CN-119979483-B - Preparation method and application of recombinant adenovirus capable of spontaneously generating virus-like particles

Abstract

The invention discloses a preparation method and application of a recombinant adenovirus capable of spontaneously generating virus-like particles, and relates to the technical field of biology. The preparation method of the recombinant adenovirus comprises the following steps of connecting a virus membrane protein gene with a bud promoting EARP gene, constructing and obtaining a recombinant exogenous virus membrane protein gene, inserting the recombinant exogenous virus membrane protein gene into an E3 region of an adenovirus skeleton plasmid to obtain a recombinant skeleton plasmid, inserting the recombinant exogenous virus membrane protein gene into an E1 region of an adenovirus shuttle plasmid to obtain a recombinant shuttle plasmid, and co-transfecting the recombinant skeleton plasmid and the recombinant shuttle plasmid into a host cell to obtain the recombinant adenovirus capable of spontaneously generating virus-like particles (VLPs). The recombinant adenovirus constructed by the preparation method can spontaneously assemble VLP containing virus outer membrane proteins in an in vivo exogenous infection process, so that the immunogenicity of the vaccine can be greatly improved, the VLP production process is simplified, and the production cost of the vaccine is reduced.

Inventors

• ZHAO LING
• FU ZHENFANG
• ZHANG YUAN
• WANG CAIQIAN
• Fang Daiying
• WANG ZONGMEI
• ZHENG YANHONG
• CHEN FEIYU
• FENG YAYA
• ZHOU MING

Assignees

• 华中农业大学

Dates

Publication Date

20260512

Application Date

20250210

Claims (5)

1. 1. A method for preparing a recombinant adenovirus that spontaneously produces virus-like particles, comprising the steps of: connecting an exogenous viral membrane protein gene with an EARP gene to construct a recombinant exogenous viral membrane protein gene; Inserting the recombinant exogenous viral membrane protein gene into an E3 region of an adenovirus backbone plasmid to obtain a recombinant backbone plasmid; Inserting the recombinant exogenous viral membrane protein gene into an E1 region of an adenovirus shuttle plasmid to obtain a recombinant shuttle plasmid; the spontaneously virus-like particle-producing recombinant adenovirus was prepared as follows: co-transfecting the recombinant backbone plasmid and the recombinant shuttle plasmid into a host cell; The nucleotide sequence of the EARP gene is shown as SEQ ID NO. 17; the adenovirus skeleton plasmid is pBHGcre/loxp plasmid; the adenovirus shuttle plasmid is a pDC315 plasmid; the nucleotide sequence of the recombinant exogenous virus membrane protein gene is shown in any one of SEQ ID NO. 29-31.
2. 2. A recombinant adenovirus spontaneously producing virus-like particles, said recombinant adenovirus being prepared by a process comprising the steps of: connecting an exogenous viral membrane protein gene with an EARP gene to construct a recombinant exogenous viral membrane protein gene; Inserting the recombinant exogenous viral membrane protein gene into an E3 region of an adenovirus backbone plasmid to obtain a recombinant backbone plasmid; Inserting the recombinant exogenous viral membrane protein gene into an E1 region of an adenovirus shuttle plasmid to obtain a recombinant shuttle plasmid; the spontaneously virus-like particle-producing recombinant adenovirus was prepared as follows: co-transfecting the recombinant backbone plasmid and the recombinant shuttle plasmid into a host cell; The nucleotide sequence of the EARP gene is shown as SEQ ID NO. 17; The nucleotide sequence of the recombinant exogenous virus membrane protein gene is shown in any one of SEQ ID NO. 29-31; the adenovirus skeleton plasmid is pBHGcre/loxp plasmid, and the adenovirus shuttle plasmid is pDC315 plasmid.
3. 3. Use of the recombinant adenovirus according to claim 2 for the preparation of a recombinant adenovirus vaccine.
4. 4. A recombinant adenovirus vaccine comprising the recombinant adenovirus of claim 2.
5. 5. The recombinant adenovirus vaccine according to claim 4, further comprising a pharmaceutically acceptable adjuvant.

