US-12618083-B2 - Recombinant adenovirus expressing African swine fever virus EP153R-EP402R protein and construction method thereof

Abstract

The present disclosure provides a recombinant adenovirus for expressing African swine fever virus (ASFV) EP153R-EP402R protein and a construction method thereof, and belongs to the technical field of genetic engineering. In the present disclosure, a recombinant adenovirus vector pAD-CMV-EGFP-EP153R-EP402R is obtained through a series of intermediate processes using a recombinant adenovirus shuttle vector pENTRE-EGFP-TOPO; the recombinant adenovirus vector is linearized to transfect AD293 cells, a recombinant virus is screened according to cytopathy formed by adenovirus infection, an adenovirus packaging process is achieved, and the recombinant adenovirus for expressing ASFV EP153R-EP402R protein is obtained, laying a foundation for the construction of a recombinant adenovirus vaccine for expressing the ASFV EP153R-EP402R protein.

Inventors

• Hong Jia
• Hongfei Zhu
• Xiaoyu Guo
• Weifeng Yuan
• Shuai CUI
• Yang Wang
• Ting Xin

Assignees

• INSTITUTE OF ANIMAL SCIENCES OF CHINESE ACADEMY OF AGRICULTURAL SCIENCES

Dates

Publication Date

20260505

Application Date

20221122

Priority Date

20211126

Claims (5)

1. 1 . A recombinant adenovirus vector expressing African swine fever virus (ASFV) EP153R-EP402R protein, wherein based on a pAD-CMV-3×FLAG adenovirus vector, EP153R-EP402R gene is introduced to construct a recombinant adenovirus vector pAD-CMV-EGFP-EP153R-EP402R; the EP153R-EP402R gene has a nucleotide sequence shown in SEQ ID NO: 1.
2. 2 . A construction method of the recombinant adenovirus vector expressing ASFV EP153R-EP402R protein according to claim 1 , comprising the following steps: step 1, synthesizing an EP153R-EP402R gene, and adding four bases, CACC, before a start codon of the EP 153R-EP402R gene; step 2, conducting TOPO cloning on the EP153R-EP402R gene obtained in step 1 and a pENTRE-EGFP-TOPO vector to obtain pENTR-EGFP-ASFV-EP153R-EP402R; and step 3, recombining the pENTR-EGFP-ASFV-EP153R-EP402R obtained in step 2 on an adenovirus backbone vector pAD-CMV-3×FLAG through LR recombination reaction to obtain the recombinant adenovirus vector pAD-CMV-EGFP-EP153R-EP402R.
3. 3 . A recombinant adenovirus packaging method, wherein the recombinant adenovirus vector pAD-CMV-EGFP-EP153R-EP402R according to claim 1 is single-digested with PacI, and a linearized plasmid is used for transfection; 293t cells are transfected to achieve recombinant adenovirus packaging.
4. 4 . The recombinant adenovirus packaging method according to claim 3 , comprising the following steps: step 1, single-digesting the recombinant adenovirus vector pAD-CMV-EGFP-EP153R-EP402R with a restriction endonuclease PacI, and transfecting the linearized plasmid into AD293 cells; step 2, after transfection until cell detachment, rounding and space enlargement, collecting cells and a supernatant, namely P1 recombinant adenovirus; and step 3, infecting 293t cells with the P1 recombinant adenovirus, and observing cell status.
5. 5 . The recombinant adenovirus packaging method according to claim 4 , wherein in step 2, the cells and the supernatant are collected, freeze-thawed three times at −80° C., and centrifuged at 12,000×g for 10 min to collect a supernatant.

