CN110438054A - Vp19 transgenic blue algae and its purposes in preparation prevention and treatment shrimp white spot syndrome virus drug - Google Patents

Abstract

The invention discloses vp19 transgenic cyanobacteria and its application in the preparation of medicines for preventing and treating prawn white spot syndrome virus. The vp19 transgenic cyanobacteria is constructed by transferring the vp19 shuttle plasmid into cyanobacteria, and the genomic DNA of prawn white spot syndrome virus is used as a template to design amplification primers. Perform PCR amplification, and connect the vp19 gene obtained with the pRL489 plasmid to construct a vp19 shuttle expression vector and transform it into Escherichia coli. After the type cyanobacteria are cultured, they are mixed in proportion, and the three-parent conjugation is transferred to obtain cyanobacteria transformants, and the culture is continued. The obtained vp19 transgenic cyanobacteria is used as an active ingredient to prepare a drug for preventing and treating white spot syndrome virus of prawns, and the effective dosage is 1-20 μg/tail, the effect is remarkable and the cost is easy to control.

Description

vp19 transgenic cyanobacteria and its use in the preparation of medicines for preventing and treating white spot syndrome virus in prawns the use of

technical field

The invention belongs to the technical field of cyanobacteria bioengineering, and specifically relates to obtaining vp19 transgenic cyanobacteria through the methods of plasmid vector recombination construction, primer synthesis, enzyme digestion, common PCR, three-parent conjugation transfer method, and RT-PCR, and is used for preparing and preventing white spot syndrome of prawns Viral drugs.

Background technique

White spot disease is caused by white spot syndrome virus (White Spot Syndrome Virus, WSSV), which causes huge economic losses to the global prawn farming industry every year. WSSV virus is a kind of baculoform DNA virus, and shrimp will develop into white spot syndrome once infected with WSSV, then dies rapidly in 2-7 days, and the mortality rate is as high as 100%, so far there is no effective drug control of scale application. In recent years, the prevention and control of WSSV has made great progress in immune research. vp19 is one of the most abundant envelope proteins on WSSV. The research mainly focuses on fusing vp19 protein into E. coli, yeast, animal cells and other vectors, and purifying the small Large-scale indoor experiments, but bacteria cannot be directly fed to shrimp as bait, which limits its large-scale commercial application in WSSV control.

Contents of the invention

The main purpose of the present invention is to overcome the high mortality rate of white spot syndrome virus infection of prawns and there is no effective drug control for large-scale application so far, to construct a vp19 shuttle plasmid and transfer it into blue-green algae for the first time, to obtain vp19 transgenic blue-green algae, which can be used for large-scale prevention and treatment of prawns White spot syndrome virus, to achieve the effect of "medicine and food from the same source".

The technical solution adopted by the present invention to solve its technical problems is:

In the first aspect, the construction method of vp19 transgenic cyanobacteria comprises the following steps:

(i) Using the genomic DNA of prawn white spot syndrome virus as a template, design the amplification primer pair as shown in SEQ ID.NO.1 and SEQ ID.NO.2, and obtain the vp19 gene by PCR amplification;

(ii) connecting the vp19 gene obtained in step (i) with the pRL489 plasmid, constructing a vp19 shuttle expression vector and transforming it into Escherichia coli to obtain Escherichia coli containing the pRL489-vp19 plasmid;

(Ⅲ) the Escherichia coli containing the pR4-pRL-542 plasmid, the Escherichia coli containing the pRL489-vp19 plasmid obtained in step (ii) and the purified wild-type cyanobacteria are cultivated to the mid-logarithmic growth phase, and the cell number ratio is 5 : 5:1 mixing, transforming cyanobacteria with a vector with vp19 gene by triparental conjugative transfer, and screening cyanobacteria transformants;

(iv) step (iii) obtained cyanobacteria transformants continue to cultivate to obtain vp19 transgenic cyanobacteria; and/or also include

(v) post-processing steps of sedimentation, centrifugation, collection, washing, freeze-drying and pulverization after vp19 transgenic cyanobacteria are cultivated.

