Recombinant spirulina and application thereof in preparation of white spot syndrome virus oral vaccine

Abstract

The invention provides recombinant spirulina and application thereof in preparation of a white spot syndrome virus (WSSV) oral vaccine. The recombinant spirulina expression system is obtained by introducing an expression cassette containing a white spot syndrome virus envelope protein gene into starting spirulina to stably express the virus envelope protein. According to the present invention, the spirulina strain suitable for genetic transformation is screened, and the key structure protein of the white spot syndrome virus is efficiently expressed in the spirulina strain, such that the obtained recombinant spirulina cell can induce the shellfish such as shrimp to produce the immunocompetence against the white spot syndrome virus, and has the function of oral vaccine. Besides, the spirulina is a natural additive commonly used in crustacean feeds, so that when the recombinant spirulina is added into the feeds, the white spot syndrome virus can be effectively prevented on the premise that the cost is not obviously increased, and an economical and convenient virus prevention and control means is provided for the breeding industry of crustacean animals such as shrimps and the like.

Description

Technical Field

[0001] This invention belongs to the field of microalgae metabolic engineering, specifically relating to a recombinant spirulina and its application in the preparation of an oral vaccine for vitiligo syndrome virus. Background Technology

[0002] White spot syndrome virus (WSSV) is the causative agent of white spot disease (WSD) and can infect many commercially valuable decapod species. WSSV virus particles are oval-shaped, enveloped, and are large, enveloped dsDNA viruses with a genome of approximately 300 kbp. WSSV is currently one of the most damaging viral pathogens to shrimp globally. In shrimp ponds infected with WSSV, shrimp mortality rates can reach 100% within one week.

[0003] Oral vaccine development is one of the effective means of controlling WSSV infection. Many biological vector systems have been developed for expressing the WSSV envelope protein VP28, including *Escherichia coli*, *Bacillus subtilis*, and *Saccharomyces cerevisiae*, to prevent white spot syndrome virus (WSSV). However, because crustaceans lack mature acquired immunity, vaccination only produces temporary immunity, requiring frequent supplementation of the aforementioned oral vaccines in feed to prevent infection. This approach is not only cumbersome but also significantly increases farming costs. Therefore, oral vaccines prepared using the aforementioned recombinant expression systems cannot provide effective prevention against WSSV under normal cost control conditions; hence, there are currently no commercially available oral vaccines containing WSSV proteins.

[0004] Spirulina, as a highly efficient photosynthetic microorganism, boasts advantages such as rapid growth, ease of cultivation, and large-scale industrial aquaculture. Furthermore, spirulina is frequently used as a feed additive for crustaceans. Reports indicate that adding spirulina to crustacean feed can increase protein content, polyunsaturated fatty acid content, and minerals, significantly promoting fish and shrimp growth, enhancing their immunity, and, due to its rich content of natural pigments, enhancing the vibrancy of these animals' body colors. Therefore, if the genetic modification barriers of spirulina can be overcome, and it can be developed into a highly efficient cell factory for producing vitiligo symptomatic virus proteins, achieving a "food and medicine homology" approach in crustacean farming, vitiligo symptomatic syndrome could be effectively prevented without significantly increasing costs.

[0005] However, since the 1980s, scientists have been dedicated to the research of spirulina genetic modification, but have yet to achieve a substantial breakthrough. This invention constructs a spirulina germplasm resource bank and, based on the feasibility screening of genetic transformation of a large number of spirulina strains, obtains a genetically modifiable spirulina. Using the modified spirulina mutant strain as a chassis, a recombinant spirulina expression system is constructed to prepare an oral vaccine containing vitiligo syndrome virus protein for the effective prevention and treatment of vitiligo syndrome. Summary of the Invention

[0006] To address the above problems, this invention provides the application of Spirulina in the preparation of a white spot syndrome virus vaccine. The Spirulina is strain MMECC-020 or a derivative thereof; strain MMECC-020 was deposited on June 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 46457.

