阅读说明：本技术 表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒载体、重组腺病毒及构建方法 (Recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, recombinant adenovirus 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p72、B602L蛋白的重组腺病毒载体、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p72、B602L蛋白,B602L通过自裂解2A信号肽与p72串联表达。本发明构建了能同时表达p72、B602L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p72、B602L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 vector for expressing African swine fever virus p72 and B602L proteins, a recombinant adenovirus and a construction method thereof, wherein the recombinant adenovirus can synchronously express African swine fever p72 and B602L proteins after infecting cells, and B602L is expressed in series with p72 through self-cleavage 2A signal peptide. The recombinant adenovirus vector capable of expressing the p72 and B602L proteins is constructed, the recombinant adenovirus capable of expressing the African swine fever virus p72 and B602L proteins is obtained after the vector and an adenovirus skeleton system are used for cotransfecting cells, and after the recombinant adenovirus is used for immunizing a test animal, the test animal can be stimulated to generate specific antibodies, so that new test data are provided for the development of an African swine fever vaccine.)

1. A nucleotide sequence for coding African swine fever virus p72 and B602L proteins, wherein the nucleotide sequence is shown as SEQ ID NO: 1 is shown.

2. A recombinant adenoviral vector for expressing p72, B602L protein of african swine fever virus, comprising the nucleotide sequence of claim 1.

3. The method for constructing the recombinant adenovirus vector for expressing the African swine fever virus p72 and B602L proteins according to 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 p72, B602L protein 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 African swine fever virus p72 and B602L proteins according to claim 4, which 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.

6. The use of the recombinant adenovirus expressing the proteins p72 and B602L 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 p72 and B602L proteins of African swine fever virus 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 p72 and B602L 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 p72 and B602L 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 p72 and B602L proteins is shown as SEQ ID NO: 1 is shown.

A recombinant adenovirus vector for expressing p72 and B602L proteins of african swine fever virus, comprising SEQ ID NO: 1.

The construction method of the recombinant adenovirus vector for expressing the proteins p72 and B602L of the African swine fever virus 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 p72, B602L protein of african swine fever virus, comprising SEQ ID NO: 1 or the recombinant adenovirus vector.

The construction method of the recombinant adenovirus for expressing the proteins of African swine fever virus p72 and B602L 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 and the recombinant adenovirus expressing the African swine fever virus p72 and B602L proteins and the construction method thereof provided by the invention have the following beneficial effects:

the invention constructs a recombinant adenovirus vector capable of simultaneously expressing proteins of African swine fever virus p72 and B602L, obtains the recombinant adenovirus capable of expressing proteins of African swine fever virus p72 and B602L 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 using the adenovirus, and provides new test data for the development of an African swine fever vaccine.

Drawings

FIG. 1 is a diagram showing the construction of the vector pDC316-p 72-B602L.

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

Detailed Description