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Preparation method and application of recombinant vaccine host protein antibody

A host protein and vaccine technology, which is applied to the preparation method of peptides, immunoglobulins, antiviral immunoglobulins, etc., can solve the problems of low actual measured value, inability to fully reflect HCP residues, and reduced sensitivity, and achieve detection Improve sensitivity, save test operation time and cost, and have good applicability

Active Publication Date: 2019-02-26
BEIJING MINHAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using the antibody produced by this preparation method will greatly reduce the sensitivity when detecting HCP residues in biological products by ELISA (enzyme-linked immunoassay), and the actual measured value is low because some HCP components with low immunogenicity cannot be detected , can not fully reflect the actual residue of product HCP

Method used

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  • Preparation method and application of recombinant vaccine host protein antibody
  • Preparation method and application of recombinant vaccine host protein antibody
  • Preparation method and application of recombinant vaccine host protein antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 Construction of Hansenula empty vector expression strain

[0050] The Hansenula expression vector PMV-05 (CN 201210592813.5) was transformed into Hansenula uracil-deficient host strain AU-0501 by electroporation (the construction method of Hansenula expression vector and Hansenula uracil-deficient host strain can be See CN 201210592813.5), the recombinant Hansenula empty vector expression strain was obtained through stable culture and screening.

Embodiment 2

[0051] The preparation of embodiment 2 Hansenula HCP

[0052] 1. Fermentation culture of recombinant Hansenula empty vector expression strain

[0053] The recombinant Hansenula empty vector expression strain was inoculated into 100ml primary seed medium (yeast extract 6.7g / L, ammonium sulfate 5g / L, glucose 20g / L), and cultured at 33°C and 200rpm shaking table for 20~ 24 hours. Then inoculate the whole amount in 1000ml secondary seed medium (yeast extract 6.7g / L, ammonium sulfate 5g / L, glycerol 20g / L), inoculate at 33°C, 200rpm shaker for 20-24 hours and then inoculate to 30L In the fermentation tank, 12L fermentation medium (glycerol 23.333g / L, ammonium dihydrogen phosphate 11.667g / L, 3.333g / L potassium chloride, calcium chloride 2.500g / L, sodium chloride 0.333g / L , magnesium sulfate 2.500g / L, sodium edetate 0.167g / L), ammonia water to adjust the pH value of the fermentation broth to maintain at 5.0, the fermentation temperature is 30°C, the speed is controlled at 350-750rpm...

Embodiment 3

[0064] Example 3 Preparation of Hansenula HCP Antibody

[0065] 1 animal immunity

[0066] 1.1 Rabbit Immunization

[0067] First immunization: take 6ml of host protein diluted to 1mg / ml, add 6ml of Freund's complete adjuvant (FCA) for emulsification, take one drop until the water surface does not disperse, and the emulsification is complete. Use a 2ml syringe to draw 2ml of Freund's complete adjuvant (FCA) emulsion (containing 1mg of host protein), and inject 0.2mL subcutaneously at multiple points on the neck and back.

[0068] The second immunization: After an interval of 10-14 days, the second immunization is carried out. Take 6ml of host protein diluted to 0.5mg / mL, add 6ml of Freund's incomplete adjuvant (FIA) for emulsification, take one drop until the water surface does not disperse, and the emulsification is complete. Use a 2ml syringe to draw 2ml of Freund's incomplete adjuvant (FIA) emulsion (containing 0.5mg of host protein), and inject 0.2mL at multiple points ...

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Abstract

The invention provides a preparation method and application of a recombinant vaccine host protein antibody, an expression vector which is used for preparing a recombinant vaccine and does not carry any exogenous gene is introduced into prokaryotic or eukaryotic host cells completely same as that used for preparing the recombinant vaccine to construct a transformant, the transformant is cultured and expanded, and extracted and purified by a method completely same as that used for preparing the recombinant vaccine to obtain a recombinant vaccine host protein; and the recombinant vaccine host protein antibody is obtained by animal immunization and antibody purification. The antibody provided by the invention can be applied to detection of host protein residues in recombinant hand-foot-mouth disease vaccines, is high in sensitivity, good in repeatability, and good in specificity, and is suitable for detection of the host protein residues of a biological product expressed by hansenula polymorpha, so that the application prospect is very wide.

Description

technical field [0001] The invention relates to a vaccine quality control method, in particular to a preparation method and application of a recombinant vaccine host protein antibody. Background technique [0002] Recombinant hand, foot and mouth disease vaccine is a genetic engineering product developed by applying Hansenula expression system, mainly used to prevent hand, foot and mouth disease in infants and young children. Residual host cell proteins in genetically engineered products are an important factor affecting the purity of products. Repeated use of products containing host cell proteins may cause side effects of vaccination and affect the efficacy of drugs. The composition of host cell protein (HCP) is complex and various, and will change due to different production processes and purification processes. The residual amount of HCP in biological products not only reflects the consistency between batches of products, but also is an important indicator to measure th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/085C07K16/10C07K1/36C07K1/34C07K1/30C07K1/18C07K16/06C12N15/41C12N15/81G01N33/569
CPCC07K14/005C07K16/065C07K16/1009C12N15/815C12N2770/32322G01N33/56983
Inventor 顾美荣李国顺张改梅陈磊赵丽丽郭林肖海峰刘司航肖霞刘建凯郑海发
Owner BEIJING MINHAI BIOTECH
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