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Preparation method of recombinant novel coronavirus subunit vaccine

A technology of subunit vaccine and coronavirus, which is applied in the field of preparation of recombinant new coronavirus subunit vaccine, can solve the problems of changing protein structure and lower expression efficiency of recombinant new coronavirus subunit vaccine, and achieves strong immunogenicity and low The effect of cost and high productivity

Active Publication Date: 2020-12-18
ZHEJIANG PUKANG BIOTECH +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, intramolecular adjuvants will change the protein structure and reduce the expression efficiency of the recombinant new coronavirus subunit vaccine

Method used

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  • Preparation method of recombinant novel coronavirus subunit vaccine
  • Preparation method of recombinant novel coronavirus subunit vaccine
  • Preparation method of recombinant novel coronavirus subunit vaccine

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preparation example Construction

[0030] The present invention relates to a preparation method of a recombinant novel coronavirus subunit vaccine, comprising:

[0031] a) Transfect the plasmid containing the nucleic acid fragment shown in SEQ ID NO: 2 into Escherichia coli, and the nucleic acid fragment can express the RBD-TT fusion protein;

[0032] b) culturing the Escherichia coli to express the RBD-TT fusion protein, disrupting the bacteria and isolating the crude inclusion body extract of the RBD-TT recombinant protein;

[0033] c) dissolving the crude inclusion body extract in a denaturing solution containing urea, and then purifying it by anion exchange chromatography to obtain a crude sample of RBD-TT recombinant protein;

[0034] d) diluting the crude pure sample of the RBD-TT recombinant protein with a diluent;

[0035] Each liter of the diluent contains: 450-510 grams of urea, 70-86 grams of arginine, 0.3-0.7 grams of reduced glutathione, 15-25 grams of glycerin, and buffer substances, pH=9-10;

...

Embodiment 1

[0069] Example 1 Optimization of diluent

[0070] (1) Utilizing the codon merger principle and the codon usage frequency of Escherichia coli, using the amino acid sequence described in SEQ ID NO: 1 as a template, design the nucleotide sequence SEQ ID NO: 2 for expressing the RBD-TT recombinant protein, And the synthesized gene was inserted into pET28a Escherichia coli expression plasmid by the method of seamless cloning to obtain the recombinant expression plasmid. The resulting recombinant expression plasmid was transformed into E. coli BL21 (DE3) competent cells by 42-degree heat activation method, and monoclonal screening was carried out on LB solid medium plates to obtain recombinant E. coli monoclonal colonies expressing the target protein. Escherichia coli monoclonal colonies were cultivated overnight at 37°C with 5 ml of LB liquid medium to obtain recombinant Escherichia coli seed liquid expressing the target protein.

[0071] (2) Take recombinant Escherichia coli seed...

Embodiment 2

[0106] The screening of embodiment 2 recombination conditions

[0107] Operate according to steps (1)~(5) in Example 1, choose diluent 4 as the diluent, and compare the refolding conditions through three schemes: scheme 1, use refolding solution 1 and refolding solution 3 in turn for tangential flow ultrafiltration refolding; plan 2, tangential flow ultrafiltration refolding with refolding solution 2 and refolding solution 3 in sequence; Refolding by directional flow ultrafiltration; the replacement ratio of renaturation liquid and protein liquid used for each tangential flow ultrafiltration renaturation is 1:3, and the feeding pressure of each tangential flow ultrafiltration is 25bar, and the tangential flow ultrafiltration The pore size of the ultrafiltration membrane cassette is 10 kd).

[0108] Take the following step (6):

[0109] (6) The resulting RBD-TT recombinant protein refolding sample was diluted to 0.1 mg / mL, and the antigen titer under different refolding condi...

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Abstract

The invention relates to the field of vaccine preparation, in particular to a preparation method of a recombinant novel coronavirus subunit vaccine. The method comprises the following steps: transfecting plasmids into which a nucleic acid fragment corresponding to RBD-TT recombinant protein is inserted into Escherichia coli; culturing the Escherichia coli to enable the Escherichia coli to expressthe RBD-TT fusion protein, and breaking thalli and separating an inclusion body crude extract of the RBD-TT recombinant protein; dissolving the inclusion body crude extract with a urea-containing denaturing solution, and then performing purifying through anion exchange chromatography to obtain an RBD-TT recombinant protein crude pure sample; diluting the RBD-TT recombinant protein crude pure sample with a diluent; performing filtering, and performing renaturation with a renaturation solution to obtain renaturation protein; and purifying the recombinant protein renaturation protein through anion exchange chromatography.

Description

technical field [0001] The invention relates to the field of vaccine preparation, in particular to a method for preparing a recombinant novel coronavirus subunit vaccine. Background technique [0002] The novel coronavirus (SARS-CoV-2) is a newly discovered human coronavirus that can cause acute infectious pneumonia, which can lead to acute respiratory distress syndrome or septic shock, and even death in severe cases. At present, there is no clinically effective drug that has been proven to be effective in the treatment of new coronavirus infection. Prophylactic vaccination is the most economical and effective means of virus prevention and control. Therefore, it is necessary to develop a safe and effective new coronavirus vaccine Research. [0003] According to the characteristics of the genome, the SARS-CoV-2 virus belongs to the genus β of the subfamily Coronavirus (CoV) Sarbecovirus Subgenus members. The length of the SARS-CoV-2 virus genome is about 29.8Kb, 2 / 3 of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/215A61K39/39A61P31/14C12N15/62C12N15/70C07K19/00C07K1/36C07K1/34C07K1/18
CPCA61K39/12A61K39/39A61P31/14C07K14/005C12N15/70C12N2770/20022C12N2770/20034C07K2319/00A61K2039/55505C12N2770/20051A61K39/215C07K1/145C07K14/165C07K2319/55
Inventor 毕胜利侯云德毛江森高福武桂珍秦川许文波毛子安苏秋东伊瑶高孟陈刚庄昉成吕杭军陆绍红
Owner ZHEJIANG PUKANG BIOTECH
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