Phaeodactylum tricornutum capable of resisting white spot syndrome virus and construction method and application of phaeodactylum tricornutum

Abstract

The invention belongs to the field of diatom genetic engineering, and mainly discloses phaeodactylum tricornutum capable of resisting white spot syndrome virus as well as a construction method and application thereof. According to the invention, the expression vector is constructed by expressing the codon-optimized VP28 gene in phaeodactylum tricornutum, and the expression vector is transformed into algae cells by a bacterial transformation method to obtain the transgenic algae strain capable of stably expressing the VP28, and the transgenic algae strain can be directly used as bait for feeding prawns, does not need to extract protein, and is safe, non-toxic, low in cost and convenient to operate. The survival rate of prawns infected with the white spot syndrome virus is remarkably improved by feeding the prawns infected with the white spot syndrome virus with the algae powder of the transgenic algae strain.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of diatom genetic engineering, and particularly relates to a Phaeodactylum tricornutum capable of resisting white spot syndrome virus and a construction method and application thereof. BACKGROUND

[0002] White Spot Syndrome Virus (WSSV) is a disease that seriously harms global shrimp aquaculture, has the characteristics of fast transmission, high mortality rate, etc., causes a large decrease in shrimp production and serious economic losses every year, and currently there is no effective treatment method. Research shows that the capsid protein VP28 of WSSV plays a key role in the virus infection process. The recombinant VP28 protein expressed by a prokaryotic or eukaryotic system can effectively stimulate the immune response of shrimp. However, the common prokaryotic expression system cannot perform post-transcriptional and translational modification of proteins, and the expression product is prone to form insoluble inclusion bodies, which limits its application. Although the eukaryotic expression system (yeast, animals and plants) has the advantages of large-scale production and functional protein modification, it itself and the expression product are prone to pollute the environment, and the difficulty of expression and purification is high, and there are certain limitations. Therefore, establishing a new type of expression system that can efficiently express WSSV proteins with complete antigen activity, has low cost and high safety has become the key to promoting the prevention and control of white spot disease of shrimp.

[0003] Phaeodactylum tricornutum is a single-celled eukaryotic diatom that has a eukaryotic expression system with a cell nucleus and can express foreign proteins. Its genome was sequenced in 2008, and the genetic background is clear, and the transformation method is mature. Phaeodactylum tricornutum can process and modify proteins and correctly express foreign proteins, so it can successfully express VP28 gene by using genetic engineering method. Phaeodactylum tricornutum has the advantages of fast growth, photosynthesis, short photoperiod, low cultivation cost and environmental protection; most importantly, it can be directly used as an additive for shrimp feed, without the need for protein purification, easy operation, safety, low cost and no toxicity. SUMMARY

[0004] In view of the problems existing in the current prokaryotic and eukaryotic expression systems for expressing foreign genes, the present application provides a Phaeodactylum tricornutum capable of resisting white spot syndrome virus and a construction method and application thereof. The transgenic algae strain capable of stably expressing VP28 is cultured on a large scale, the algae liquid is collected, the algae liquid is freeze-dried to obtain algae powder, and the algae powder is directly used as an additive for shrimp feed to feed shrimp infected with WSSV, so that the survival rate can be significantly improved.

[0005] The present application is completed in the following four steps: a construction method and application of Phaeodactylum tricornutum capable of resisting white spot syndrome virus. (1) Synthesize the codon-optimized VP28 gene cDNA sequence. Construct the VP28 expression vector, named P0521s-VP28.

[0006] (2) Transform the expressed vector into the recipient Phaeodactylum tricornutum by bacterial transformation method, and screen the positive algae strains.

[0007] (3) Verify the genomic DNA of the positive algae strains, and also verify the expression of VP28 from the RNA level and the protein level, to obtain the VP28 transgenic algae strains capable of stable expression.

[0008] (4) Large-scale culture of the obtained transgenic algae strains, freeze-drying into algal powder as an additive for prawn feed. Feed the prawns for 12 days, then inject the prawns with WSSV, and compare with the wild type and the feed group, to count the survival rate of the prawns.

