阅读说明：本技术 表达非洲猪瘟病毒p54、p30、E248R蛋白的重组腺病毒及构建方法 (Recombinant adenovirus expressing African swine fever virus p54, p30 and E248R proteins and construction method ) 是由 周远成 邝声耀 阴文奇 于 2020-06-15 设计创作，主要内容包括：本发明公开了一种表达非洲猪瘟病毒p54、p30和E248R蛋白的重组腺病毒及构建方法,该重组腺病毒感染细胞后可以同步表达非洲猪瘟p54、p30和E248R蛋白,其中p54和p30蛋白融合表达,E248R通过自裂解2A信号肽与p54和p30串联表达。本发明构建了能同时表达p54、p30和E248R蛋白的重组腺病毒载体,使用该载体与腺病毒骨架系统共转染细胞后获得可表达非洲猪瘟病毒p54、p30和E248R蛋白的重组腺病毒,使用该重组腺病毒免疫试验动物后可以刺激试验动物产生特异性的抗体,为非洲猪瘟疫苗的开发提供了新的试验数据。(The invention discloses a recombinant adenovirus for expressing African swine fever virus p54, p30 and E248R proteins and a construction method thereof, the recombinant adenovirus can synchronously express African swine fever p54, p30 and E248R proteins after infecting cells, wherein the p54 and p30 proteins are fused and expressed, and the E248R is expressed in series with p54 and p30 through self-cleavage 2A signal peptide. The invention constructs a recombinant adenovirus vector capable of simultaneously expressing p54, p30 and E248R proteins, obtains the recombinant adenovirus capable of expressing the proteins of African swine fever virus p54, p30 and E248R after the vector and an adenovirus skeleton system are used for cotransfecting cells, can stimulate a test animal to generate a specific antibody after the test animal is immunized by the recombinant adenovirus, and provides new test data for the development of an African swine fever vaccine.)

1. A nucleotide sequence for coding African swine fever virus p54, p30 and E248R proteins, wherein the nucleotide sequence is shown as SEQ ID NO: 1 is shown.

2. A recombinant adenovirus vector expressing the p54, p30 and E248R proteins of african swine fever virus, comprising the nucleotide sequence of claim 1.

3. The method for constructing the recombinant adenovirus vector for expressing the proteins of African swine fever virus p54, p30 and E248R of claim 2, which comprises the following steps: artificially synthesizing the amino acid sequence shown in SEQ ID NO: 1, and inserting the nucleotide sequence into a pDC316-mCMV-EGFP vector to obtain the vector.

4. A recombinant adenovirus expressing p54, p30 and E248R proteins of african swine fever virus, comprising the nucleotide sequence of claim 1 or the recombinant adenovirus vector of claim 3.

5. The method for constructing the recombinant adenovirus expressing the proteins of African swine fever virus p54, p30 and E248R of claim 4, comprising the steps of:

(1) artificially synthesizing the amino acid sequence shown in SEQ ID NO: 1, and inserting the nucleotide sequence into a pDC316-mCMV-EGFP vector;

(2) and (2) co-transfecting the vector constructed in the step (1) and a pBHGloxdel E13cre vector into 293A cells, and then culturing to obtain the recombinant vector.

6. Use of the recombinant adenovirus expressing proteins p54, p30 and E248R of African swine fever virus according to claim 4 in the preparation of an African swine fever vaccine.

7. An African swine fever vaccine, comprising the recombinant adenovirus expressing the proteins of African swine fever virus p54, p30 and E248R of claim 4.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a recombinant adenovirus vector for expressing African swine fever virus p54, p30 and E248R proteins, a recombinant adenovirus and a construction method.

