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Recombinant adenovirus carrier for expressing African swine fever virus B646L gene, construction method and preparation method of recombinant adenovirus

An African swine fever virus, B646L technology, applied in the field of B646L gene overexpression adenovirus vector and its construction

Inactive Publication Date: 2018-07-31
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no vaccine that can prevent African swine fever virus infection in the world. In the prior art, there is a lack of research on vaccines or other preventive measures against African swine fever.

Method used

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  • Recombinant adenovirus carrier for expressing African swine fever virus B646L gene, construction method and preparation method of recombinant adenovirus
  • Recombinant adenovirus carrier for expressing African swine fever virus B646L gene, construction method and preparation method of recombinant adenovirus
  • Recombinant adenovirus carrier for expressing African swine fever virus B646L gene, construction method and preparation method of recombinant adenovirus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] African swine fever virus B646L gene Gene ID: KJ195685.1, the African swine fever virus B646L gene was synthesized by Shanghai Sangong Co., Ltd. and added restriction sites XhoI and AsisI, and the synthesized B646L gene was connected to the T vector to obtain T vector of the B646L fragment.

Embodiment 2

[0069] The gene synthesis sequence B646L (that is, the T vector with the B646L fragment) and the adenovirus shuttle plasmid vector pKO-FH ( Figure 4 ) to obtain the linearized B646L gene fragment and the pKO vector fragment respectively. The enzyme digestion reaction system of the double enzyme digestion reaction is as follows in Table 1:

[0070] Table 1 Double enzyme digestion system

[0071] B646L gene fragment or pKO-FH vector

20uL

10×Buffer

3μL

AsisⅠ

1μL

MLuⅠ

1μL

wxya 2 o

5μL

total

30μL

[0072] The temperature of the double enzyme digestion is 37° C., and the time of the double enzyme digestion is 1 hour.

[0073] After the reaction, use 1% agarose gel electrophoresis to detect the size of the digested band, and use a gel recovery kit to recover the target fragment. The vector pKO-FH was digested in the same way, and the vector sequence was recovered from the gel.

Embodiment 3

[0075] The linearized B646L gene fragment and the pKO vector fragment were ligated with T4 ligase to obtain pKO-B646L. The connection system is shown in Table 2:

[0076] Table 2 connection system

[0077] Target gene B646L fragment

2~6μL

pKO vector fragment

2~4μL

10×T4Buffer

1μL

T4 DNA ligase (10U / μL)

1μL

total

10 μL

[0078] After mixing, microcentrifuge and connect at 22°C for 2h.

[0079] The ligation product was transformed into Escherichia coli DH5α competent cells. Spread on the LB plate containing kanamycin resistance for screening, pick a single colony, extract the plasmid after expanding the culture, Asis Ⅰ and MLu Ⅰ double enzyme digestion to identify positive clones, and sequence verification to obtain the correct pKO-B646L clone, Sequencing was performed using CMV-F (SEQ No. 2: 5'CAATGGGAGTTTGTTTTGGCACCA-3') and FH-R (SEQ No. 3: 5'CTTATTAGTGGTGGTGGTGGTGGTGCTCG-3').

[0080] The conversion process is...

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Abstract

The invention provides a recombinant adenovirus carrier for expressing an African swine fever virus B646L gene, a construction method and a preparation method of the recombinant adenovirus, belongingto the technical field of genetic engineering. According to the method, adenovirus shuttle vectors pKO-FH and pAD-EF1a-GFP are utilized to be subjected to a series of intermediate processes to obtaina recombinant adenovirus expression plasmid pAD-B646L. An HEK293 cell is transfected to an obtained linear recombinant adenovirus vector plasmid; according to cytopathy caused by adenovirus infection,a recombinant virus is screened to finally realize an adenovirus packaging process, the recombinant adenovirus capable of directly infecting eukaryocyte can be obtained through amplification, concentration and authentication, so that the purpose of normally expressing a B646L gene in the eukaryocyte is realized, and the foundation is laid for researching an adenovirus vector vaccine based on expression of B646L.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an adenoviral vector for B646L gene overexpression and a construction method thereof. Background technique [0002] African swine fever (African swine fever, ASF) is an acute and severe infectious disease of pigs caused by African swine fever virus (ASFV). Clinically, it is characterized by high fever, systemic hemorrhage and high mortality. The disease is very harmful to the pig industry and is listed as a class A disease by OIE. Although my country has not yet introduced the disease, it is always facing the risk of introduction. Therefore, it is of great significance to carry out reserve research on this disease for the prevention of this disease. [0003] ASFV is a regular 20-hedron with a diameter of about 175nm to 215nm. It consists of five components: outer envelope, viral capsid, inner envelope, nucleocapsid, and viral nucleic acid. Viral nucle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/66C12N7/01
CPCC07K14/005C12N7/00C12N15/66C12N15/86C12N2710/10021C12N2710/10043C12N2710/12022
Inventor 张泉孙靖谕钱淼王涛
Owner YANGZHOU UNIV
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