Culture medium additive for recombinant protein expression of humanized HEK293 cell line, recombinant hepatitis B vaccine and expression method

A medium additive, recombinant protein technology, applied in tissue culture, artificial cell constructs, chemical instruments and methods, etc., can solve the problem of low protein yield and achieve the effect of improving the expression level

Active Publication Date: 2021-11-09
河南普诺易生物制品研究院有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although HEK293 cells have some advantages in the production of recombinant therapeutic proteins, the problem of low protein production still needs to be solved urgently, and it is particularly important to further increase the expression of recombinant proteins in the expression system of HEK293 cells

Method used

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  • Culture medium additive for recombinant protein expression of humanized HEK293 cell line, recombinant hepatitis B vaccine and expression method
  • Culture medium additive for recombinant protein expression of humanized HEK293 cell line, recombinant hepatitis B vaccine and expression method
  • Culture medium additive for recombinant protein expression of humanized HEK293 cell line, recombinant hepatitis B vaccine and expression method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Optimal design of HBsAg gene

[0027] Codon optimization was performed on the amino acid sequence disclosed in Genbank no: AAS20191.1 to obtain the optimized hepatitis B surface antigen gene (HBsAg gene), whose sequence is shown in SEQ ID NO.1.

Embodiment 2

[0028] Example 2 Construction of EGFP recombinant expression vector pIRES-EGFP

[0029] The present embodiment provides the construction method of the engineered bacterium that contains recombinant expression vector, comprises the following steps:

[0030] 1) PCR amplification of EGFP gene

[0031] Referring to the Enhanced green fluorescent protein (EGFP) gene sequence of the pEGFP-C1 vector (GenBank: U55763.1, bases 613-1332), design primers P1 and P2 (for amplifying 720bp EGFP gene DNA), primer 5 EcoRV and NheI restriction sites were introduced at the ' ends respectively, and the primer sequences were as follows (the restriction sites are underlined):

[0032] P1: 5′-CCG GATATC ATGGTGAGCAAGGGCGAGGAG-3'; as shown in SEQ ID NO.2;

[0033] P2: 5′-CTA ACCGGT GGACTTGTACAGCTCGTCCATGC-3'; shown in SEQ ID NO.3.

[0034] Using the pEGFP-C1 plasmid (purchased from Clontech, USA) as a template, primers P1 and P2 were used to amplify the EGFP gene. The reaction system is shown i...

Embodiment 3

[0044] Embodiment 3 Contains the construction of different promoter expression vectors

[0045] This embodiment provides a method for constructing recombinant expression vectors comprising different promoters, including the following steps:

[0046] Synthetic PGK (GenBank: KJ175229.1), CAG enhancer (GenBank: AJ575208.1), mCMV (GenBank: KT343252.1), CHEF-1α (GenBank: KY447299.1), CMV mutant, HEF-1α (GenBank: AY188393.1) and CAG (GenBank: GU299216.1) seven promoter fragments. Use seamless cloning technology to replace the original promoter on pIRES-EGFP, such as figure 1 shown. The correctly constructed plasmids were named pIRES-PGK, pIRES-CAGen, pIRES-mCMV, pIRES-CHEF, pIRES-CMVmut, pIRES-HEF, pIRES-CAG, respectively.

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Abstract

The invention belongs to the technical field of gene recombinant vaccines and particularly relates to a culture medium additive for recombinant protein expression of a humanized HEK293 cell line, a recombinant hepatitis B vaccine and an expression method. According to the culture medium additive for the recombinant protein expression of the humanized HEK293 cell line, provided by the invention, sodium butyrate and hydrocinnamic acid are added in a suspended serum-free culture process of HEK293 cells transfected with a recombinant expression vector, preferably, the addition amount of the sodium butyrate is 1.0-3.0 mol / L, the addition amount of the hydrocinnamic acid is 0.2-1.0 mol / L, synergy is achieved, and the expression level of a recombinant protein is increased. Tests show that through carrying out culturing under a low-temperature condition by utilizing the culture medium additive provided by the invention, the expression level of a hepatitis B surface antigen (HBsAg) target gene in an HEK293 cell is improved.

Description

technical field [0001] The invention belongs to the technical field of gene recombinant vaccines, and specifically relates to a culture medium additive for expressing recombinant proteins in a humanized HEK293 cell line, a recombinant hepatitis B vaccine, and a recombinant hepatitis B vaccine expression method. Background technique [0002] Hepatitis B virus infection is one of the major public health problems to be solved urgently in the world today. It is estimated that about 60% of people in China have been infected with hepatitis B virus, although the World Health Organization proposes that people should be vaccinated against hepatitis B virus effectively in infants and young children , but more than 350 million people worldwide are still chronically infected with hepatitis B virus (HBV), and are at high risk of developing liver decompensation, cirrhosis and even liver cancer. The current clinical antiviral treatment is difficult to effectively eliminate the virus. The m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/073C12N5/071C07K14/02C12N15/85A61K39/29A61P31/20A61P1/16
CPCC12N5/0686C12N5/0603C07K14/005C12N15/85A61K39/12A61P31/20A61P1/16C12N2730/10134C12N2500/30C12N2730/10122C12N2510/02Y02A50/30
Inventor 王天云林艳窦媛媛张俊河樊振林曹祥祥耿少雷李波
Owner 河南普诺易生物制品研究院有限公司
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