[0009] The present application has the following advantages in using Phaeodactylum tricornutum as a cell factory to produce VP28 antigen: (1) The genetic engineering method is mature, and can successfully express heterologous active substances in vivo, such as monoclonal antibodies, precursors of biological plastics, etc. (2) The growth cycle of Phaeodactylum tricornutum is short, and it can perform photosynthesis, so it can be directly used as a bait additive for prawns, without the need for protein extraction and purification, with low cost and simple operation.

[0010] VP28 gene sequence table SEDIQ No. 1 Information of SEQ ID No. 1 (a) Sequence characteristics Length: 615 nucleotides Type: nucleotide Chain type: single strand (b) Molecular type: DNA Sequence description: SEQ ID NO. 1 ATGGACCTCTCCTTCACCCTCTCCGTTGTCTCCGCCATTCTCGCCATTACCGCCGTCATTGCTGTTTTCATTGTCATTTTTCGTTACCACAACACCGTCACCAAGACCATTGAAACCCACACCGACAACATTGAAACCAACATGGATGAAAACTTGCGTATTCCCGTCACCGCCGAAGTCGGATCGGGATACTTCAAGATGACCGATGTCTCCTTTGATTCCGACACCTTGGGAAAGATTAAGATTCGTAACGGAAAGTCCGATGCCCAGATGAAGGAAGAAGATGCCGACTTGGTCATTACCCCCGTCGAAGGACGTGCCTTGGAAGTCACCGTCGGACAAAACTTGACCTTTGAAGGAACCTTTAAGGTCTGGAACAACACTTCTCGTAAGATTAACATTACTGGTATGCAGATGGTCCCTAAGATTAACCCTTCCAAGGCCTTCGTTGGATCTTCCAACACCTCCTCCTTTACCCCCGTTTCCATTGATGAAGATGAAGTCGGAACCTTCGTCTGCGGAACCACCTTCGGAGCCCCCATTGCCGCCACCGCCGGAGGAAACCTCTTCGACATGTACGTCCACGTCACCTACTCCGGAACCGAAACCGAATAA VP28 gene sequence Information for SEQ ID No. 2 (a) Sequence characteristics Length: 204 amino acids Type: amino acid Chain type: single chain (b) Molecule type: amino acid Sequence description: SEQ ID NO. 2 MDLSFTLSVVSAILAITAVIAVFIVIFRYHNTVTKTIETHTDNIETNMDENLRIPVTAEVGSGYFKMTDVSFDSDTLGKIKIRNGKSDAQMKEEDADLVITPVEGRALEVTVGQNLTFEGTFKVWNNTSRKINITGMQMVPKINPSKAFVGSSNTSSFTPVSIDEDEVGTFVCGTTFGAPIAATAGGNLFDMYVHVTYSGTETE BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Construction of VP28 expression vector in the present application.

[0012] Figure 2 Verification of VP28 positive algal strain at DNA and RNA levels in the present application.

[0013] Figure 3 Verification of VP28 positive algal strain at protein level in the present application. DETAILED DESCRIPTION

[0014] The present application is further described in conjunction with the following examples. It should be understood, however, that these examples are intended to illustrate the present application and are not intended to limit the scope of the present application. Furthermore, since modifications to this application will occur to those of ordinary skill in the art, it will be understood that all such modifications are included within the scope of the present application.

[0015] Unless otherwise indicated, the reagents used in the following examples were obtained from common commercial sources.

[0016] Example 1 Culture of Phaeodactylum tricornutum Phaeodactylum tricornutum was used as the recipient algal strain for transgene. It was cultured in f / 2 artificial seawater medium according to the research report of Guillard and Ryther (1962) (Reference: Guillard RRL, Ryther JH. Studies of marine planktonic diatoms: I. Cyclotella na Hustedt, and Detonula confervacea (Cleve) Gran [J]. Canadian Journal of Microbiology, 1962, 8 (2): 229-239.) in a constant temperature and light incubator (GXZ-380C, Jiangnan Instrument Factory, Ningbo, China). The culture was statically incubated and the flask was manually shaken 3 times a day. The light intensity of the culture was 80 mmol photons m-2s-1.-2 s -1 Phaeodactylum tricornutum was obtained by culturing the above-mentioned P. tricornutum in a 250 mL flask containing 100 mL of F / 2 medium under light and dark cycles of 12 h: 12 h at a temperature of 20 ± 1 °C.

