Signal peptide for improving antibody yield

A signal peptide and antibody technology, applied in the field of molecular biology, can solve problems such as differences in antibody secretion efficiency

Pending Publication Date: 2021-09-17
VACDIAGN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibodies are a type of protein secreted by mammalian immune B cells. Due to the polymorphism of antibodies, the sequence of each antibody molecule is different. Therefore, the signal peptide sequence of each antibody is also different, resulting in differences in antibody secretion efficiency.

Method used

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  • Signal peptide for improving antibody yield
  • Signal peptide for improving antibody yield
  • Signal peptide for improving antibody yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of antibody fusion protein 3D6-SpyTag heavy chain expression vector

[0045]SpyTag (polypeptide segment) amino acid sequence (SEQ ID NO: 1) mammalian codon optimized sequence (SEQ ID NO: 2) and 3D6 (anti-human DEC205 monoclonal antibody) heavy chain expression vector phIgG1-3D6 (signal peptide is mouse IgG1 signal peptide, amino acid sequence SEQ ID NO: 3) and light chain expression vector phIgK-3D6 (signal peptide is mouse IgG1 signal peptide, amino acid sequence SEQ ID NO: 3) were provided by Suzhou Industrial Park Weida Biotechnology Co., Ltd. Using phIgG1-3D6 as a template, design primers (Table 1), amplify the hIgG1-SpyTag gene fragment by PCR, then recover the corresponding fragment from the gel, and clone it into the multiple cloning site between Sal I and HindIII on the phIgG1-3D6 vector. Construct the heavy chain expression vector phIgG1-3D6-SpyTag that can express fusion protein 3D6-SpyTag (plasmid map such as figure 1 ), which were cor...

Embodiment 2

[0050] Example 2 Construction of antibody fusion protein 3D6-SpyTag (N) heavy chain expression vector

[0051] The mammalian codon-optimized sequence (SEQ ID NO: 7) of the amino acid sequence (SEQ ID NO: 6) of the signal peptide of the heavy chain of 1G11 was provided by Suzhou Industrial Park Weida Biotechnology Co., Ltd. Using phIgG1-3D6-SpyTag (self-constructed in Example 1) as a template, design primers (Table 3), amplify the 1G11SP-3D6-SpyTag gene fragment by PCR, then recover the corresponding fragment from the gel, and clone it into the phIgG1-3D6-SpyTag vector Between the multiple cloning sites on EcoR I and Hind III, a heavy chain expression vector phIgG1-3D6-SpyTag (N) capable of expressing the fusion protein 3D6-SpyTag (N) was constructed (plasmid map as shown in image 3 ), which were correctly identified by sequencing and entered into the library. The carrier phIgG1-3D6-SpyTag (N) was identified with restriction endonucleases EcoR I and Hind III (enzyme digestion...

Embodiment 3

[0056] Example 3 Construction of antibody fusion protein 3D6-SpyTag(N) light chain expression vector

[0057] 1G11 light chain signal peptide amino acid sequence (SEQ ID NO: 11) mammalian codon optimized sequence (SEQ ID NO: 12) and 3D6 (anti-human DEC205 monoclonal antibody) light chain expression vector phIgK-3D6 (signal peptide is mouse IgG1 signal peptide, amino acid sequence (SEQ ID NO: 3) was provided by Suzhou Industrial Park Weida Biotechnology Co., Ltd. Using phIgK-3D6 as a template, design primers (Table 5), amplify the 1G11SP-3D6VL-hIgkappa gene fragment by PCR, then recover the corresponding fragment from the gel, and clone it into the multiple cloning site EcoR I and HindIII on the phIgK-3D6 vector. In between, the light chain expression vector phIgK-3D6(N2) that can express the fusion protein 3D6-SpyTag(N) was constructed (plasmid map as Figure 5 ), which were correctly identified by sequencing and entered into the library. The carrier phIgK-3D6(N2) was identi...

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Abstract

The invention relates to the technical field of molecular biology, and particularly relates to a signal peptide for improving antibody yield. The amino acid sequence of the signal peptide is MNFGLSWVFLVLVLKGVQC or MVFTPQILGLMLFWISASRG, or the amino acid sequence of the signal peptide is from a heavy chain and a light chain of an antibody 1G11, the signal peptide is used for improving secretory expression of target protein, and the signal peptide is secretory expression of the antibody. According to the signal peptide, PCR amplification is conducted on the signal peptide to obtain different gene segments, gel recovery is conducted, and the yield of target genes, especially antibody secretion can be improved by identifying a vector through restriction endonuclease.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a signal peptide for improving antibody production. Background technique [0002] Signal peptide is a unique amino acid sequence located at the N-terminus of newly secreted proteins and cell membrane proteins, which can position the protein on the cell membrane or transmembrane to the outside of the cell. The length of the signal peptide is generally about 30 amino acids, and it consists of a positively charged N domain, an H domain rich in hydrophobic amino acids, and a negatively charged C domain. Antibodies are a type of protein secreted by mammalian immune B cells. Due to the polymorphism of antibodies, the sequence of each antibody molecule is different. Therefore, the signal peptide sequence of each antibody is also different, resulting in differences in antibody secretion efficiency. When the secretion and expression efficiency of antibodies or antibody fusion p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/11C12N15/85
CPCC07K7/08C12N15/85C12N2800/22
Inventor 徐建青郜明泉黄杨
Owner VACDIAGN BIOTECH
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