A recombinant phaeodactylum tricornutum stably expressing shRNA targeting ostrea gigas herpesvirus OsHV-1 and application thereof

Abstract

The application relates to a recombinant Phaeodactylum tricornutum stably expressing shRNA targeting oyster herpesvirus OsHV-1 and an application thereof. The recombinant Phaeodactylum tricornutum stably expressing shRNA targeting oyster herpesvirus OsHV-1 is obtained by introducing a recombinant expression vector containing shRNA sequences for targeting and silencing oyster herpesvirus OsHV-1 gene expression into wild-type Phaeodactylum tricornutum (recipient strain). The recombinant expression vector is obtained by inserting shRNA sequences for targeting and silencing oyster herpesvirus OsHV-1 gene expression into a p35-sh vector plasmid. The shRNA sequences are one of shRNA-1, shRNA-2 or shRNA-3. The constructed recombinant expression vector is transformed into wild-type Phaeodactylum tricornutum, and the recombinant Phaeodactylum tricornutum expressing shRNA targeting oyster herpesvirus OsHV-1 is obtained by sequencing verification. The recombinant Phaeodactylum tricornutum contains shRNA targeting oyster herpesvirus OsHV-1, and can be used as a biological carrier for orally delivering antiviral RNA molecules to oysters, thereby providing a new technical path for the prevention and control of OsHV-1.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and molecular biology, and more specifically to a recombinant brown finger algae that stably expresses shRNA targeting oyster herpesvirus OsHV-1 and its applications. Background Technology

[0002] Ostreid herpesvirus 1 (OsHV-1) is one of the most important viral pathogens seriously endangering the oyster farming industry in recent years. It can infect a variety of economically important shellfish, and is particularly pathogenic to oyster larvae, juveniles, and some growth stages. Outbreaks of OsHV-1 infection often cause large-scale mortality in farmed populations within a short period, resulting in decreased seedling survival rates, interrupted farming cycles, and significant economic losses. It has become one of the important factors restricting the stable development of the oyster industry.

[0003] Currently, the main control measures for oyster herpesvirus disease include seedling quarantine, aquaculture environment control, biosecurity management, and breeding of disease-resistant strains. However, these methods are still mainly passive control measures and are difficult to implement in a rapid, specific, and efficient manner during a virus outbreak.

[0004] RNA interference (RNAi) can inhibit pathogen replication or key pathogenic processes by specifically recognizing target gene sequences and inducing the degradation of corresponding mRNA. RNAi technology has advantages such as strong targeting, flexible design, and theoretical applicability to a variety of viral targets, making it an important direction in antiviral research for aquatic animals. However, existing nucleic acid delivery methods mostly rely on injection, encapsulation, or artificial addition, which are not only complex to operate but also difficult to meet the practical needs of high-throughput, low-cost, and large-scale aquaculture of shellfish. Especially for filter-feeding aquatic animals like oysters, developing a nucleic acid vector system that can be delivered via feeding, possesses biocompatibility, stability, and scalable production potential, is crucial for the industrial application of RNAi antiviral technology.

[0005] Phaeodactylum tricornutum is a model diatom with advantages such as rapid growth, large-scale cultivation, mature genetic transformation, and stable expression of exogenous genes, and has been extensively studied in the field of microalgae biotechnology. As an aquatic feed ingredient, Phaeodactylum tricornutum can directly enter the aquaculture system in the form of live algal cells, thus showing promise as a "feedable nucleic acid delivery vector." Summary of the Invention

[0006] The purpose of this invention is to construct an engineered *Phaeodactylum tricornutum* that stably expresses shRNA targeting oyster herpesvirus OsHV-1, enabling it to serve as a biological vector for oral delivery of antiviral RNA molecules to oysters. This provides a new technical approach for the prevention and control of OsHV-1 and lays the foundation for the development of oral microalgal RNAi vaccines suitable for aquaculture.