[0017] Example 2 Synthesis of VP28 gene The VP28 gene sequence (NCBI Genbank Accession No. ABG75923.1) corresponding to the amino acid sequence MDLSFTLSVVSAILAITAVIAVFIVIFRYHNTVTKTIETHTDNIETNMDENLRIPVTAEVGSGYFKMTDVSFDSDTLGKIKIRNGKSDAQMKEEDADLVITPVEGRALEVTVGQNLTFEGTFKVWNNTSRKINITGMQMVPKINPSKAFVGSSNTSSFTPVSIDEDEVGTFVCGTTFGAPIAATAGGNLFDMYVHVTYSGTETE, see SEQ ID NO. 1 was downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ).The gene sequence of VP28 gene expressed in Phaeodactylum tricornutum according to the codon preference of Phaeodactylum tricornutum and codon optimization by Shengong Company is SEQ ID NO. 1: ATGGACCTCTCCTTCACCCTCTCCGTTGTCTCCGCCATTCTCGCCATTACCGCCGTCATTGCTGTTTTCATTGTCATTTTTCGTTACCACAACACCGTCACCAAGACCATTGAAACCCACACCGACAACATTGAAACCAACATGGATGAAAACTTGCGTATTCCCGTCACCGCCGAAGTCGGATCGGGATACTTCAAGATGACCGATGTCTCCTTTGATTCCGACACCTTGGGAAAGATTAAGATTCGTAACGGAAAGTCCGATGCCCAGATGAAGGAAGAAGATGCCGACTTGGTCATTACCCCCGTCGAAGGACGTGCCTTGGAAGTCACCGTCGGACAAAACTTGACCTTTGAAGGAACCTTTAAGGTCTGGAACAACACTTCTCGTAAGATTAACATTACTGGTATGCAGATGGTCCCTAAGATTAACCCTTCCAAGGCCTTCGTTGGATCTTCCAACACCTCCTCCTTTACCCCCGTTTCCATTGATGAAGATGAAGTCGGAACCTTCGTCTGCGGAACCACCTTCGGAGCCCCCATTGCCGCCACCGCCGGAGGAAACCTCTTCGACATGTACGTCCACGTCACCTACTCCGGAACCGAAACCGAATAA, see SEQ ID NO. 1. First, Shanghai Shengong Company Limited was entrusted to synthesize VP28 gene, and according to the instructions of Infusion kit (Code# CU201-02, Quansheng Biotech Co., Ltd., Beijing), the synthesized gene sequence was inserted into the diatom overexpression P0521S plasmid (literature: Karas, B., Diner, R., Lefebvre, S. et al. Designer diatom episomes delivered by bacterial conjugation[J]. Nature Communications 6, 6925 (2015)) to construct VP28 expression vector (e.g. Figure 1VP28 gene was amplified by PCR using Taq DNA polymerase (Cat. CW0690H, CWBIO, Beijing) according to the manufacturer's instructions, using primers VP28-F and VP28-R with the sequences: ATTTGTCTGCCGTTTCGAATGGACCTCTCCTTCA and TTCGATAGCACGCTTCTGTTATTCGGTTTCGGTTCC, respectively. The amplified VP28 target fragment and linearized P0521S plasmid were recovered by gel recovery kit (D2500-02, omega, Japan). The target fragment and linearized plasmid were ligated according to the instructions of the Infusion kit (Code# CU201-02, Genmark Biotech Co., Ltd., Beijing). The Infusion reaction system was as follows: 2x Basic Assemble Mix was 5 ml, linearized plasmid was 1 ml, and target fragment was 4 ml. The reaction system was mixed gently, and the reaction was performed at 50°C for 15 min. After the reaction, the centrifuge tube was placed on ice to cool for 2 min to obtain the ligation product. Then, transformation was performed. 50 ml of DH5a competent cells (Shanghai Generay Biotech Co., Ltd.) were added to a 1.5 ml centrifuge tube, and 2.5 ml of the ligation product was added. The mixture was mixed gently and placed on ice for 30 min. Then, the mixture was heated at 42°C for 60 s in a water bath, and then transferred to ice for 2 min. Then, 450 ml of LB medium (LB medium was purchased from Shenguo Bioengineering Co., Ltd., NO. A507002) was added to a 37°C shaker at 220 rpm for 1 h. Then, 100 ml of the mixture was evenly coated on an LB plate containing Amp (Cat# A8180, Solarbio, 100 mg / ml) (15 g agar / L, 250 g LB medium / L), and then cultured in a 37°C incubator overnight. The next day, a single colony was picked (sequencing was performed by Shenguo Bioengineering Co., Ltd.), and the P0521S-VP28 plasmid with correct sequencing was used for the next experiment.

