Shrimp white spot syndrome virus VP28 recombinant protein and its preparation method and application

Abstract

The present invention relates to a shrimp white spot syndrome virus VP28 recombinant protein and a preparation method and application thereof. The amino acid sequence of the VP28 recombinant protein is shown in SEQ ID NO: 1. The present invention also provides a coding gene, an expression vector and a host cell thereof, so that the VP28 recombinant protein can be obtained by heterologous expression. The VP28 recombinant protein provided by the present invention can play a certain role in preventing shrimp white spot syndrome virus infection and has good application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a shrimp white spot syndrome virus VP28 recombinant protein and a preparation method and application thereof. Background Art

[0002] White spot disease is a serious viral disease caused by white spot syndrome virus (WSSV). Its typical symptom is a large number of white spots ranging in size from 0.5 to 3 mm on the cephalothorax of diseased shrimp. WSSV has a wide host range. Studies have found that about 98 species of crustaceans, including shrimps and crayfishes, can become its hosts. The virus develops quickly, has a high mortality rate, and spreads quickly. After shrimps are infected with white spot syndrome virus, the mortality rate is 100% within 3 to 10 days, causing huge economic losses to the shrimp farming industry worldwide. It has become one of the main factors restricting the development of shrimp farming worldwide.

[0003] WSSV virus particles are long rod-shaped and are double-stranded DNA viruses. The structure of its virus particles consists of three parts from the outside to the inside, including a three-layer envelope structure, a bridge structure, and a nucleocapsid structure. So far, more than 39 structural proteins have been discovered through genomic and proteomic methods. Among them, VP28 accounts for about 60% of the WSSV envelope protein, is the main envelope protein of WSSV, and is considered to be the main factor in systemic infection. So far, there is a lack of effective strategies to prevent and control the virus. In recent years, it has been found that introducing the virus envelope protein into shrimp can protect the host, and the envelope protein VP28 has become a key factor in defending against WSSV. Summary of the invention

[0004] The purpose of the present invention is to provide a shrimp white spot syndrome virus VP28 recombinant protein and its preparation method and application. The VP28 recombinant protein is optimized and designed based on the wild-type VP28 protein (amino acid sequence is SEQ ID NO: 3). The VP28 recombinant protein can be obtained by heterologous expression and has good application prospects in the prevention and treatment of shrimp white spot disease.

[0005] To this end, in a first aspect, the present invention provides a VP28 recombinant protein, whose amino acid sequence is shown in SEQ ID NO:1.

[0006] The second aspect of the present invention provides a gene encoding the VP28 recombinant protein described in the first aspect of the present invention.

[0007] Furthermore, the nucleotide sequence of the gene is SEQ ID NO: 2.

[0008] The third aspect of the present invention provides an expression vector, which comprises the gene described in the second aspect of the present invention.

[0009] The fourth aspect of the present invention provides a host cell, which expresses the VP28 recombinant protein described in the first aspect of the present invention and / or contains the gene described in the second aspect of the present invention or the expression vector described in the third aspect.

[0010] Furthermore, the host cell is a prokaryotic cell.

[0011] Furthermore, the host cell is Escherichia coli.

[0012] The fifth aspect of the present invention provides a method for preparing the VP28 recombinant protein, comprising: culturing the host cell described in the fourth aspect of the present invention under conditions allowing the expression of the VP28 recombinant protein, and purifying the VP28 recombinant protein from the obtained culture.

[0013] The sixth aspect of the present invention provides the use of the VP28 recombinant protein in preparing a product for preventing and / or treating white spot disease in shrimp.

[0014] Furthermore, the product is a feed additive.

[0015] Furthermore, in the product, the dosage of the VP28 recombinant protein is 0.5-2% (w / w).

[0016] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0017] The prokaryotic expression vector recombinant bacteria prepared by the present invention successfully express the shrimp white spot syndrome virus VP28 recombinant protein. A large number of studies have shown that VP28 is the main envelope protein of the shrimp white spot syndrome virus, which can independently bind to the host cell surface. It can be inferred that this protein plays an important role in the initial stage of infecting shrimp. The VP28 recombinant protein provided by the present invention can play a certain role in preventing shrimp white spot syndrome virus infection and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only used for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. In the accompanying drawings:

[0019] Figure 1 : SDS-PAGE electrophoresis detection results after induced expression of VP28 recombinant protein; wherein, lane 1 is the control group without induced expression, lane 2 is the sample after 4 hours of induction, and lane 3 is the sample after overnight induction. DETAILED DESCRIPTION

[0020] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0021] Unless otherwise indicated, the examples herein employ conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology within the skill of the art.

[0022] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0023] In this article, the words "include" and "comprise" represent open-ended or closed-ended expressions. For example, the words "include" or "comprise" may represent that other components, steps or other elements not listed may also be included or comprised, or may only include or comprise the listed components, steps or other elements.

[0024] In this article, the term "recombinant protein" refers to a protein produced by transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a suitable host cell. It is well known to those skilled in the art that in gene cloning operations, it is often necessary to design suitable restriction sites, which will inevitably introduce one or more irrelevant residues at the end of the expressed protein, and this does not affect the activity of the target protein. For example, in order to promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acid sequences to the N-terminus, C-terminus or other suitable regions in the protein of the recombinant protein. Such amino acid sequences include but are not limited to linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, tags (such as histidine tags or Flags), or suitable proteolytic enzyme sites, etc. It should be understood that the presence of these amino acid sequences will not affect the activity of the resulting polypeptide.

