effects, and wide indications, and have become a hot spot for various investments, especially for large pharmaceutical companies to invest in research and development. The production methods of biopharmaceuticals can be divided into two categories. One is to introduce specific biopharmaceutical coding gene sequences into engineered bacteria or biological cells, and obtain them through fermentation and cultivation of bacteria, yeast, or mammalian and human cells, and separation and purification. Microbial and yeast fermentation cultures are on...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C12N5/10C07K16/18A23C9/00A61K39/395A61P35/00C12N15/873A01K67/027
Inventor 黄跃进
Owner NANJING JENOMED BIOTECH CO LTD
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