[0007] The present invention also provides a recombinant spirulina capable of synthesizing the structural protein of vitiligo syndrome virus, which is prepared by introducing the envelope protein gene expression cassette of vitiligo syndrome virus into the spirulina.

[0008] In one specific implementation, the protein encoded by the envelope protein gene of the vitiligo syndrome virus is the VP28 structural protein.

[0009] In one specific embodiment, the DNA sequence of the envelope protein gene of the vitiligo syndrome virus is shown in SEQ ID NO:2.

[0010] In one specific embodiment, the amino acid sequence of the envelope protein gene of the vitiligo syndrome virus is shown in SEQ ID NO:3.

[0011] In one specific implementation, the starting Spirulina is strain MMECC-020 or a derivative thereof;

[0012] The algal strain MMECC-020 was deposited at the China General Microbiological Culture Collection Center on June 18, 2025, with accession number CGMCC No. 46457.

[0013] The present invention also provides the application of the above-mentioned recombinant spirulina in the preparation of vitiligo syndrome virus vaccine.

[0014] In one specific implementation, the vaccine is an oral vaccine.

[0015] The present invention also provides a crustacean feed (e.g., for shrimp, crab, lobster, crayfish or copepods) that can prevent white spot syndrome virus, said crustacean feed containing the above-mentioned recombinant spirulina culture.

[0016] The culture of the recombinant spirulina can be used to obtain algal mud, algal powder, algal cell lysate, or protein extracts, etc.

[0017] This invention screens Spirulina strains suitable for genetic transformation and efficiently expresses key structural proteins of white spot syndrome virus (WSSV) within them. The resulting recombinant Spirulina cells can induce immune activity against WSSV in crustaceans such as shrimp, thus functioning as an oral vaccine. Since Spirulina is a commonly used feed additive in aquaculture, the recombinant Spirulina of this invention can provide preventative protection against WSSV while feeding crustaceans, eliminating the need for separate medication or new additives and significantly reducing the cost of preventing WSSV infection.

[0018] The Spirulina culture of the present invention can not only be used directly as a feed additive for shrimp such as prawns to prevent white spot syndrome, but also as a feed additive for shrimp such as prawns (such as some copepods) to prevent these copepods from being infected with white spot syndrome virus, thereby preventing shrimp such as prawns from being infected with white spot syndrome virus by consuming feed carrying the virus.

[0019] Microbial Preservation

[0020] The Spirulina described in this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46457, deposit date June 18, 2025, address of the depository: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Its taxonomic name is Spirulina platensis. Attached Figure Description

[0021] Figure 1 pLX009 plasmid map

[0022] Figure 2 This is the pLX012 plasmid map.

[0023] Figure 3 Electrophoresis diagram of PCR amplification products of the LX003 algal strain with the endogenous kanamycin gene knocked out.

[0024] Figure 4 Electrophoresis image of the PCR amplification product of the LX004 algal strain overexpressing the vp28 gene.

[0025] Figure 5 The image shows the RT-qPCR results of the LX004 algal strain overexpressing the vp28 gene.

[0026] Figure 6 Western blot electrophoresis image of the LX004 algal strain overexpressing the vp28 gene.

[0027] Figure 7 To and Figure 6Western blotting electrophoresis of algal strain LX004 after a 10-month interval. Detailed Implementation

[0028] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0029] The spirulina cell factory was obtained by introducing the WSSV-vp28 gene into the host genome. The vp28 gene is a structural protein gene derived from white spot syndrome virus (WSSV). Using Spirulina MMECC-020 as the starting strain, the endogenous kanamycin resistance gene of the starting strain was knocked out to obtain the chassis strain LX003. Preferably, the vp28 gene of WSSV was overexpressed in the LX003 strain.