[0025] As used herein, the term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector can be transformed, transduced or transfected into a host cell to express the genetic material elements carried in the host cell. Expression vectors are well known to those skilled in the art, including but not limited to plasmids (e.g., pET expression vectors, pCW expression vectors, pUC expression vectors or pPIC9k expression vectors), bacteriophages, cosmids, artificial chromosomes, bacteriophages, animal viruses, and the like. The vector may contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain an origin of replication.

[0026] In this article, the term "host cell" refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include initially transformed cells and progeny derived therefrom (regardless of the number of generations). In some embodiments, the host cell can be a unicellular microorganism or a non-unicellular microorganism, for example, including but not limited to Escherichia coli cells, Bacillus subtilis cells, Lactobacillus cells, Actinomycete cells, yeast cells or algae cells.

[0027] The embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the art (for example, reference to "Molecular Cloning Experiment Guide" by J. Sambrook, Huang Peitang, etc., the third edition, Science Press) or according to the product instructions. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased and obtained on the market.

[0028] Example 1

[0029] The protein structure of the VP28 wild-type protein was analyzed and optimized to obtain the amino acid sequence shown in SEQ ID NO: 1. According to the amino acid sequence and after the codon preference design of Escherichia coli, the nucleotide sequence of the gene encoding the VP28 recombinant protein was provided (SEQ ID NO: 2). EcoR I and Hind III restriction sites were added to both ends of the sequence of SEQ ID NO: 2 and sent to Shanghai Shenggong Biotechnology Co., Ltd. for synthesis. The coding gene of the VP28 recombinant protein was connected to the PET28a vector by the EcoR I / Hind III restriction ligation method, and a monoclonal strain containing the correct recombinant plasmid was obtained after transformation of BL21 competent cells, resistance screening, restriction verification, and sequencing verification. The recombinant plasmid was named PET28a-VP28', and its expression strain was named BL21-VP28'.

[0030] Example 2

[0031] Inducing the expression of VP28 recombinant protein specifically includes the following steps:

[0032] The recombinant strain BL21-VP28' was inoculated into LB medium and cultured at 30°C overnight. The next day, the culture solution was taken and inoculated into fresh LB medium containing ampicillin resistance again. The culture was cultured at 30°C for about 4 hours. When OD 600 reached about 0.6, IPTG inducer was added. The final concentration of IPTG inducer was 1mM. The bacterial solution of expression 0h, 4h and overnight was collected respectively, centrifuged at 4°C and 12000rpm for 1min, the bacterial cells were collected and ultrasonically disrupted under ice bath conditions, the supernatant after ultrasonic disruption was collected, and the samples of induction 0h, 4h and overnight were subjected to SDS-PAGE electrophoresis. The results are shown in Figure 1 The sample induced overnight was purified by Ni2+ affinity chromatography column, and the eluted protein was collected to prepare the VP28 recombinant protein.

[0033] Example 3

[0034] Healthy Penaeus vannamei weighing 2-5g were selected and randomly divided into 4 treatment groups, each with 40 shrimps. The groups were fed with the following feeds: Group 1, conventional feed; Group 2, conventional feed; Group 3, conventional feed with 1% (w / w) wild-type VP28 protein (amino acid sequence of SEQ ID NO: 3); Group 4, conventional feed with 1% (w / w) VP28 recombinant protein prepared in Example 2. Except for the above-mentioned difference in feed, the same feeding conditions were adopted for 7 days. Then, the same amount of shredded shrimp infected with WSSV was put into Group 2, Group 3 and Group 4 respectively. After 5 days of continuous feeding, the mortality rate of each group was counted. The results are shown in Table 1.

[0035] Table 1

[0036] Group 1 Group 2 Group 3 Group 4 Deaths 0 38 21 8 mortality rate 0 95％ 52.5％ 20％

[0037] According to the above results, the VP28 recombinant protein provided by the present invention has a good effect in preventing white spot disease in shrimp.

[0038] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A VP28 recombinant protein, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. A gene, characterized in that It encodes the VP28 recombinant protein described in claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

2.

4. An expression vector, characterized in that: The expression vector comprises the gene according to claim 2 or 3.

5. A host cell, characterized in that The host cell expresses the VP28 recombinant protein according to claim 1, and / or contains the gene according to claim 2 or 3 or the expression vector according to claim 4.

6. The host cell according to claim 5, characterized in that The host cell is a prokaryotic cell.

7. The host cell according to claim 5, characterized in that The host cell is Escherichia coli.

8. A method for preparing VP28 recombinant protein, characterized in that: include: The host cell according to any one of claims 5 to 7 is cultured under conditions that allow the expression of the VP28 recombinant protein according to claim 1, and the VP28 recombinant protein is purified from the obtained culture.

9. Use of the VP28 recombinant protein according to claim 1 in preparing a product for preventing and / or treating white spot disease in shrimp.

10. The use according to claim 9, characterized in that The product is a feed additive.

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