[0018] Example 3: The constructed P0521S-VP28 plasmid was transferred to Phaeodactylum tricornutum by bacterial transformation.

[0019] 1. Screening of positive algae strains by bacterial transformation The plasmid was transferred into Phaeodactylum tricornutum by conjugation with E. coli using the method reported in the literature (H. Wang, S.S. Slattery, B.J. Karas, D.R. Edgell, Delivery of the Cas9 or TevCas9 system into Phaeodactylum tricornutumvia conjugation of plasmids from a bacterial donor, Bio-protocol, 8 (2018)e2974.).

[0020] (1) Phaeodactylum tricornutum was cultured in a light incubator to the exponential growth phase, with light intensity of 80 pmol m -2 s -1 , and the algal cells were concentrated to 1 x 10 8 cells / mL by centrifugation, and spread on solid plates containing 50% ASW and cultured for 2 days.

[0021] (2) The algal cells were scraped from the plates with sterile ASW containing f / 2 medium, and the algal cells were concentrated to 5 x 10 8 cells / mL.

[0022] (3) E. coli containing the plasmid with VP28 was inoculated into 1 mL of LB medium (containing 40 pg / mL of gentamicin and 50 pg / mL of chloramphenicol) and cultured overnight at 37°C in a shaker to activate the bacteria. 1 mL of the bacteria was inoculated into 35 mL of LB medium for expansion culture, and shaken at 37°C for about 4-5 h until the OD600nmof the bacteria was 0.8-1.0.

[0023] (4) The bacteria were collected by centrifugation at 3000 g for 10 min, and the supernatant was discarded (poured onto toilet paper and the supernatant was sucked dry), and the precipitate was suspended with 200 pL of SOC medium.

[0024] (5) 200 pL of the algal cells concentrated in step (2) were mixed with 200 pL of the bacterial solution in the previous step, and spread on a plate (50% ASW, 1% agarose, 5% LB), and placed in a 30°C incubator for 90 min, and then placed in a 20°C light incubator for 2 days.

[0025] (6) After two days, the algal cells on the plate were scraped with ASW (100 pg / mL of bleomycin) and evenly spread on a plate containing 50 pg / mL of bleomycin (50% ASW, 1% agarose), and placed in a light incubator for 3-4 weeks until single algal colonies were grown.

[0026] 2. Screening of positive algal strains (1) 10 single algal colonies grown on the resistance plate were picked and cultured in 10 ml of culture tubes, and 2 ml of f / 2 liquid medium containing bleomycin (100 pg / mL) was added and cultured for 4-5 days.

[0027] (2) Take out 500 ml of algal liquid from the culture tube into a 1.5 ml centrifuge tube, centrifuge at 8000g for 1 min, discard the supernatant, add 20 ml of TE lysis solution to lyse the algal cells, and perform lysis at 95°C for 10 min to obtain the DNA of the algal cells.