Preferably, the cyanobacteria is Synechococcus.

In the second aspect, the use of vp19 transgenic cyanobacteria in the preparation of medicines for preventing and treating shrimp white spot syndrome virus.

Preferably, the use of the vp19 transgenic cyanobacteria in the preparation of oral vaccines or immune enhancers for the prevention and treatment of prawn white spot syndrome virus.

In the third aspect, the pharmaceutical composition for preventing and treating prawn white spot syndrome virus uses vp19 transgenic cyanobacteria as an effective active ingredient, and its effective dosage is 1-20 μg/tail, and also includes fishery pharmaceutically acceptable carriers or auxiliary materials.

Preferably, the effective dosage of the vp19 transgenic cyanobacteria is 10-20 μg/tail.

Preferably, the dosage form of the pharmaceutical composition is powder, granule, capsule, tablet or pill.

Compared with prior art, the beneficial effect of the present invention is:

According to the high content of vp19 protein in WSSV, the expression of the fusion protein is detected from the level of transcription and translation, and the vp19 gene expression shuttle vector is successfully constructed, and successfully expressed in Synechococcus sp. , and the effect is remarkable, it can be used as a medicine for preventing and treating white spot syndrome virus of prawns, such as oral vaccine or immune booster, and it is an effective and cost-effective immunization method.

Description of drawings

Figure 1 is a map of the approximate restriction positions of the pRL489 plasmid.

Figure 2 is a vp19 gene fragment.

Figure 3 is the PCR amplification product of vp19 gene; wherein, 1 and 2 are vp19 gene fragments; 3 is DL2000Marker.

Figure 4 is the ligation product of the pRL489-vp19 plasmid.

Figure 5 is the electrophoresis diagram of pRL-489-vp19 recombinant plasmid; 1 is DL15000Marker; 2, 3, 4 are pRL-489-vp19 recombinant plasmid; 5, 6, 7 are pRL-489 empty vector.

Figure 6: (a) is PCR identification of recombinant plasmid pRL489-vp19, 1 is DL2000Marker; 2 is pRL489-vp19 gene fragment; (b) is KpnⅠ/XhoI double enzyme digestion identification of recombinant plasmid pRL489-vp19, 1 is the result of enzyme digestion; 2 is DL10000Marker.

Fig. 7 is the partial sequencing result of the recombinant plasmid pRL489-vp19 compared by the Blast program.

Figure 8 is the growth of vp19 transgenic cyanobacteria on the plate containing resistance; a1, a2 are pRL489 in the first week, a3, a4 are pRL489-vp19 in the first week; b1, b2 are pRL489 in the second week; b3, b4 is pRL489-vp19 in the second week.

Figure 9 is the growth of vp19 transgenic cyanobacteria in conical flasks containing resistance; c1, c2 are the pRL489-vp19 plate a3 in the first week; c3, c4 are the pRL489-vp19 plate a4 in the first week; d1, d2 are the second Weekly pRL489-vp19 plate b3; d3 and d4 are the second week pRL489-vp19 plate b4.

Fig. 10 is a control chart of the mortality rate in the oral challenge test of vp19 transgenic cyanobacteria.

Detailed ways

In the following examples, the cyanobacterium is Synechococcus 7942. The construction method of vp19 transgenic cyanobacteria comprises the following steps:

(i) Using the genomic DNA of prawn white spot syndrome virus as a template, design the amplification primer pair as shown in SEQ ID.NO.1 and SEQ ID.NO.2, and obtain the vp19 gene by PCR amplification;

(ii) connecting the vp19 gene obtained in step (i) with the pRL489 plasmid, constructing a vp19 shuttle expression vector and transforming it into Escherichia coli to obtain Escherichia coli containing the pRL489-vp19 plasmid;

(Ⅲ) the Escherichia coli containing the pR4-pRL-542 plasmid, the Escherichia coli containing the pRL489-vp19 plasmid obtained in step (ii) and the purified wild-type cyanobacteria are cultivated to the mid-logarithmic growth phase, and the cell number ratio is 5 : 5:1 mixing, transforming cyanobacteria with a vector with vp19 gene by triparental conjugative transfer, and screening cyanobacteria transformants;

(iv) step (iii) obtained cyanobacteria transformants continue to cultivate to obtain vp19 transgenic cyanobacteria; and/or also include

(v) post-processing steps of sedimentation, centrifugation, collection, washing, freeze-drying and pulverization after vp19 transgenic cyanobacteria are cultivated.