Description

CROSS REFERENCE TO RELATED APPLICATION This patent application claims the benefit and priority of Chinese Patent Application No. 202111425085.4, filed on Nov. 26, 2021, the disclosure of which is incorporated by reference herein in its entirety as part of the present application. REFERENCE TO SEQUENCE LISTING A computer readable XML file entitled “GWP20220700545”, that was created on Nov. 10, 2022, with a file size of about 3,612 bytes, contains the sequence listing for this application, has been filed with this application, and is hereby incorporated by reference in its entirety. TECHNICAL FIELD The present disclosure relates to the technical field of genetic engineering, in particular to a recombinant adenovirus expressing African swine fever virus (ASFV) EP153R-EP402R protein and a construction method thereof. BACKGROUND ART African swine fever (ASF) is an acute, severe and highly contagious infectious disease caused by African swine fever virus (ASFV). Clinical manifestations include fever, skin cyanosis, obvious bleeding in lymph nodes, kidneys, and gastrointestinal mucosa. ASFV is the sole member of the genus Asfivirus within the family Asfarviridae, and the only DNA arbovirus at present. It is generally considered to have only one serotype. Internationally, ASFV is divided into 24 genotypes according to a 478 bp nucleic acid sequence at the terminal of ASFV B646L gene, most of which belong to genotype II in China. ASFV can be transmitted by domestic pigs, wild boars and soft ticks, and mainly attacks swine monocytes and macrophages. The virion has a diameter of 175-215 nm. It is icosahedral and enveloped, structurally comprising, from the inside out, a nucleoid, a nucleocapsid, an inner envelope, a capsid, and external envelope. The genome is double-stranded linear DNA with a size of 170-193 kb and 150-167 open reading frames, encoding more than 50 structural proteins and more than 100 non-structural proteins. The main substances involved in the structural composition of virions include structural proteins, gene transcription and RNA modification enzymes. As the main components of virions, structural proteins play important roles in ASFV adsorption, invasion and replication and other infectious processes. The encoding proteins mainly include p72, p49, p30, p54, EP153R, CD2v, and the like. The EP153R protein is encoded by the EP153R gene located in the ASFV EcoRI E9 genome fragment, is approximately 18 kDa in size, and is transcribed at the early and late stages of viral infection. The insertion of the marker gene LacZ into EP153R does not change the growth rate of the virus in vitro and the sensitivity and drug resistance of the virus, but it eliminates the hemosorption induced by ASFV-infected cells. EP153R stabilizes the interaction between EP402R and other ligands and participates in mediating apoptosis. CD2v protein is named because it is similar to the adhesion molecule CD2 on the surface of T cells. It is encoded by the EP402R gene and has a size of approximately 45.3 kDa. It is embedded in the outer surface of the viral envelope and is a glycoprotein composed of a signal peptide, a transmembrane domain, and a plasmic tail domain containing 147 amino acids. The CD2v protein enables virions to attach to erythrocytes and acts as an essential protein for erythrocytes to bind to infected cells and extracellular virions. The expression of CD2v is associated with the spread of ASFV among domestic pigs, which can disrupt the function of lymphocytes. At present, there is no recombinant adenovirus against EP153R-EP402R and a feasible method for preparing the recombinant adenovirus. SUMMARY An objective of the present disclosure is to provide a recombinant adenovirus expressing ASFV EP153R-EP402R protein and a construction method thereof, so as to solve the above-mentioned problems existing in the prior art and lay a technical foundation for the study of ASF candidate vaccines. To achieve the above objective, the present disclosure provides the following solutions: The present disclosure provides a recombinant adenovirus vector expressing ASFV EP153R-EP402R protein, where based on a pAD-CMV-3×FLAG adenovirus vector, EP153R-EP402R gene is introduced to construct a recombinant adenovirus vector pAD-CMV-EGFP-EP153R-EP402R; the EP153R-EP402R gene has a nucleotide sequence shown in SEQ ID NO: 1. The present disclosure further provides a construction method of the foregoing recombinant adenovirus vector expressing ASFV EP153R-EP402R protein, including the following steps: step 1, synthesizing an EP153R-EP402R gene, and adding four bases, CACC, before a start codon of the EP153R-EP402R gene; step 2, conducting TOPO cloning on the EP153R-EP402R gene obtained in step 1 and a pENTRE-EGFP-TOPO vector to obtain pENTR-EGFP-ASFV-EP153R-EP402R; and step 3, recombining the pENTR-EGFP-ASFV-EP153R-EP402R obtained in step 2 on an adenovirus backbone vector pAD-CMV-3×FLAG through LR recombination reaction to obtain the reco