Novel flow method adeno-quadruplet virus-like particle and preparation method and application thereof

A flow method adeno-virus-like technology, applied in the field of new flow method adeno-quadruplet virus-like particles and their preparation, can solve the problems of inability to quantify anchored proteins, difficult to control multi-gene expression, etc., and achieve high release rate and strong immunity. Original, immunogenic effect

Active Publication Date: 2022-07-26
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the existing chimeric virus-like particle transformation strategy is the extracellular domain replacement strategy, which is mainly based on the gene transfection level of the fusion sequence, which has the problems of difficulty in controlling the expression of multiple genes and quantification of anchoring proteins

Method used

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  • Novel flow method adeno-quadruplet virus-like particle and preparation method and application thereof
  • Novel flow method adeno-quadruplet virus-like particle and preparation method and application thereof
  • Novel flow method adeno-quadruplet virus-like particle and preparation method and application thereof

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preparation example Construction

[0054] The preparation method of the novel flow method adeno-quadrup virus-like particle of the present invention specifically comprises the following steps:

[0055] Step 1. Optimization and synthesis of target gene

[0056] According to the codon preference of insect cells, the M and F genes of NDVNA-1 strain, the HA gene of H9N2AIV strain, the VP2 gene of IBD SH99 strain, and the Fiber-2 gene of HB 1510 strain were codon-optimized; the optimized sequence of M gene It is shown in SEQ ID NO.1; the optimized sequence of F gene is shown in SEQ ID NO.2; the optimized sequence of HA gene is shown in SEQ ID NO.3; the optimized sequence of VP2 gene is shown in SEQ ID NO.4; The optimized sequence of Fiber-2 gene is shown in SEQ ID NO.5;

[0057] Step 2. Construction of Recombinant Shuttle Plasmid

[0058] According to the optimized sequence of each gene and the information of pFastbac-1 vector, specific primers and M13 identification primers were designed, plasmids were synthesized ...

Embodiment 1

[0070] Example 1 Optimization and synthesis of target gene

[0071] According to the M gene and F gene of NDV NA-1 strain published in GenBank (GenBank accession number: DQ659677), the HA gene of A / chicken / Jilin / SJ150 / 2012 (H9N2) strain (GenBank accession number: KF886457.1), The VP2 gene of IBDSH99 strain (GenBank accession number: LM651365.1) and the Fiber-2 gene of HB 1510 strain (GenBank accession number: KU587519.1), in order to increase the expression of chimeric virus-like particles, according to the codon preference of insect cells Codon-optimized for sex. At the same time, according to the information of the pFastbac-1 vector, the propolis signal peptide sequence, HIS tag sequence, TEV cleavage sequence and GPI signal peptide sequence were sequentially introduced into the HA gene, VP-2 gene and Fiber-2 gene.

[0072] The optimized sequence was synthesized by Shanghai Sangon Bioengineering (Shanghai) Co., Ltd.

[0073] The optimized sequence of M gene is shown in SEQ...

Embodiment 2

[0078] Example 2 Construction of Recombinant Shuttle Plasmid

[0079] (1) Primer design

[0080] According to the optimized sequence of M gene, SEQ ID NO.1, the optimized sequence of F gene, SEQ ID NO.2, the optimized sequence of HA gene, SEQ ID NO.3, the optimized sequence of VP2 gene, SEQ ID NO.4, and the optimized sequence of Fiber-2 gene Design specific primers based on the post sequence SEQ ID NO.5 and pFastbac-1 vector information, and design M13 identification primers. The specific primer sequences are as follows:

[0081] M-Xba I-F: 5'-TCTAGAATGGATAGCAGTCGTACGATTGGC-3',

[0082] M-HindIII-R: 5'-AAGCTTTTATTTTCTGAATGGATTGTATTTCGCG-3';

[0083] F-Xba I-F: 5'-TCTAGAGGATCCTTGTGGAAGGTTTTGATCCCAT-3',

[0084] F-Kpn I-R: 5'-GGTACCTTAAGCACGAGTAGTTGCGCG-3';

[0085] HA-Xba I-F: 5'-TCTAGAATGGAACAGGTGTCGCTC-3',

[0086] HA-Kpn I-R: 5'-GAGCTCTTAGATACAGATGTTACACCTACACG-3';

[0087] VP2-Sac I-F: 5'-GAGCTCGCCACCATGAAGTTCC-3',

[0088] VP2-HindIII-R: 5'-AAGCTTTTAGGTCAGCAGACCCATG...

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Abstract

A new flow method adeno-quadruplet virus-like particle and its preparation method and application belong to the field of quadruple vaccine research and development. Rescue, preparation and purification of chimeric virus-like particles, and finally obtain chimeric virus-like particles NDV‑AIV‑IBDV‑FAdV4 cVLPs. The new flow method AAV-like particle prepared by the preparation method of the present invention has the following advantages: the new flow method AAV-like particle can repeatedly display foreign antigens with high density, and has strong immunogenicity; It does not contain viral nucleic acid, green and safe, which is conducive to the purification of new flow glands, and conforms to the modern green and safe breeding concept; the vaccine seed virus is matched with the popular virus strain; one shot for multiple prevention, simplifying the vaccine immunization procedure, avoiding a variety of Mutual interference of vaccines; suitable for large-scale suspension culture and mass production.

Description

technical field [0001] The invention belongs to the technical field of quadruple vaccine research and development, and in particular relates to a novel flow method adeno-quadruplex virus-like particle and a preparation method and application thereof. Background technique [0002] Newcastle disease (ND), avian influenza (AI), infectious bursal disease (IBD) and fowl adenovirus type 4 (FAd) are the current threats to domestic poultry farming. The major animal diseases in the industry have caused serious economic losses. According to recent molecular epidemiological survey data, the main epidemic strain of Newcastle disease in my country is genotype V II, and the main vaccine strain is genotype II LaSota attenuated strain. The mismatch of genotypes and the insecurity of live vaccines lead to The purification of Newcastle disease is difficult. The results of avian influenza serological investigations show that the isolation rate of H9N2 subtype ranks first among all AIV subtype...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/866C12N5/10C12N15/45C12N15/44C12N15/34C12N15/40A61K39/295A61K39/17A61K39/12A61K39/145A61K39/235A61P31/14A61P31/16
CPCC12N15/86C07K14/005A61K39/12A61P31/14A61P31/16C12N2710/14043C12N2760/18122C12N2760/16122C12N2760/16123C12N2710/10222C12N2760/18123C12N2710/10223C12N2720/10022C12N2720/10023A61K2039/5258A61K2039/70A61K2039/552
Inventor 丁壮李金斗丁佳欣邵亚男郭春红冯嘉轩辛梅
Owner JILIN UNIV
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