[0028] 3. DNA level verification PCR amplification was performed using a PCR instrument (TaKaRa, Japan), and the 25 ml PCR reaction system was as follows: 12.5 ml Taq DNA polymerase (Cat. CW0690H, CWBIO, Beijing), 1 μL of each verification primer (YZP0521F: GACACTTTCAGTGAGGACA AGAAG, YZP0521R: CACCCGCTCGCGGGTGGGCCTACT, 2 ml of lysed algal cells, and 8.5 ml of water. The reaction process was as follows: 94°C for 4 min, 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, for 35 cycles. After 2.5 ml of PCR product was electrophoresed on a 1% agarose gel for 15 min, it was found by a gel imaging instrument that three VP28 transgenic algal strains had a band of about 1500 bp, and WT had no band (such as Figure 2 A), the PCR product was entrusted to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the sequencing results were compared. The correct comparison indicated that VP28 was successfully expressed at the DNA level of Phaeodactylum tricornutum.

[0029] 4. RNA level verification (1) Collect 50 ml of algal liquid from each of the three VP28 transgenic algal strains and the WT algal strain, centrifuge, suspend the centrifuged algal liquid, transfer it to a 1.5 ml centrifuge tube, centrifuge at 8000g for 1 min, discard the supernatant, and place it in liquid nitrogen for RNA extraction.

[0030] (2) Extract the RNA of the three VP28 transgenic algal strains and the WT algal strain.

[0031] RNA was extracted according to the instructions of the Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Cat.#DP441, Tiangen Biotech Co., Ltd., Beijing): The collected algal solution was rapidly ground into powder in liquid nitrogen. After grinding, the powder was transferred to a centrifuge tube, and 500 ml of lysis buffer (475 ml) was added. Add SL + 25ml β-mercaptoethanol, immediately vortex for 2 min, centrifuge at 12000 rpm for 2 min, transfer the supernatant to the CS filter column, centrifuge at 1200 rpm for 2 min, carefully aspirate the supernatant from the collection tube into a new RNase-Free centrifuge tube, avoiding contact between the pipette tip and cell debris precipitate in the collection tube as much as possible, add 0.4 times the volume of supernatant anhydrous ethanol and mix well, transfer the resulting solution and precipitate together into the CR3 adsorption column, centrifuge at 12000 rpm for 15 s, discard the waste liquid in the collection tube, put the CR3 adsorption column back into the collection tube, add 350ml of protein removal solution RW1 to the CR3 column, centrifuge at 12000 rpm for 15 s, discard the waste liquid in the collection tube, prepare the working solution: take 10ml of DNaseI and add 70ml of RDD buffer, mix gently, add 80ml of working solution to the center of the CR3 adsorption column, and incubate at room temperature for 15 min. Add 350 ml of protein removal buffer RW1 to the adsorption column CR3, centrifuge at 12000 rpm for 15 s, discard the waste liquid in the collection tube, put the adsorption column CR3 back into the collection tube, add 500 ml of washing buffer RW to the adsorption column CR3, centrifuge at 12000 rpm for 15 s, discard the waste liquid in the collection tube, repeat this operation once, centrifuge at 12000 rpm for 2 min, put the adsorption column CR3 into a new RNase-Free centrifuge tube, add 35 ml of RNase-Free ddh2o to the middle of the adsorption column, incubate at room temperature for 2 min, centrifuge at 12000 rpm for 1 min to obtain the RNA solution.

[0032] (3) RT-PCR Following the instructions of the reverse DNA ionization kit (Code.#RR047A, Takara, Japan), the DNA was reversed to cDNA, and then used to remove genomic DNA from the total RNA. The reaction system is as follows:

[0033] Using RNA with genomic DNA removed as a template, a reverse reaction was performed using TakaRa's PrimerScrip™ RT reagent Kit with gDNA Eraser.