[0007] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows: A recombinant brown finger algae stably expressing shRNA targeting oyster herpesvirus OsHV-1, wherein the recombinant brown finger algae is obtained by introducing a recombinant expression vector containing an shRNA sequence that targets and silences the expression of the oyster herpesvirus OsHV-1 gene into a recipient algal strain.

[0008] The shRNA sequence that targets and silences the OsHV-1 herpesvirus gene expression in oysters is one of shRNA-1, shRNA-2, or shRNA-3; The target nucleotide sequence of the shRNA-1 sequence is shown in SEQ ID NO.1, the target nucleotide sequence of the shRNA-2 sequence is shown in SEQ ID NO.2, and the target nucleotide sequence of the shRNA-3 sequence is shown in SEQ ID NO.3.

[0009] The forward primer sequence for preparing the shRNA-1 sequence is shown in SEQ ID NO.4, and the reverse primer sequence is shown in SEQ ID NO.5.

[0010] The forward primer sequence for preparing the shRNA-2 sequence is shown in SEQ ID NO.6, and the reverse primer sequence is shown in SEQ ID NO.7.

[0011] The forward primer sequence for preparing the shRNA-3 sequence is shown in SEQ ID NO.8, and the reverse primer sequence is shown in SEQ ID NO.9.

[0012] The recipient algal strain was wild-type *Phaeodactylum tricornutum*.

[0013] The recombinant expression vector containing the shRNA sequence that targets and silences the OsHV-1 herpesvirus gene expression of oysters is p35-sh, and the nucleotide sequence of the p35-sh vector plasmid is shown in SEQ ID NO.10.

[0014] Application of recombinant *Phaeodactylum tricornutum* in the preparation of an oyster herpesvirus OsHV-1 vaccine. The vaccine is an oral vaccine.

[0015] A microalgae feed for preventing oyster herpesvirus OsHV-1, said microalgae feed comprising recombinant *Phaeodactylum tricornutum* that stably expresses shRNA targeting oyster herpesvirus OsHV-1 according to any one of claims 1-7. Attached Figure Description

[0016] Figure 1 The image shows the p35 plasmid vector obtained by structural optimization based on the pPtGE35 plasmid framework.

[0017] Figure 2 This is a PCR verification diagram of algal strains transformed with shRNA.

[0018] Figure 3 The sequencing results are for the recombinant positive transformed strain of *Phaeodactylum tricornutum*.

[0019] Figure 4 The expression level of shRNA in recombinant Brownian finger algae cells is shown. Detailed Implementation

[0020] Cultivation of Phaeodactylum tricornutum *Phaeodactylum triangularis* (WT) was used as the recipient strain for transgenic research. Following the research report of Guillard and Ryther (1962) (Reference: Guillard RRL, Ryther JH. Studies of marine planktonic diatoms: I. *Cyclotella nana* Hustedt, and *Detonula confervacea* (Cleve) Gran[J]. Canadian Journal of Microbiology, 1962, 8 (2):229-239.), artificial seawater medium (ASW) containing f / 2 was prepared for cultivation. The culture was placed in a constant temperature and light incubator (GXZ-380C, Jiangnan Instrument Factory, Ningbo, China) and cultured statically, with manual shaking three times daily. The light intensity for cultivation was 80 μmol photons. -1 m -2 s -1 The light-dark cycle is 12h:12h, and the temperature is 20±1℃.

[0021] Example 1: Construction of Recombinant Expression Vector 1. Design and construction of shRNA oligonucleotide sequences for the OsHV-1 gene: The mRNA sequence of OsHV-1 was obtained from the GenBank database (GenBank accession number: AY509253). Target gene regions closely related to OsHV-1 viral replication were screened from the obtained Ostreid herpesvirus 1 (OsHV-1) mRNA sequence, namely ORF7 and ORF100.

[0022] Within the two target gene regions of ORF7 and ORF100 mentioned above, specific shRNAs were designed and generated using the online shRNA design tool (Kay LabsiRNA / shRNA / Oligo Optimal Design, https: / / web.stanford.edu / group / markkaylab / cgi-bin / ), and their target nucleotide sequences are shown in Table 1.

