CN104043111B - The application of rainbow trout IFN-γ 2 in the anti-IHN viral product of preparation - Google Patents

Abstract

The invention discloses the application of rainbow trout IFN-γ2 in preparing anti-IHNV virus products. The invention provides the application of rtIFN-γ2 protein or its coding gene or the recombinant vector containing its coding gene in the preparation of products for inhibiting infectious hematopoietic necrosis virus; the amino acid sequence of the rtIFN-γ2 protein is the sequence in the sequence list 2. The present invention finds that rainbow trout IFN-γ2 can resist IHNV activity, and the present invention can supplement the current situation that there is no effective anti-IHNV preparation in my country. Meanwhile, gamma interferon also has immunoregulatory activity, and rtIFN-γ2 can be further developed as a natural anti-IHNV agent for rainbow trout. Immunopotentiators or adjuvants.

Description

Application of rainbow trout IFN-γ2 in the preparation of anti-IHN virus products

technical field

The invention relates to the field of biotechnology, in particular to the application of rainbow trout IFN-γ2 in the preparation of anti-IHN virus products.

Background technique

In recent years, the research enthusiasm of fish interferon has been increasing year by year, and the antiviral effect of interferon has always been the focus of this research. Earlier, in 2003, through the plaque reduction experiment, the artificially prepared Atlantic salmon type I interferon α1 subtype was confirmed to have the effect of resisting infectious pancreatic necrosis virus infection. With the deepening of research, zebrafish, rainbow trout, seven-banded grouper, and grass carp have been successively confirmed to have the ability to resist virus replication. Among them, it is worth noting that the prepared seven-banded grouper and grass carp injected with interferon can make them resistant to virus infection, which provides strong evidence for the effectiveness of artificially prepared fish interferon in clinical applications. In 2005, the rainbow trout interferon gamma was cloned for the first time, and it was confirmed that the active recombinant rainbow trout interferon gamma could be successfully prepared by using the prokaryotic expression system. In 2009, research showed that there was a second subtype of rainbow trout interferon gamma (rtIFN-γ2 ), and its expression level was significantly higher than that of rtIFN-γ1 in the early stage of vaccine immunity and virus infection. In 2011, researchers compared the antiviral effects of IFN-γ of rainbow trout and type I interferon α1 subclass of Atlantic salmon, The results show that IFN-γ can up-regulate the expression of Mx protein and ISG15 protein gene, and can well inhibit the plaque formation caused by infectious pancreatic necrosis virus. In order to effectively induce the production of antiviral protein GBP, interferon regulation transcription factor and chemokine IP-10. Therefore, the use of low-cost, easy-to-operate, and high-efficiency prokaryotic expression system to prepare active rainbow trout IFN-γ2 will have a good development prospect.

With the development of my country's fish farming industry, due to the increase of breeding density and the change of environment, the occurrence of fish diseases is more and more frequent. As a natural antiviral agent, fish interferon has pointed out a new application prospect for the prevention and cure of fish virus diseases. The artificial preparation of fish interferon in my country has gradually attracted attention, and there are 3 related invention patents, of which there is 1 invention patent for the preparation and purification of rainbow trout α-interferon (2012). rtIFN3) was artificially prepared. Infectious Hematopoietic Necrosis (IHN) often causes high mortality in fry or juveniles, and can be insidiously infected. It is one of the threats to the rainbow trout aquaculture industry in my country, but so far there is no literature that clearly states the infectious hematopoietic necrosis. Sensitivity of Infectious Hematopoietic Necrosis Virus (IHNV) to interferon in rainbow trout.

Contents of the invention

One object of the present invention is to provide the use of rtIFN-γ2 protein or its coding gene or the recombinant vector containing its coding gene.

The application of the rtIFN-γ2 protein or its encoding gene or the recombinant vector containing its encoding gene in the preparation of products for inhibiting infectious hematopoietic necrosis virus provided by the present invention;

The amino acid sequence of the rtIFN-γ2 protein is sequence 2 in the sequence list.

In the above application, the virus inhibition is the activity of virus inhibition; the rtIFN-γ2 protein is derived from rainbow trout.

In the above application, the nucleotide sequence of the gene encoding rtIFN-γ2 is sequence 1 in the sequence listing.