Below in conjunction with accompanying drawing 1-10 further explain the construction and verification method of vp19 transgenic cyanobacteria, specifically as follows:

The first step: construction of recombinant expression vector. The complete sequence of the pRL489 plasmid was sequenced, and the restriction sites in the pRL489 plasmid were sorted out. The main restriction sites are shown in Table 1, and then the prawn white spot syndrome virus vp19 and vp1 base sequences registered on the GenBank database were retrieved ( AJ937860.1), designed a pair of amplification full-length primers 383F/R as shown in Table 2, the underlined places in the table are the restriction sites of XhoI and KpnI respectively, and the expected DNA amplification fragment length is 383bp. Then use the prawn white spot syndrome virus (WSSV) as template, carry out PCR amplification vp19 gene with primer, after PCR product detects by 2% agarose gel electrophoresis, reclaim with gel recovery kit, product connects to pMD19-T, transforms Into Escherichia coli TOP10, after the colonies grew out, the recombinants were selected for bacterial liquid PCR identification, as shown in Figure 3.

Table 1: Main restriction sites of plasmid 489

wxya C^TCGAG 6 5' (4nucleotides) 1cut positions: 4767 KpnI GGTAC^C 6 3' (4 nuucleotides) 1cut positions: 6212 SacI GAGCT^C 6 3' (4 nuucleotides) 1cut positions: 6292

Table 2: vp19 primer sequences

Primer name Primer sequence(5′-3′) Size(bp) wxya CGCTCGAG-ATGGCCACCACGACTAACACTC 30 wxya TCGGGTACC-TTACTGCCTCCTCTTGGGGTAAGACA 35

E. coli containing the pRL489-vp19 plasmid were prepared.

① Transformation and screening of Escherichia coli: Add 5 μL of plasmid to every 50 μL of competent cells, flick and mix well. After 30 minutes of ice bathing, heat shock at 42°C for 90 seconds, and quickly ice bath for 2 minutes to cool down the system. Do not shake the centrifuge tube during the process. Add 450 μL of LB culture solution to the centrifuge tube, centrifuge at 37°C, 45 min, 150 rpm, take 100 μL of the bacterial solution and add it to the LB plate containing the corresponding antibiotic, place it upright for about 1 hour to fully absorb the bacterial solution, and incubate it upside down for 12 hours for screening.

②Escherichia coli plasmid preparation: Ligate the target gene and pRL489 empty vector by T4DNA ligase, as shown in Figure 3, then overnight at 16°C, transfer the ligated product into Escherichia coli TOP10 competent cells, take 50 μL of bacterial liquid and add 5 mL of sterile LB for culture solution, cultivated overnight at 37°C, 160rpm. The transformed Escherichia coli cultured overnight was washed twice with clean LB culture medium, single clones were picked for expansion and cultured, and the recombinant plasmid pRL489-vp19 was extracted using the Tiangen small plasmid kit. The recombinant pRL489-vp19 plasmid was verified by PCR, and an empty vector was used as a control for 1% agarose gel electrophoresis detection. The results showed that the quality of the extracted plasmid was good, the purity was high, and there was no pollution of protein and RNA, as shown in FIG. 5 .

Referring to Figures 6 and 7, positive clones were picked and verified, and the bacterial liquid PCR product of the recombinant expression vector pRL489-vp19 was subjected to agarose gel electrophoresis, and the results showed that a specific target band was visible at 109bp (Figure 6a); The purified recombinant expression vector pRL489-vp19 was identified by KpnI and XhoI double enzyme digestion, 1% agarose gel electrophoresis detection showed two fragments of a vector band with a size of about 11.3kb and a target band of 380bp (Figure 6b). The exogenous DNA in the multiple cloning site on the plasmid pRL489-vp19 was sequenced and identified, as shown in Figure 7, the sequence result was 100% homologous to the coding sequence retrieved by GenBank through the Blast program, indicating that the constructed recombinant The plasmid is correct.