[0034] The specific reaction system is as follows:

[0035] The three VP28 transgenic algal strains obtained from the above RT reaction and the cDNA from the WT type were used as templates for PCR amplification to verify the RNA expression level of the three transgenic algal strains. RPS was used as an internal control (RPSF: CGAAGTCAACCAGGAAACCAA and RPSR: GTGCAAGAG). PCR was performed using the verification primers YZVP28F: ATGGACCTCTCCTTCACC and YZVP28R: TTATTCGGTTTCGGTTCCG. Using RPS primers WT and the three VP28 transgenic algal strains, bands were observed (e.g., ACCGGACATACC). Figure 2 (B) The three VP28 transgenic algal strains using the validation primers showed a band of approximately 600 bp, while WT did not (e.g., Figure 2 C).

[0036] 5. Verification of protein levels Algal solutions of the VP28 transgenic algae were collected, and total protein was extracted using SDS extraction buffer (50 mM Tris, 2% SDS, adjusted to pH 6.8) for protein level verification. Coomassie Brilliant Blue staining was used; 20 μL of extracted total protein was added to 5× SDS-PAGE protein loading buffer (Biosharp, Guangzhou), and heated at 95°C for 10 min to denature the proteins. Electrophoresis was performed on a 12% SDS-PAGE gel in Tris-MOPS-SDS electrophoresis buffer (GenScript, Nanjing) for half an hour at a constant voltage of 180V. The protein gel was then transferred to a container containing 25 ml of staining solution, ensuring complete coverage. The gel was shaken on a shaker for approximately 10 min, after which clear protein bands were observed. After staining, the gel was placed in water for destaining for approximately 15 min, and the target VP28 band (approximately 28 kDa) was cut off. Mass spectrometry identification was performed by Shanghai Sangon Biotech Co., Ltd., and the results showed the presence of VP28 protein. The abundance of proteins identified at this location (Lhcf9, Rbcl, atpB, and psbA) was compared (e.g., Figure 3 ).

[0037] 6. Verification through challenge experiments First, Litopenaeus vannamei was divided into four boxes and cultured normally in seawater. The first group was fed a mixture of feed and VP28-positive *Phaeodactylum tricornutum* powder (5:1 ratio). The second group was fed a mixture of feed and wild-type *Phaeodactylum tricornutum* powder (5:1 ratio). The third and fourth groups were fed only feed. Water was changed daily during feeding, and this process was repeated for 12 days. Then, the shrimp in the first, second, and third groups were injected with white spot syndrome virus (prepared with PBS buffer at a concentration of 600 copies). Seventeen shrimp were injected in the first group, seventeen in the second group, and nineteen in the third group, with each shrimp receiving 10 μL. The fourth group served as a blank control, receiving only PBS buffer. A challenge experiment was then conducted, and the shrimp survival rate was statistically analyzed (as shown in Table 1). Table 1. Survival rate of Litopenaeus vannamei shrimp after 4 days of WSSV challenge.

[0038] Note: The first group was fed with feed and VP28 transgenic algae powder (5:1), the second group was fed with feed and wild-type algae powder (5:1), the third group was fed with feed, and the fourth group was fed with feed as a control.

Claims

1. A method of constructing a white spot syndrome virus-resistant Phaeocystis globosa, characterized in that: The codon-optimized VP28 gene sequence is connected and cloned into a plasmid to construct a VP28 expression vector, and the constructed expression vector is transformed into a recipient Phaeodactylum tricornutum by a bacterial transformation method to obtain a VP28 transgenic algae strain capable of stably expressing VP28, wherein the VP28 gene sequence is codon-optimized, and the VP28 gene sequence is a sequence of SEQ ID NO.

1.

2. The construction method of the Phaeodactylum tricornutum capable of resisting white spot syndrome virus according to claim 1, wherein an amino acid sequence in the VP28 gene sequence is a sequence of SEQ ID NO.

2.

3. The construction method of the Phaeodactylum tricornutum capable of resisting white spot syndrome virus according to claim 1, wherein the constructed expression vector is named P0521S-VP28.

4. A VP28 transgenic algae strain capable of stably expressing VP28, obtained by the construction method of claim 1.

5. The VP28 transgenic algae strain capable of stably expressing VP28 of claim 4 is applied to white spot syndrome virus-infected prawns as a feed additive.

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