[0023] Table 1. Target nucleotide sequences of shRNA sh-20 SEQ ID NO. 1 GTGAACAAACTGATTGGACAT sh-21 SEQ ID NO. 2 GAAATTTAACGCGAAAGAAGT sh-25 SEQ ID NO. 3 GGTGTCATGATTTCGGAGAAT To ensure the designed shRNA could form a stable and expected secondary structure, it was submitted to the RNAfold online prediction platform (http: / / rna.tbi.univie.ac.at / / cgi-bin / RNAWebSuite / RNAfold.cgi?PAGE=3&ID=YWYuxUl4fF) for structural validation. The prediction results confirmed that the hairpin structure of the above shRNA was complete and met the requirements for subsequent expression and functional analysis.

[0024] Based on the nucleotide sequence of the shRNA target site mentioned above, two shRNA DNA template single-stranded sequences were designed and synthesized. Restriction sites XbaI and HindIII, along with their protective bases, were added to both ends of the shRNA DNA template single-stranded sequences to synthesize shRNA oligo sequences, as shown in Table 2 below. Table 2. ShRNA oligo sequences of the OsHV-1 gene. sh-20-R TCTCTAGAGTGAACAAACTGATTGGACATTATGGGTCAGGTAATGTCCAATCAGTTTGTTCACGGATCCGCG (SEQ ID NO. 5) sh-21-F CGCGGATCCGAAATTTAACGCGAAAGAAGTtacctgacccataACTTCTTTCGCGTTAAATTTCTCTAGAga (SEQ ID NO. 6) sh-21-R TCTCTAGAGAAATTTAACGCGAAAGAAGTTATGGGTCAGGTAACTTCTTTCGCGTTAAATTTCGGATCCGCG (SEQ ID NO. 7) sh-25-F CGCGGATCCGGTGTCATGATTTCGGAGAATtacctgacccataATTCTCCGAAATCATGACACCTCTAGAga (SEQ ID NO. 8) sh-25-R TCTCTAGAGGTGTCATGATTTCGGAGAATTATGGGTCAGGTAATTCTCCGAAATCATGACACCGGATCCGCG (SEQ ID NO. 9) The designed shRNA oligonucleotides were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0025] 2. Construction of recombinant plasmid vectors Based on the pPtGE35 plasmid framework (https: / / www.addgene.org / 107999 / ), its structure was optimized through PCR amplification and homologous recombination ligation: the Cas9 expression module (including the 3×FLAG tag, nuclear localization signal NLS, 40SRPS8 promoter, and Cas9 and 40SRPS8 terminators) was deleted, while the gRNA backbone (retaining the U6 promoter and terminator) and scaffold region were removed to eliminate the influence of the original CRISPR-Cas9 elements. The modified recombinant plasmid vector was named p35-sh, and its nucleotide sequence is shown in SEQ ID NO.10. A schematic diagram of the plasmid vector is shown below. Figure 1 As shown.

[0026] The synthesized shRNA oligo was annealed to form a double-stranded structure: 20 μL of annealing reaction system was prepared according to Table 3, and then placed in a PCR instrument for denaturation at 95℃ for 5 min. After that, it was slowly cooled to 50℃ at a rate of -1℃ per minute to anneal the upstream and downstream primer chains of the shRNA oligo to form a double strand.

[0027]

[0028] The annealing product and the p35-sh vector plasmid were subjected to double digestion with XbaI and HindIII, respectively. The double digestion reaction system is shown in Table 4. Digestion was performed at 37℃ for 30 min, and the target band was recovered by gel extraction (D2500-02, OMEGA, China).

[0029] (1) T4 ligation: The enzyme-digested target band is ligated with the p35-sh vector plasmid. The ligation system is configured according to Table 5 and ligated at 16℃ for 2h.