The third step: triparental conjugation transfer. It is to mix the activated donor bacteria, auxiliary bacteria and recipient bacteria together, so that the bacteria are in close contact, and the plasmid of the donor bacteria can be introduced into the recipient bacteria through conjugation and transfer with the help of the assistant bacteria. The donor bacteria contain The Escherichia coli containing the pRL489-vp19 plasmid, the auxiliary bacterium is the Escherichia coli containing the PR4-PRL-542 plasmid, and the recipient bacterium is Synechococcus sp. 7942. Specifically: Synechococcus 7942 in the logarithmic growth phase was prepared, centrifuged at 4000 rpm for 2 minutes, washed twice with culture medium, and then resuspended in 1 mL of culture medium to make the cell density 10 ⁸ /mL. Shake the bacteria one night before inoculation, including pRL489-vp19 strains, pRL489 strains and pR4-pRL-542 strains, inoculate 1 mL of bacterial liquid into 40 mL of LB culture medium and activate at 200 rpm for 3 hours, then centrifuge at 7000 rpm for 5 minutes to collect precipitated bacteria and resuspend In 400 μL LB culture medium, mix pRL489-vp19 and pR4-pRL-542 bacterial solution, pRL489 and pR4-pRL-542 bacterial solution in equal proportions. Then mix the algae and bacteria according to the ratio of 1:10, and then gently spread it on the filter membrane of the BG-11 culture plate without antibiotics, place it upright for 45mins, put it into the incubator and cultivate it upside down, and obtain the transformed algae strain of cyanobacteria .

After the three parents were mixed, they were evenly spread on the filter membrane, and initially placed on solid BG-11 medium without antibiotics. After the cyanobacteria on the filter membrane grow up, they are moved to the solid BG-11 medium (25ml BG11+50ul (100mg/ml) kana) with antibiotics for screening. After culturing for 2 days, carefully transfer the filter membrane from the antibiotic-free plate to the plate containing Kanna antibiotics (25mL+50μL), and transfer the filter membrane again to the resistant plate (25mL+100μL) after two weeks, It can be clearly seen that pRL489-vp19 grows better than pRL489.

Pick out the green single algae colony from the plate containing antibiotics, take out the algae colony and culture it aseptically in a test tube containing about 2mL of 25μg/mL culture solution; after 1 week, transfer it to a 30mL medium containing 25-50μg/mL antibiotic Cultured in BG-11 medium (50mL Erlenmeyer flask). After one week, expand to culture in a 250mL Erlenmeyer flask containing 150mL culture solution, and gradually increase the concentration of antibiotics from 25μg/mL to 250μg/mL through 50, 75, 100, 150, 200. Cut off the filter membrane and screen in different concentrations of antibiotic liquid culture solution, cut a small piece from each filter membrane, put it into a 100 mL Erlenmeyer flask containing 50 mL of liquid culture solution, and add 100 μL of 100 mg/mL card The growth situation of Namycin is shown in Figure 9c. After another 10 days, 150 μL of 100 mg/mL Kanamycin was added to the conical flask, and the growth situation was shown in Figure 9d. The growth state of the membrane cut from the top is better.

Take the transformed algal strains cultured to the logarithmic growth phase (cell concentration ³ ×105 cells/mL), extract the algal cell RNA with an RNA extraction kit, reverse transcribe the cDNA, detect the expression of vp19 by RT-PCR, and use the RT-PCR primers See Table 3.

Table 3: RT-PCR Primers

vp19_157-F ATGGATGGGACCAAAGAA vp19_157-R GGAAACGAGGAACAGAAGA vp19_378-F GGTACCATGGCCACCACGACTAACAC vp19_378-R CTCGAGCTGCCTCCTCTTGGGGT

Below in conjunction with specific example further illustrate vp19 transgenic cyanobacteria is used for the medicine effect of preparation prevention and treatment prawn white spot syndrome virus medicine.

