SARS-CoV-2 recombinant protein subunit vaccine

A coronavirus and protein technology, applied in the field of genetic engineering, can solve the problems of difficult expression and low yield

Active Publication Date: 2021-03-26
ZHEJIANG VBIOSCI INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the total length of the S1 protein is 75kd, which is not easy to express and the yield is very low. It has become the top priority of research to modify the S1 protein to make it easy to express while retaining its receptor binding activity, so as to increase its yield.

Method used

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  • SARS-CoV-2 recombinant protein subunit vaccine
  • SARS-CoV-2 recombinant protein subunit vaccine
  • SARS-CoV-2 recombinant protein subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Construction of expression vector and screening of S protein-Ferritin fusion gene

[0057] 1.1 Acquisition and optimization of S protein gene sequence

[0058] The native S protein nucleotide sequence was obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / protein / YP_009724390.1?feature=any). The S1 and S2 regions were analyzed (https: / / zhanglab.ccmb.med.umich.edu / C-I-TASSER / 2019-nCov / ) to obtain the amino acid fragments of the original S1 protein. One of the bases was mutated for the original S1 nucleotide sequence to obtain the S1 sequence of the present invention (amino acid sequence: as shown in SEQ ID No.1, nucleotide sequence: as shown in SEQ ID No.8), and in On this basis, the protein sequences of SSO, SS1, SS2, and SS3 were respectively designed in a truncated manner. The amino acid sequences of the SS0, SS1, SS2 and SS3 proteins are respectively shown in SEQ ID No. 2-5, and the nucleotide sequences are shown in SEQ ID No. 9-12 in turn.

[0059] The o...

Embodiment 2

[0117] Example 2: Transferring pcDNA3.1-S protein particles into CHO cells.

[0118] 2.1 Large-scale extraction of pcDNA3.1-S protein Ferritin plasmid

[0119] The five pcDNA3.1-S1 protein Ferritin plasmids finally identified in Example 1 were subjected to plasmid extraction. The plasmid extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd.

[0120] 2.3 Plasmid transfection based on electroporation

[0121] A, culture medium preparation: Dialyze FBS (purchased from U.S. Gibco Company) with a 3500 dialysis bag, then configure 1L of CSC-03 medium containing 10% dFBS, and preheat it in the incubator after the configuration is completed. The temperature was set to 37°C.

[0122] B. Host cell preparation: the initial cell concentration for inoculation is 0.5×10 6 cells / ml CHO cell line (introduced from ATCC by Beijing Dingzhi Biotechnology Co., Ltd., introduction time: May 1, 2018, ATCC number: CCL61. After the cells were expanded and cultured in Beijing Di...

Embodiment 3

[0138] Example 3: Protein Purification

[0139] Chromatographic purification of fusion proteins

[0140] (1) The cell supernatants obtained in Example 2 containing five groups of optimized S protein-Ferritin were purified by chromatography with core 400 of GE. The specific steps are: centrifuge the five groups of corresponding cell supernatants obtained in Example 2 at 10,000×g for 30 min, collect the supernatant, filter at 0.22um, and concentrate the filtered supernatant by 30kDa membrane bag ultrafiltration for 10-20 times. as a sample solution.

[0141] (2) Wash the medium with 5 times column volume of distilled water.

[0142] (3) Wash the medium with equilibration buffer (PBS, pH 7.4) of 5 times column volume.

[0143] (4) Load 1 / 3CV each time.

[0144] (5) Collect the external water volume peak.

[0145] (6) Wash the chromatographic column with 1M NaOH+30% isopropanol, then equilibrate to neutral pH with PBS, and store the chromatographic column with 20% ethanol.

...

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Abstract

The invention relates to a polypeptide, a fusion protein of the polypeptide and helicobacter pylori ferritin and a subunit vaccine prepared from the fusion protein for preventing novel coronavirus (SARS-CoV-2) infection. Specifically, the polypeptide comprises an S1 protein or a fragment thereof (SS0, SS1, SS2 or SS3 protein), and the amino acid sequences of the S1 protein or the fragment thereofare derived from the sequence of an S protein of SARS-CoV-2, and optimization is carried out. The polypeptide can be expressed as a fusion protein by fusion with the helicobacter pylori ferritin performed with codon optimization via a hinge. The fusion protein of the polypeptide and the helicobacter pylori ferritin has the advantages of being high in expression level in CHO cells and easy to purify, and a high-titer neutralizing antibody against the SARS-CoV-2 can be generated after animals are immunized.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a recombinant protein subunit vaccine of a novel coronavirus (SARS-CoV-2) and a preparation method thereof. Background technique [0002] Coronaviruses are non-segmented single-stranded positive-sense RNA viruses that belong to the Nidovirales Coronaviridae subfamily Orthocoronavirinae. According to serotype and genome characteristics, the Coronaviridae subfamily is divided into α , β, γ and δ four genera. So far, a total of 7 coronaviruses can infect humans: including 229E and NL63 of the genus α, OC43 and HKU1 of the genus β, Middle East respiratory syndrome-associated coronavirus (MERSr-CoV), severe acute respiratory syndrome-associated coronavirus (SARSr-CoV) -CoV) and novel coronavirus (SARS-CoV-2). At present, it is of great significance to provide a vaccine against the new coronavirus that can effectively induce the body's immune response, block the spread of the virus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/215A61K39/385A61P31/14
CPCC07K14/005C12N15/85C12N5/0682A61K39/12A61P31/14A61K39/385C12N2770/20022C12N2770/20034C07K2319/735C12N2800/107C12N2800/22C12N2510/02A61K2039/6068A61K2039/53Y02A50/30
Inventor 宋春雨陈艳兰青陈小娟司欢欢侯野
Owner ZHEJIANG VBIOSCI INC
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