Recombinant Kluyveromyces sp. expressing antibody or antibody analogue, and construction method and use thereof

An antibody analog, Kluyveromyces technology, applied to recombinant Kluyveromyces expressing antibody or antibody analog and its construction and application fields, can solve the problems of long construction period, cumbersome operation and high production cost

Inactive Publication Date: 2009-04-22
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mammalian cell expression system is the mature and most widely used system for expressing complete antibodies and antibody analogs containing Fc fragments, but mammalian cell expression system has long construction period, cumbersome operation, relatively low expression yield, and high production cost. High disadvantage
However, Kluyveromyces, which is low in cost, easy to scale up, and has good industrial fermentation scale characteristics, has so far not been used to express complete antibodies and antibody analogs containing Fc fragments of antibodies.

Method used

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  • Recombinant Kluyveromyces sp. expressing antibody or antibody analogue, and construction method and use thereof
  • Recombinant Kluyveromyces sp. expressing antibody or antibody analogue, and construction method and use thereof
  • Recombinant Kluyveromyces sp. expressing antibody or antibody analogue, and construction method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1, Expression of anti-HER2 humanized monoclonal antibody in Kluyveromyces lactis

[0052] 1. Obtaining the whole gene of anti-HER2 humanized monoclonal antibody

[0053]According to the light and heavy chain sequences of anti-HER2 humanized monoclonal antibodies reported on Genbank (Genbank: AAB49171.4 and AAB48814.1) and yeast preferred codons, synonymously mutate the commonly used enzyme cutting sites. Using the whole gene synthesis method, one-step PCR method to obtain antibody light chain gene (Light chain) L fragment (sequence 2 in the sequence listing) and heavy chain gene H (heavy chain) fragment (sequence 1 in the sequence listing), and with Alpha mating factor signal peptide.

[0054] For the one-step PCR method, refer to "Molecular Cloning Experiment Guide" (Second Edition, Science Press, 1995) by J. Sambrook et al. The PCR amplification conditions are as follows: after denaturation at 94°C for 5 minutes, 55 cycles of denaturation at 94°C for 30 sec,...

Embodiment 2

[0106] Example 2, Expression of anti-HER2 humanized monoclonal antibody in Kluyveromyces lactis variant

[0107] According to the method described in Example 1, the recombinant vector pYES2-och1-LAC4-CL and pPICZαA-ura3-LAC4-H were transformed into Kluyveromyces lactis KLGE02 CGMCC No.2400, and the recombinant vector pYES2-och1-LAC4-CL and pPICZαA-ura3-LAC4-H were transformed into K. lactis KLGE03CGMCC No.2401. Anti-HER2 monoclonal antibodies were expressed in K. lactis KLGE0 2CGMCC No.2400 and K. lactis KLGE03 CGMCC No.2401. Purified samples HER2-1 and HER2-2 were obtained according to the method of 4 in Example 1. Purified samples were subjected to 12% SDS-PAGE electrophoresis, and sent to the Instrument Center Laboratory of the Academy of Military Medical Sciences for N-terminal amino acid residue sequence analysis of the protein band of the heavy chain (methods refer to "Protein Technology Manual" edited by Wang Jiazheng and Fan Ming (Science Press, 2000)), the sequencin...

Embodiment 3

[0109] Example 3, Expression of Antibody Analog sTNFRII-IgGFc in Kluyveromyces lactis

[0110] 1. Fishing of sTNFRII gene and IgGFc gene

[0111] Take acid citrate dextrose (ACD) anticoagulated venous blood from healthy people, dilute it with Hanks' balanced salt solution (Invitrogen Corp. The 1640 medium (GIBCO, USA) of 10% fetal bovine serum (Hyclone Company, USA) adjusted the number of cells to be 5×10 6 cells / mL, placed in a cell incubator and incubated for 2 hours, then replaced with 1640 fresh medium containing lipopolysaccharide (LPS, 20 μg / mL) (Sigma) and 10% fetal bovine serum, continued to culture for 5 hours, and centrifuged to enrich the cells; TRIzol (Shanghai Sangon Bioengineering Technology Service Co., Ltd.) method to extract total RNA (operate according to the instructions of TRIzol); total RNA was subjected to RT-PCR to obtain cDNA (operate according to the instructions of the RT-PCR kit), and then use cDNA as a template , to amplify the human sTNFRII gene ...

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Abstract

The invention discloses a recombinant yeast Kluyveromyces for expressing antibodies or antibody analogues and application thereof. The invention provides a method for constructing the recombinant yeast Kluyveromyces for expressign antibodies or antibody analogues. The method comprises the following steps: encoding genes of the antibodies or the antibody analogues are guided into the yeast Kluyveromyces to obtain recombinant bacteria; the antibody analogues are fusion proteins formed by Fc fragments of the antibodies and proteins or protein subunits; the encoding genes of the antibodies are heavy chain and light chain encoding genes of the antibodies. The recombinant yeast Kluyveromyces for expressing antibodies or antibody analogues can be cultured to produce complete antibodies or antibody analogues. The invention uses the yeast Kluyveromyces to prepare the antibodies or antibody analogues to preliminarily overcome the difficulties of difficult antibody production, high purity and large quantity requirements for products and provide good application prospects.

Description

technical field [0001] The invention relates to a recombinant Kluyveromyces expressing an antibody or an antibody analogue, a construction method and application thereof. Background technique [0002] Antibody (Ab) is a glycoprotein that specifically binds to an antigen. The natural antibody is about 150,000 daltons, composed of two identical light chains (Light Chain, L) and two identical heavy chains (Heavy Chain, H). The light chain is linked to the heavy chain by a covalent disulfide bond. Each heavy chain consists of a variable region (VH) and constant regions. Each light chain consists of a variable region (VL) and a constant region; the constant region of the light chain is opposed to the first constant region of the heavy chain, and the variable region of the light chain is opposed to the variable region of the heavy chain. Papain digestion of antibodies yields two identical antigen-binding fragments (termed "Fab" fragments, each with a single antigen-binding site...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/13C12N15/62C12N1/19C07K16/00C07K19/00C12R1/645
Inventor 刘波吴军唱韶红巩新马清钧
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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