Shrimp seedlings in the larval stage were used, and they were divided into 4 groups for treatment:

(1) Negative control (neither feeding oral agent, nor using WSSV challenge);

(2) Positive control (do not feed oral agent, but challenge virus with WSSV);

(3) feed the wild-type cyanobacteria group (challenge after feeding the wild-type cyanobacteria);

(4) Feed the transgenic algae group (challenge after feeding the transgenic algae).

The 2 groups of feeding algae treatment are: feeding with wild-type or transgenic algae for 10 days respectively, and then (2), (3), and (4) three groups were challenged with WSSV for 10 days to detect the survival rate, calculated as a percentage, the results are shown in Fig. 10 shows that the Synechococcus sp. 7942 expressing the vp19 protein was fed to Penaeus vannamei for 10 days, and the mortality rate of the vp19 group was 42.22%, that of the wild type was 88.89%, and that of the positive control group was 96.67%. After 10 days, the mortality rate in each group leveled off.

In summary, the vp19 transgenic cyanobacteria can significantly enhance the resistance of Penaeus vannamei to WSSV, and oral administration of drugs to shrimp has broad application prospects.

sequence listing

<110> Shanghai Ocean University

<120> vp19 transgenic cyanobacteria and its use in the preparation of medicines for preventing and treating white spot syndrome virus in prawns

<141> 2019-07-26

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 30

<212>DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 1

cgctcgagat ggccaccacg actaacactc 30

<210> 2

<211> 35

<212>DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 2

tcgggtacct tactgcctcc tcttggggta agaca 35

Claims (9)

1.vp19 transgenic blue algae, it is characterised in that: cyanobacteria is transferred to by vp19 shuttle plasmid and is obtained, construction method include with Lower step:

(I) designs amplimer to such as SEQ ID.NO.1 and SEQ using the genomic DNA of White Spot Syndrome Virus as template Shown in ID.NO.2, is expanded with PCR method and obtain vp19 gene；

Vp19 gene obtained by (II) step (I) is connected with pRL489 plasmid, is constructed vp19 shuttle expression carrier and is transferred to large intestine bar Bacterium obtains the Escherichia coli containing pRL489-vp19 plasmid；

(III) contains the big of pRL489-vp19 plasmid for what the Escherichia coli containing pR4-pRL-542 plasmid, step (II) obtained Enterobacteria and wild type cyanobacteria after purification are cultivated to mid log phase, and are that 5:5:1 is mixed according to cell quantity ratio, pass through Three parent's engagement transfers convert cyanobacteria with the carrier with vp19 gene, and screen cyanobacteria transformant；

Cyanobacteria transformant obtained by (IV) step (III) continues to cultivate, and obtains vp19 transgenic blue algae.

2. vp19 transgenic blue algae as described in claim 1, it is characterised in that: further include gained vp19 transgenic blue algae through heavy It forms sediment, centrifugation, collect, washing, the post-processing step for being freeze-dried and crushing.

3. vp19 transgenic blue algae as described in claim 1, it is characterised in that: the cyanobacteria is Synechococcus.

4. the pharmaceutical composition for preventing and treating shrimp white spot syndrome virus, it is characterised in that: described in any one of claims 1 to 3 Vp19 transgenic blue algae be effective active composition, effective dosage be 1~20 μ g/ tail, further include that fishery pharmaceutically may be used The carrier or auxiliary material of receiving.

5. pharmaceutical composition as claimed in claim 4, it is characterised in that: the effective of the vp19 transgenic blue algae uses agent Amount is 10~20 μ g/ tails.

6. pharmaceutical composition as claimed in claim 4, it is characterised in that: dosage form is pulvis, granule, capsule, tablet or ball Agent.

7. the described in any item vp19 transgenic blue algaes of claims 1 to 3 prevent and treat shrimp white spot syndrome virus drug in preparation In purposes.

8. purposes as claimed in claim 7, it is characterised in that: administration mode is oral.

9. purposes as claimed in claim 7, it is characterised in that: the drug is oral vaccine or immunopotentiating agent.