[0030] 1. Starting algal strain and its genetic transformation and integration platform

[0031] Our team collected over 40 Spirulina strains from geographically representative locations including Lake Chad in Africa, Lake Tecicoco in Mexico, Japan, the United States, and Yunnan and Inner Mongolia in my country. From these strains, we selected Spirulina strain MMECC-020. This strain can form single colonies, is highly sensitive to antibiotics such as erythromycin, chloramphenicol, spectinomycin, and streptomycin, and can readily absorb exogenous DNA for genetic transformation.

[0032] Our team has deposited MMECC-020 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46457, on June 18, 2025. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Its taxonomic name is Spirulina platensis.

[0033] Spirulina MMECC-020 exhibits kanamycin resistance, and we identified a kanamycin resistance gene in its genome, the DNA sequence of which is shown in SEQ ID NO:1. Studies have shown that knocking out this gene only affects its resistance to kanamycin, making it sensitive to kanamycin, without affecting other functional genes in the algae. Therefore, it can serve as a platform for the integration of exogenous genes in genetic transformation operations.

[0034] 2. Plasmid Construction

[0035] The kanamycin resistance gene knockout plasmid pLX009 was obtained by amplifying the upstream homologous arm (Km-Up) and downstream homologous arm (Km-Down) of the kanamycin resistance gene using the MMECC-020 genome as a template. The recombinant plasmid pLX009 was then integrated into the pUC19 backbone by inserting streptomycin, spectinomycin, and green fluorescent protein gene fragments between the upstream and downstream homologous arms of the kanamycin resistance gene.

[0036] Construction of WSSV-vp28 gene overexpression plasmid pLX012: First, the vp28 gene fragment (DNA sequence as shown in SEQ ID NO:2, amino acid sequence as shown in SEQ ID NO:3) and the endogenous promoter P of MMECC-020 algal strain were obtained through gene synthesis. rbcL (DNA sequence as shown in SEQ ID NO.4) Next, using the MMECC-020 genome as a template, the kanamycin resistance gene fragment and its upstream homologous arm (Km-Up), i.e., Km-Up+Km, and the downstream homologous arm (Down) were amplified. By inserting the overexpression cassette of the vp28 gene between the two fragments, using kanamycin resistance as a selection marker, it was integrated into the pUC19 backbone to obtain the recombinant plasmid pLX012. The plasmid map is shown below. Figure 2 As shown.

[0037] 3. Construction of transgenic algal strains

[0038] 3.1 Plasmid pLX009 was transformed into the MMECC-020 algal strain to obtain the kanamycin-resistant knockout algal strain LX003.

[0039] Spirulina strains were cultured using Zarrouk medium. The strains were then cultured at OD... 730 ≈0.1g was inoculated into fresh, sterile Zarrouk medium and cultured in 50 mL shake flasks (20 mL total volume). The culture was continuously incubated on a shaker for 5-7 days under the following conditions: light intensity 30-50 μmol·photons. -1 ·m -2 ·s -1 The shaker speed was 150 rpm, the culture temperature was 30℃, and the culture was carried out until the logarithmic growth phase (OD2). 730 =0.8-1.2).

[0040] (1) Take 5 mL of the above logarithmic phase algal solution, centrifuge at 1,600 g for 5 min and discard the supernatant, add the same volume of Zarrouk medium to resuspend, centrifuge at 1,600 g for 5 min again and discard the supernatant, resuspend the centrifuged algal cell volume to 180 μL and divide it into 6 1.5 mL sterile Ep tubes.

[0041] (2) Add 300 ng of pLX009 plasmid DNA to each Ep tube individually and gently tap the bottom of the Ep tube several times to mix. One portion of the algal solution is used as a negative control and no plasmid is added.

[0042] (3) After all the plasmids and algal solutions of the samples are mixed, the Eppendorf tubes are laid flat under a light intensity of 30-50 μmol·photons. -1 ·m -2 ·s -1 Incubate on a light-incubated rack for 3-4 hours.

[0043] (4) After incubation, transfer the mixture to a 12 mL sterile shaking tube and add 600 μL of antibiotic-free Zarrouk medium to each shaking tube.