[0030]

[0031] (2) Transformation: The ligated product was transformed into DH5α (10 μL ligation system + 30 μL DH5α competent cells, ice bath for 30 min, heat shock at 42℃ for 90 s, ice bath for 2-3 min, 500 μL LB medium, shaken at room temperature for 1 h). 100 μL of bacterial culture was spread on LB agar plates containing ampicillin resistance (0.1 g / mL Amp), and cultured overnight at 37℃. Single clones on the plates were picked and transferred to LB liquid medium containing ampicillin resistance and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing verification.

[0032] Using a plasmid extraction kit ( EasyPure ®Recombinant plasmids containing gRNA were extracted using the Plasmid MiniPrep Kit (TransGold, China). These plasmids were then transformed into *E. coli* containing Mob elements (10 μL recombinant plasmid + 30 μL *E. coli* containing Mob elements, incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, incubated on ice for 2-3 min, then added to 500 μL LB medium and incubated at room temperature for 1 h). 100 μL of the bacterial culture was plated onto LB agar plates containing ampicillin resistance (0.1 g / mL Amp), incubated overnight at 37℃, and single clones were picked from the plates and transferred to LB liquid medium containing ampicillin resistance to obtain *E. coli* containing the recombinant plasmid.

[0033] Example 2: Transformation of recombinant plasmids into *Phaeodactylum tricornutum* 1) *Phaeodactylum tricornutum* (WT) grew to the exponential stage under white LED light at 80 μmol / m³. 2 Centrifuge at 20°C for collection and concentration to 10 μL / s, 20°C. 8 Cells / mL, transferred to plates (50% ASW, 1% agarose), and cultured for 4 days.

[0034] 2) After 4 days, wash the algae off the plate with f / 2 medium and concentrate to 5×10⁻⁶. 8 cells / mL.

[0035] 3) Inoculate the *E. coli* containing the recombinant plasmid into 3 mL of LB medium (containing 100 μg / mL ampicillin and 40 μg / mL gentamicin) and incubate overnight. Then, take 1 mL of the inoculum and inoculate it into 50 mL of LB medium, shaking until the OD of the bacterial culture reaches [value missing]. 600 =0.8-1.0.

[0036] 4) Centrifuge at 3000 g for 10 min to collect the bacterial cells, discard the supernatant, and suspend the precipitate in 500 μL of SOC.

[0037] 5) Take 200 μL of the algae obtained in step 2) and mix it with 200 μL of the bacterial solution obtained in step 4). Spread the mixture on a plate (50% ASW, 1% agarose, 5% LB) and incubate at 30 ℃ for 90 min. Then, allow it to recover at 20 ℃ for 2 days.

[0038] 6) Two days later, wash the algae on the plate with ASW containing 100 μg / mL, and transfer it evenly to 3 plates containing 50 μg / mL bleomycin (50% ASW, 1% agarose). Incubate under white light for 3-4 weeks until single algal colonies grow.

[0039] Example 3 Screening of transformed algal strains 1) After 3-4 weeks, pick the monoclonal algal strains that have grown on the plate and put them into liquid culture medium containing 50 μg / mL bleomycin, and culture for one week.

[0040] 2) Take 200 μL of algal solution, centrifuge at 5000 g for 2 min to remove the supernatant, add 20 μL of LTE buffer (10 mM Tris-HCl, 1 mM EDTA-Na2, pH = 8.0) to resuspend the algal cell pellet, and lyse the algal cells at 95℃ for 10 min.

[0041] 3) Using lysed algal cells as templates, the target band was identified by 1% agarose gel electrophoresis using primers across the shRNA site (yz-F: GACACGCCAAGTATTCATTG, (SEQ ID NO.11), yz-R: CACTCGTGTTCCAAACTGTAAA, (SEQ ID NO.12). The results are as follows: Figure 2 As shown, subsequent first-generation sequencing verification (Shanghai Sangon Biotech Co., Ltd.) yielded three positive transformed algal strains: Pt20, Pt21, and Pt25. The sequencing results are shown below. Figure 3 As shown.