Description

Preparation method and application of recombinant adenovirus capable of spontaneously generating virus-like particles Technical Field The invention relates to the technical field of biology, in particular to a preparation method and application of a recombinant adenovirus capable of spontaneously generating virus-like particles. Background New emergent infectious diseases are continuously emerging to promote urgent demands of safe and efficient novel vaccines. In recent decades, with the rapid development of fields of genetic engineering, molecular cellular immunology, structural biology, bioinformatics, computational biology, nanotechnology, synthetic biology, etc., new vaccine platforms such as mRNA, synthetic DNA, recombinant viral vectors, virus-like particles (viruses-LIKE PARTICLE, VLPS), etc., have been gradually established and matured, and have been increasingly colored during the fight against new coronary epidemic. Virus-like particle (VLPs) technology provides an alternative platform for developing effective vaccines to control infectious diseases, in parallel with mRNA and viral vector vaccines. VLPs are mostly nanoparticles formed by in vitro self-assembly of one or more viral structural proteins, free of genetic material, greatly improving the safety of such vaccines. VLPs are highly similar in their antigenic structure, i.e., spatial distribution, to their viral origin, and thus can effectively elicit both humoral and cellular immune responses. In addition, their morphology, size, and dimensions are highly variable, varying from 20 to 200 nanometers in size. This size range allows the body to freely drain them into lymph nodes and be more easily taken up by Antigen Presenting Cells (APCs), particularly Dendritic Cells (DCs), and then antigen processed and presented by Major Histocompatibility Complex (MHC) class II molecules. In the current state of the art, only a few structural proteins of non-enveloped viruses (e.g. HPV L1 protein, HBV surface antigen HBsAg, norovirus VP 1) are able to spontaneously assemble into VLPs. In contrast, most enveloped viruses cannot spontaneously form VLPs through only a single viral structural protein. The influenza virus VLP is formed by coexpression of matrix protein M1 and surface glycoprotein HA (hemagglutinin) or NA (neuraminidase), the novel coronavirus is formed into an intact VLP by coexpression of spike protein S, membrane protein M and envelope protein E, the Zika virus prM and E proteins are coexpression to form the VLP, and the rabies virus VLP is formed by coexpression of glycoprotein G and matrix protein M. Co-expression of the multi-structural proteins complicates the VLP formation process. VLPs of the prior art have been developed primarily by cloning viral structural protein genes into expression vectors and then expressing these genes in vitro in bacterial, yeast, mammalian and insect cell systems, with each of the above-described expression systems having advantages but being somewhat deficient in some respects, some of the key challenges associated with VLPs include lower stability, difficulty in downstream processing, poor batch-to-batch stability, high production costs and high sensitivity to storage environmental conditions. Mass production and purification of VLPs requires different processes, such as density gradients and even chromatography, to make the final formulation. These complex processes are costly and time consuming. This also results in difficulties in the industrial scale production of VLPs and the need for multiple quality control measures. Compared with the complex construction process, high cost, complicated production quality control steps and severe storage and transportation conditions of the in-vitro self-assembled VLPs, the adenovirus vector has the remarkable advantages of high virus yield, low production cost, simple purification steps, stable physicochemical properties, easy storage and transportation and the like. With the intensive research and continuous exploration of adenovirus biology and immunology, various adenovirus vector vaccines are continuously developed. In the prior art, adenovirus vector vaccines integrate exogenous genes into the E1 or E3 region of the adenovirus genome mainly by reverse genetic technology, and express exogenous genes in target cells after infecting the cells so as to stimulate the organism to generate immune response or perform gene therapy. For viral membrane proteins, the prior art only expresses intact membrane proteins to the surface of the infected cell membrane, however, the anchor site for the membrane proteins is limited, and expression of membrane protein antigens alone on the cell surface may limit the large expression of foreign genes of adenovirus vector vaccines. Furthermore, the prior art only produces membrane proteins in situ (immunization/injection site) and the antigen is not recognized by immune cells remote from the injection site, so that the prior art vect