[0044] (5) Transfer the culture tubes to a shaker and set the following conditions: 30°C temperature, 150 rpm shaking, 30-50 μmol·photons -1 ·m -2 ·s -1 Incubate overnight under light intensity.

[0045] (6) On the second day, add Zarrouk medium with streptomycin resistance to make the working volume 3 mL and the working antibiotic concentration 2.5 μg / mL. Transfer the shake tube to a shaker with the following culture conditions: 35℃, 220 rpm shaking, 50-150 μmol·photons -1 ·m -2 ·s -1 illumination.

[0046] (7) During the resistance screening period, resistance resuspension should be performed every 3-4 days. Remove the sample from the shaker and allow the shaken tube to settle naturally for about 30 minutes before removing the supernatant for resuspension. The algal strain undergoes a green-dark green-yellow-white-green process, which takes approximately 15-20 days, after which the algal strain recovers and turns green. Simultaneously, increase the streptomycin screening concentration to 5 μg / mL until the completely isolated algal strain LX003 is obtained. Figure 3 ).

[0047] 3.2 Plasmid pLX012 was transformed into algal strain LX003 to obtain algal strain LX004 that overexpresses the WSSV-vp28 gene.

[0048] The method is basically the same as above, except that the screening marker is changed to kanamycin and the working concentration of the antibiotic is 75 μg / mL.

[0049] The algal strain undergoes a process of green-dark green-yellow-white-green, taking approximately 15-20 days, after which it recovers and turns green again. The kanamycin screening concentration is increased to 90 μg / mL. After about a month, when the algal filament concentration is high, the kanamycin screening concentration can be increased to 100 μg / mL until the completely isolated algal strain LX004 is obtained.

[0050] 4. Identification of transgenic algal strains

[0051] After obtaining positive transformants, they were cultured in 50 mL Erlenmeyer flasks under the following conditions: 30℃, 30-60 μmol·photons. -1 ·m -2 ·s -1 The culture was subjected to shaking light intensity until the logarithmic growth phase, and the culture was collected for RT-qPCR and Western blot verification.

[0052] 4.1 PCR identification

[0053] Take 500 μL of the above-mentioned logarithmic-phase algal strain, centrifuge and discard the supernatant as much as possible, add 250 μL of ddH2O and a small amount of quartz sand, and shake to disrupt for 3-5 min. Take 2 μL of the disrupted algal solution as a template for PCR identification. Figure 4 As shown, the kanamycin resistance gene was reintroduced in LX004, and the vp28 gene was overexpressed.

[0054] 4.2 RT-qPCR

[0055] Total RNA was extracted from the cultures of MMECC-020 and LX004 algal strains using the Trizol method (TAKARA, RNAiso puls) and then reverse transcribed into cDNA.

[0056] RT-qPCR was performed using the ChamQ Universal SYBR qPCR Master Mix from Vazyme, with rnpB from the MMECC-020 algal strain as an internal control. The reaction system is shown in Table 1.

[0057] Table 1

[0058]

[0059] Perform qPCR reactions under the following conditions, as shown in Table 2.

[0060] Table 2

[0061]

[0062] The mRNA transcription of the vp28 gene in the LX004 algal strain was detected using the above RT-qPCR experiment, and the results are as follows: Figure 5As shown, RT-qPCR verified that the overexpressing algal strain maintained stable transcription during continuous passage, while the wild-type MMECC-020 only showed background fluorescence and did not express the gene.

[0063] 4.3 Identification of protein levels in transgenic algal strains

[0064] (1) Extract total protein from MMECC-020 and LX004 algal cultures

[0065] Cells with a biomass of approximately 10 OD730 were collected by centrifugation at 6,000 rpm for 10 min at 4°C, and the supernatant was removed as much as possible. The cells were then resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 20% glycerol, 1 mM PMSF, 2 mM β-mercaptoethanol, pH 7.5). An appropriate amount of quartz sand (particle size 150-212 μm) was added, and the mixture was shaken for 30 s, then incubated in an ice-water bath for 30 s. This cycle was repeated 5 times. After centrifugation at 10,000 g for 10 min, the protein concentration in the supernatant was determined using a Bradford protein quantification kit (Beyotime, China).

