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Method for efficiently expressing structural proteins of Senecavirus A

A structural protein and protein technology, applied in the field of genetic engineering, can solve problems such as uneven expression of structural proteins, and achieve the effects of avoiding molecular sieve chromatography purification, increasing yield, and improving expression efficiency.

Active Publication Date: 2021-08-13
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In view of the above problems, the present invention first optimizes the prokaryotic expression codons based on the gene sequences encoding the three structural proteins VPO, VP3 and VP1 of Seneca virus type A, and screens out A single plasmid solublely expresses these three structural proteins in E. coli at the same time; again, a plasmid expressing the molecular chaperone and the structural protein of Seneca virus type A is transferred into E. coli to construct a co-expression system of the target protein and the molecular chaperone , further improved the expression level and soluble ratio of Seneca virus type A virus structural protein, and improved the problem of uneven expression of the above-mentioned structural protein; the obtained target protein accounted for more than 25% of the total bacterial protein; by these three The Seneca virus disease subunit vaccine prepared by structural protein can stimulate pigs to produce a higher level of neutralizing antibodies, and has a good protective effect on the domestic A-type Seneca virus

Method used

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  • Method for efficiently expressing structural proteins of Senecavirus A
  • Method for efficiently expressing structural proteins of Senecavirus A
  • Method for efficiently expressing structural proteins of Senecavirus A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1 Soluble expression of a type A seneca virus protein

[0092] 1. Construction of the expression vector of the Sena virus protein recombinant expression vector

[0093] (1) Designing a gene sequence THS of the encoded fusion tag protein consisting of the originally described above, where T is the nucleotide sequence of the translation start zone, and H is a nucleotide sequence encoding a group-containing label, and S is encoded Nucleotide sequences of saproprine yeast small ubiquitin-like modes (SUMO); nucleotide sequences of THS, such as SEQ ID NO.11.

[0094] (2) The above THS gene sequence is respectively connected in series with the CH-FJ-2017 structural protein gene VP0, VP3, VP1, respectively, and form three-stage fusion gene sequence THS-VP0, THS-VP3. And THS-VP1.

[0095] (3) Synthesis the three-segment optimized fusion gene fragment by China Great Gene Biotechnology Co., Ltd., and cloned the above-mentioned fragment by molecular cloning technique to the sam...

Embodiment 2

[0108] Example 2 Preparation of a type A seneca virus protein

[0109] 1. Soluble expression of the Sena virus dominant protein

[0110] (1) Remove the expression of the plasmid pGTF transformant and recombinant expression plasmid PET-SVA-VP031 of the expression molecular partner, 10 hwanni-resistant 50 ml of LB liquid medium, 250 rpm, 37 ° C, and culture for about 12 hours. After entering 1L LB liquid medium, 37 ° C culture, etc. OD 600 After the value reached 0.6-0.8, IPTG was added at a final concentration of 0.5 mm, and protein expression was induced at 16 ° C.

[0111] In this example, the fusion of small ubiquitin-like renaissance protein (Sumo) tags of Sumo, SDS-PAGE identification results Figure 8 As shown, where m is a molecular weight Marker; 1 is a precipitate after inducing pre-cleavage; 2 is supernatant after inducing pre-cleavage; 3 is a precipitate after induction of whole bacterial cleavage; 4 is induced after a cleavage After the supernatant, the sample was 5 μl; ...

Embodiment 3

[0126] Example 3 A type of Seneca unit vaccine immunization animal neutralizing antibody response

[0127] Example 2 was prepared by the vaccine in Example 2, the vaccine was prepared after the vaccine was immunized, and the blood was added to 0, 7, 14, 21, 28 days, respectively, and separated serum in progress. And antibody titer detection. Test results Figure 12 As shown, Negative Control is an animal serum of immunized PBS; VLPS Vaccine is an animal serum immunized by the A-type seneca virus protein sub-unit vaccine of the present invention. The results show that the A-type seneca virus-based protein composition RE / SVA / CH-FJ-2017 constructed in animal protein gene VP0, VP3 and VP1 according to the present invention can be immunized after animal immunization Produce a high level of neutralizing antibody, improve the in vivo stability and protection effectiveness of the A-type mouth-and-mouth hypertrophic engineering vaccine.

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Abstract

The invention relates to the technical field of molecular biology, in particular to a method for efficiently expressing structural proteins of Senecavirus A. According to the method, firstly, prokaryotic expression codon optimization is carried out on genes for coding three structural proteins VP0, VP3 and VP1 of the Senecavirus A, and a single plasmid for simultaneously carrying out soluble expression of the three structural proteins in escherichia coli is obtained by means of a small ubiquitin-like fusion protein; secondly, molecular chaperone plasmids and plasmids of the structural proteins of the Senecavirus A are transferred into the escherichia coli, and thus, the soluble expression of the structural proteins of the Senecavirus A is further improved, and the problem of non-uniform expression of target proteins is solved, and the obtained target protein accounts for about 25% or more of the total protein of bacteria. A Senecavirus subunit vaccine prepared from the three structural proteins obtained by expression can stimulate a pig body to generate a high-level neutralizing antibody, and has a good protection effect on a Senecavirus epidemic strain.

Description

Technical field [0001] The present invention relates to the field of genetic engineering, and more particularly to a method of highly expressing a type A-type seneca viral structure protein. Background technique [0002] Senecavirus A, SVA is also known as Seneca Valleyvirus, SVV), belongs to small RNA virus, Seneca virus, whose genome is single-chain positively strand RNA, and the genome is about 7.2kb. SVA unique open reading box (ORF) is used to encode a polypatient protein, a typical L-4-3-4 structure having a small RNA virus, containing a P1 region of l precursor protein and structural protein and a non-structural P2. Zone and P3 districts, P1 region proteins can produce three structural proteins and seven non-structural proteins in virus 2a, 3C protease, and host protease water. VP0, VP1, and VP3 have good immunogenic proteins as the surface structural protein of the virus casing. [0003] SVA was initially considered to be contaminants in the cell culture process, speculat...

Claims

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Application Information

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IPC IPC(8): C07K14/085C12N15/70C12N15/62C07K1/22A61K39/125A61P31/14
CPCC07K14/005C12N15/70A61K39/12A61P31/14C12N2770/32022C07K2319/35A61K2039/552Y02A50/30
Inventor 郑海学茹毅伍春平马坤刘华南张贵财郝荣增李亚军李丹刘永杰杨帆田宏张克山曹伟军刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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