[0042] Example 4: Validation of shRNA expression in transformed algal strains To detect the transcription level of shRNA in algal cells, total RNA was extracted using the Trizol method, and the total RNA was reverse transcribed into cDNA using a reverse transcription kit (SPARKscript II 1st Strand cDNA Synthesis Kit, Cisco, China).

[0043] shRNA expression levels in each strain were analyzed using RT-qPCR. qRT-PCR was performed using the ChamQ UniversalSYBR qPCR Master Mix from Vazyme, with RPS (Ribosomal protein S1, Phatr3_J10847) from *Phaeodactylum tricornutum* as an internal control. The reaction system is shown in Table 6, and the reaction program is shown in Table 7.

[0044]

[0045] The results are as follows Figure 4 The results showed that the Pt25 algal strain had the highest shRNA expression level, which was about 1 / 10 of the expression level of the internal reference gene RPS (Ribosomal protein S1, Phatr3_J10847).

Claims

1. A recombinant *Phaeodactylum tricornutum* species stably expressing shRNA targeting oyster herpesvirus OsHV-1, characterized in that... The recombinant expression vector containing a shRNA sequence that targets and silences the OsHV-1 herpesvirus gene was obtained by introducing it into the recipient algal strain.

2. The recombinant *Phaeodactylum tricornutum* stably expressing shRNA targeting oyster herpesvirus OsHV-1 according to claim 1, characterized in that, The shRNA sequence that targets and silences the OsHV-1 herpesvirus gene expression in oysters is one of shRNA-1, shRNA-2, or shRNA-3; The target nucleotide sequence of the shRNA-1 sequence is shown in SEQ ID NO.1, the target nucleotide sequence of the shRNA-2 sequence is shown in SEQ ID NO.2, and the target nucleotide sequence of the shRNA-3 sequence is shown in SEQ ID NO.

3.

3. The recombinant *Phaeodactylum tricornutum* stably expressing shRNA targeting oyster herpesvirus OsHV-1 according to claim 2, characterized in that, The forward primer sequence for preparing the shRNA-1 sequence is shown in SEQ ID NO.4, and the reverse primer sequence is shown in SEQ ID NO.

5.

4. The recombinant *Phaeodactylum tricornutum* stably expressing shRNA targeting oyster herpesvirus OsHV-1 according to claim 2, characterized in that, The forward primer sequence for preparing the shRNA-2 sequence is shown in SEQ ID NO.6, and the reverse primer sequence is shown in SEQ ID NO.

7.

5. The recombinant *Phaeodactylum tricornutum* stably expressing shRNA targeting oyster herpesvirus OsHV-1 according to claim 2, characterized in that, The forward primer sequence for preparing the shRNA-3 sequence is shown in SEQ ID NO.8, and the reverse primer sequence is shown in SEQ ID NO.

9.

6. The recombinant *Phaeodactylum tricornutum* stably expressing shRNA targeting oyster herpesvirus OsHV-1 according to claim 1, characterized in that, The recipient algal strain was wild-type *Phaeodactylum tricornutum*.

7. The recombinant *Phaeodactylum tricornutum* stably expressing shRNA targeting oyster herpesvirus OsHV-1 according to claim 1, characterized in that, The recombinant expression vector containing the shRNA sequence that targets and silences the OsHV-1 herpesvirus gene of oysters used the p35-sh vector plasmid. The nucleotide sequence of the p35-sh vector plasmid is shown in SEQ ID NO.

10.

8. The use of the recombinant *Phaeodactylum tricornutum* as described in any one of claims 1-7 in the preparation of oyster herpesvirus OsHV-1 vaccine.

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

10. A microalgae feed that can prevent oyster herpesvirus OsHV-1, characterized in that, Recombinant *Phaeodactylum tricornutum* containing shRNA stably expressing oyster herpesvirus OsHV-1 as claimed in any one of claims 1-7.

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