Background

African Swine Fever Virus (ASFV) is a nucleoplasm large DNA virus and is the only arbovirus, can infect domestic pigs and wild pigs, and has stronger infectivity and pathogenicity. The clinical symptoms of swine infection African swine fever are similar to swine fever, swine erysipelas and other diseases, the severity of the symptoms is different from the virulence, infection dosage and infection route of ASFV, and the symptoms can be divided into acute infection, subacute infection, invisible infection and the like according to the severity of the clinical symptoms. Acute infections are characterized mainly by anorexia, hyperpyrexia, leukopenia, bleeding of the skin and internal organs, with a high mortality rate, some of which can reach 100%. Subacute infections are characterized by transient thrombocytopenia and cytopenia, with observable foci of bleeding that can lead to respiratory changes, abortion and death, with a lower mortality rate than acute infections. At present, no specific vaccine and therapeutic drug aiming at African swine fever exist, so that the forced catching and killing mode is adopted at home and abroad to treat the sick swine herd.

The African swine fever is introduced into China in 2018, and the epidemic situation reported nationwide at present exceeds 150. The outbreak of African swine fever causes huge economic loss to the domestic pig breeding industry. In the time of more than one year of the African swine fever in China, the stock volume of breeding pigs in China is reduced from about 4000 million before the epidemic situation appears to about 1900 million of the lowest peak, and according to the statistics of incompleteness, the direct and indirect economic losses caused by the African swine fever can exceed 1 trillion yuan. After the outbreak of the African swine fever, the research on the African swine fever vaccine including subunit vaccine, polypeptide vaccine, gene deletion vaccine and the like is carried out by a plurality of domestic units, but no effective vaccine is available at present.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a recombinant adenovirus vector for expressing African swine fever virus p54, p30 and E248R proteins, a recombinant adenovirus and a construction method.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a nucleotide sequence for coding African swine fever virus p54, p30 and E248R proteins, which is shown as SEQ ID NO: 1 is shown.

A recombinant adenovirus vector expressing african swine fever virus p54, p30, and E248R proteins, comprising SEQ ID NO: 1.

The construction method of the recombinant adenovirus vector for expressing the proteins of the African swine fever viruses p54, p30 and E248R comprises the following steps: artificially synthesizing the amino acid sequence shown in SEQ ID NO: 1, and inserting the nucleotide sequence into a pDC316-mCMV-EGFP vector to obtain the vector.

A recombinant adenovirus expressing african swine fever virus p54, p30, and E248R proteins, comprising SEQ ID NO: 1 or the recombinant adenovirus vector.

The construction method of the recombinant adenovirus for expressing the proteins of the African swine fever virus p54, p30 and E248R comprises the following steps:

(1) artificially synthesizing the amino acid sequence shown in SEQ ID NO: 1, and inserting the nucleotide sequence into a pDC316-mCMV-EGFP vector;

(2) and (2) co-transfecting the vector constructed in the step (1) and a pBHGloxdel E13cre vector into 293A cells, and then culturing to obtain the recombinant vector.

The recombinant adenovirus can be used for preparing African swine fever vaccines.

The recombinant adenovirus vector for expressing the proteins of African swine fever viruses p54, p30 and E248R, the recombinant adenovirus and the construction method have the following beneficial effects:

the invention constructs a recombinant adenovirus vector capable of simultaneously expressing p54, p30 and E248R proteins, wherein the p54 and p30 proteins are expressed in a fusion mode, and the E248R is expressed in series with p54 and p30 through self-cleavage 2A signal peptide. The recombinant adenovirus capable of expressing proteins of African swine fever viruses p54, p30 and E248R is obtained after the vector and an adenovirus skeleton system transfect cells together, and after the recombinant adenovirus is used for immunizing a test animal, the test animal can be stimulated to generate a specific antibody, so that new test data are provided for the development of an African swine fever vaccine.

Drawings

FIG. 1 is a diagram showing the construction of the constructed pDC316-54F30-2A-E248R vector.

FIG. 2 is a graph showing the result of fluorescent plaque formation by the prepared recombinant adenovirus.

Detailed Description