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Herpes virus glycoprotein gE recombinant protein, vaccine, preparation method and application

A herpes virus vaccine and protein technology, applied in the biological field, can solve the problems of unfavorable large-scale production, cumbersome cultivation process, weakened immune response, etc., and achieve the effect of quality control cost advantage, wide application prospect, and improved protein expression level

Pending Publication Date: 2022-07-05
上海博唯生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The existing recombinant herpes zoster vaccines on the market or in clinical research all use the glycoprotein gE of varicella-zoster virus prepared by mammalian cells, and there are many problems in the preparation of vaccines by mammalian cells, such as cumbersome culture process, High cost, complex cell components obtained after culture, many purification steps, not conducive to large-scale production, etc.
Since gE is a glycoprotein, the impact of glycosylation on protein function has not been clarified in the prior art research, and the contribution of the degree of glycosylation when the protein acts as an antigen to stimulate the human immune response has led researchers to give priority to the ability of glycosylation modification Better mammalian cells produce this protein, but the current accumulated research results show that the main factor of the pathogenesis of herpes zoster is the weakening of the body's cellular immune response to VZV, so the effective antigenic components are mainly T cell epitope polypeptides, Glycosylation modification has little effect on protein triggering immune response of human cells

Method used

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  • Herpes virus glycoprotein gE recombinant protein, vaccine, preparation method and application
  • Herpes virus glycoprotein gE recombinant protein, vaccine, preparation method and application
  • Herpes virus glycoprotein gE recombinant protein, vaccine, preparation method and application

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preparation example Construction

[0059] Preferably, the preparation method of the recombinant gE protein provided by the present invention uses the polynucleotide sequence shown in SEQ ID NO. Acidic recombinant engineering bacteria, culture, collect the bacteria, break the bacteria to obtain a lysate, and separate and purify the lysate to obtain gE protein. Specifically, the method includes the following steps:

[0060] 1) construct the above-mentioned gE protein recombinant expression vector;

[0061] 2) the above-mentioned recombinant expression vector is transformed into bacteria to construct recombinant engineering bacteria;

[0062] 3) Verify the recombinant engineered bacteria and obtain the correct constructed target recombinant engineered bacteria;

[0063] 4) Cultivate the target recombinant engineered bacteria under specific conditions, collect the cultured bacteria and purify the recombinant gE protein.

[0064] In step 1), primers are designed to amplify the coding sequence of the target gE pro...

Embodiment 1

[0116] Example 1 Construction of recombinant gE protein engineering strain

[0117] 1. Selection of gE protein-coding genes and optimal design of codons

[0118] The amino acid sequence encoding the gE protein refers to the gE protein sequence of the Japanese vaccine strain Oka, as shown in SEQ ID NO.2.

[0119] The corresponding nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO.2 is modified, and the codons used in the E. coli expression system are used as frequently as possible, and at the same time, the transcription factor binding region and repetition that may affect the expression are avoided. Sequence and RNA higher order structure. The gE protein coding gene sequence obtained after codon optimization is shown in SEQ ID NO.1.

[0120] 2. Construction of recombinant expression vector of gE protein

[0121] The gE protein coding gene sequence was designed and the whole gene was synthesized to obtain the nucleotide sequence shown in SEQ ID NO.1. ...

Embodiment 2

[0127] The preparation technology of embodiment 2 recombinant gE protein

[0128] 1. Fermentor culture of pET28a-gE-BL21(DE3) engineering strain

[0129] The pET28a-gE-BL21(DE3) expression strain stored in glycerol tube was inoculated into LB medium at a ratio of 1:1000 (v / v), and cultured in shake flasks for 16-24 hours to obtain seed liquid. The seed liquid was inoculated into the fermentation medium at a ratio of 1:20 (v / v) to make E. coli cells grow, and the fermentation was terminated after 4 hours of induction. The fermented cells were collected after centrifugation for subsequent purification.

[0130] The composition of the fermentation medium is: disodium hydrogen phosphate dodecahydrate 1.7%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.0225%, anhydrous calcium chloride 0.00113%, glucose 0.5%, ammonium sulfate 0.2%, yeast powder 1%, tryptone 1%, sodium chloride 0.5%. Feed (mass ratio) during fermentation and culture: 20% yeast powder.

[0131] The fer...

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Abstract

The invention belongs to the technical field of biology, discloses a recombinant expression method of varicella-zoster virus glycoprotein gE, and relates to optimized screening of a gE protein coding gene, construction of a recombinant expression vector, acquisition of a target strain and fermentation culture of the target strain, the sequence of the gE protein coding gene is as shown in SEQ ID NO.1, and the sequence of the gE protein coding gene is as shown in SEQ ID NO.2. The gE protein can be applied to development of herpes zoster vaccines. The recombinant gE protein antigen prepared by the invention is high in yield, good in purity and excellent in immunogenicity, and the problems that a large amount of gE protein cannot be directly soluble-expressed in escherichia coli and the yield is low are solved. The invention further discloses a novel adjuvant composition for vaccines and application of the novel adjuvant composition. The gE protein and a novel adjuvant composition are compounded to prepare a vaccine, so that the vaccine has an obvious and excellent immune enhancement effect and a wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a varicella-zoster virus glycoprotein gE recombinant protein, a vaccine, a preparation method and applications thereof. Background technique [0002] Varicella-zoster virus (varicella-zoster virus, VZV), also known as type 3 human herpes virus (humanherpesvirus3, HHV-3), it and type 1 and type 2 herpes simplex virus (herpes simplex virus type 1and2, HSV-1and HSV) -2) Both belong to human alpha-herpesvirus. VZV is mainly composed of four-layer structure, from inside to outside: round baculovirus core composed of double-stranded viral DNA; icosahedral symmetrical structure with a diameter of about 100nm composed of 162 capsids, namely nucleocapsid; Covering the outside of the nucleocapsid, a layer of amorphous material composed of proteins and enzymes, also known as the cortex (tugement); the outermost layer is the capsule, which is a typical lipid bilayer structure, with a large number...

Claims

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Application Information

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IPC IPC(8): C12N15/38C12N15/70C12N1/21C07K14/035C07K14/04C07K16/08G01N33/569A61K39/245A61K39/25A61P31/22
CPCC07K14/005C12N15/70C07K16/088C07K16/087G01N33/56994A61K39/12A61P31/22C12N2710/16722C12N2710/16734C12N2710/16622C12N2710/16634G01N2333/04G01N2333/035
Inventor 夏慧傅文彬茅倩宇王圆
Owner 上海博唯生物科技有限公司
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