[0066] (2) Validation of immunoblotting

[0067] Adjust the total protein concentration in the samples to the same level, add 5× protein loading buffer, boil at 95℃ for 10 min, and centrifuge. Load the samples with 30 μg of protein and run SDS-PAGE electrophoresis. The electrophoresis program is: 80 V, 20 min, then 120 V, 90 min. After electrophoresis, place one protein gel in Coomassie Brilliant Blue staining solution and gently vortex at room temperature for 1 h; keep the other gel for later use.

[0068] The protein profile obtained by electrophoresis was transferred onto a PVDF membrane, and incubated with VP28 antibody as the primary antibody. After incubation with secondary antibody and color development, a Western blot pattern was obtained.

[0069] like Figure 6 As shown, the protein profile of algal strain LX004 exhibited a specific band for the VP28 protein, with a size (~23 kDa) consistent with expectations. No specific band was observed in the wild-type MMECC-020 algal strain. After 10 months of routine continuous subculturing of LX004 algal strain, the total protein content of the culture was again analyzed by Western blot, and the results are as follows. Figure 7 As shown, the expression of VP28 protein in algal cells was not reduced.

[0070] In summary, this invention overexpresses the WSSV-vp28 gene in the wild-type MMECC-020 algal strain. Sequencing confirmed that the vp28 gene was precisely integrated into the target site, resulting in a scarless edited cell factory. RT-qPCR showed that the vp28 gene maintained stable transcription during continuous passage. Two Western blot analyses confirmed that the VP28 protein was continuously expressed under laboratory culture conditions for 10 consecutive months.

[0071] To assess its potential for industrial application, further scale-up experiments were conducted. In a 100 L photobioreactor system, the VP28 protein expression level of the recombinant algal strain was consistent with that of the laboratory shake-flask culture. These results systematically demonstrate the feasibility and stability of Spirulina as a cell factory for WSSV oral vaccine, providing a practical new approach for the development and application of microalgae-based aquatic oral vaccines.

Claims

1. Use of a Spirulina in the preparation of a white spot syndrome virus vaccine.

2. Use according to claim 1, characterized in that, The Spirulina is a strain MMECC-020 or a derivative strain thereof; The strain MMECC-020 was deposited with the China General Microbiological Culture Collection Center on June 18, 2025, and has a preservation number of CGMCC No. 46457.

3. A recombinant Spirulina capable of synthesizing a white spot syndrome virus structural protein, characterized in that, The Spirulina is prepared by introducing an expression frame of a capsid protein gene of a white spot syndrome virus into a starting Spirulina.

4. The recombinant Spirulina according to claim 3, wherein The protein encoded by the capsid protein gene of the white spot syndrome virus is a VP28 structural protein.

5. The recombinant Spirulina according to claim 4, wherein The DNA sequence of the capsid protein gene of the white spot syndrome virus is shown as SEQ ID NO:

2.

6. The recombinant Spirulina according to claim 4, wherein The amino acid sequence encoded by the capsid protein gene of the white spot syndrome virus is shown as SEQ ID NO:

3.

7. The recombinant Spirulina according to any one of claims 3 to 6, wherein The Spirulina is a strain MMECC-020 or a derivative strain thereof; The strain MMECC-020 was deposited with the China General Microbiological Culture Collection Center on June 18, 2025, and has a preservation number of CGMCC No. 46457.

8. Use of the recombinant Spirulina of any one of claims 3-7 in the preparation of a white spot syndrome virus vaccine.

9. Use according to claim 8, characterized in that, The vaccine is an oral vaccine.

10. A crustacean feed which prevents white spot syndrome virus, characterized by, A culture comprising the Spirulina of any one of claims 3-7.

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