In the above application, the recombinant vector is a recombinant vector obtained by inserting the gene encoding rtIFN-γ2 into an expression vector, and the expression vector is specifically pET32a. In an embodiment of the present invention, the recombinant vector is the sequence 1 in the sequence listing The plasmid obtained by inserting the indicated IFN-γ2 gene between the BamHI and HindIII restriction sites of vector pET32a.

In the above application, the product is a kit or a vaccine.

Another object of the present invention is to provide a recombinant vector for suppressing infectious hematopoietic necrosis virus.

The recombinant vector provided by the present invention is a recombinant vector obtained by inserting the rtIFN-γ2 coding gene into an expression vector.

Among the above recombinant vectors, the expression vector is pET32a.

The third object of the present invention is to provide a recombinant bacterium that inhibits infectious hematopoietic necrosis virus.

The recombinant bacterium provided by the present invention is a recombinant bacterium obtained by introducing the above-mentioned recombinant vector into host cells.

In the aforementioned recombinant bacteria, the host cell is Escherichia coli.

The fourth object of the present invention is to provide a vaccine for infectious hematopoietic necrosis disease.

The infectious hematopoietic organ necrosis vaccine provided by the present invention has an active ingredient of rtIFN-γ2 protein or its coding gene or a recombinant vector containing the coding gene or the above-mentioned recombinant bacteria.

The experiment of the present invention proves that the present invention finds that rtIFN-γ2 can be highly expressed in Escherichia coli, and the inhibition rate of 0.051 μg/mL rtIFN-γ2 on CHSE-214 cells to IHNV is 50%, and can significantly inhibit IHNV in CHSE The formation of plaques on -214 cells, the final determination of its anti-IHNV activity is 6.63 × 10 ⁶ U/mg, the present invention can supplement the current situation that there is no effective anti-IHNV preparation in China, and interferon-γ also has immune regulation activity, which can rtIFN-γ2 was further developed as a natural immune enhancer or adjuvant for rainbow trout.

Description of drawings

Figure 1 shows the construction of gene cloning and recombinant expression plasmids

Figure 2 shows the recombinant protein expression and purification

Figure 3 shows the effect of protein rtIFN-γ2 on inhibiting IHNV

Figure 4 is the plaque reduction experiment

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1. Obtaining of rainbow trout IFN-γ2 gene and its recombinant vector for expression

1. Cloning of the target gene

The rainbow trout (Oncorhynchus mykiss, kept and bred in the laboratory, is a conventional rainbow trout species) was killed, the head kidney and spleen were aseptically stripped on ice quickly, and an appropriate amount of MEM medium containing 10% FBS was added, and the inner column of the syringe was rolled and passed through Filter through a 70 μm nylon mesh (product of Fisher Scientific), stimulate with 5 μg/mL phytohemagglutinin, and extract total RNA using Promega’s RNA extraction kit. According to the instructions of M-MLV reverse transcriptase, use primer Oligo(dT) 18 was reverse transcribed to obtain the first strand of cDNA.

Referring to the published rainbow trout IFN-γ2 sequence (Genbank: FM864345.1), design upstream and downstream primers for amplifying rtIFN-γ2, upstream primer: 5'-ATC GGATCC GCTCAGTACACATCAATTAAC-3' (underlined for BamHI digestion site), downstream primer: 5'-GA CAAGCTT TTACATGATGTGTGATTTGAG-3' (underlined HindIII restriction site).

Using the above cDNA as a template, PCR amplification was carried out with the above upstream and downstream primers.

The PCR reaction conditions were pre-denaturation at 94°C for 7 min, denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec. After a total of 25 cycles, a final extension at 72°C was performed for 10 min.

Electrophoresis detection of PCR products, the results are shown in Figure 1A, A clone of the target gene: M.DL2000DNAMarker; 1. RT-PCR amplification of head kidney cDNA; 2. RT-PCR of spleen cDNA; 3. Negative control , Figure 1A shows that the target gene PCR product can be successfully amplified by using the PHA-stimulated head kidney and spleen lymphocyte cDNA as a template, and the size is about 489bp.

The PCR product was recovered, connected to the pMD18Tsimple vector, transformed into Escherichia coli DH5α, and the plasmid obtained from the transformant was extracted and sent for sequencing. As a result, the PCR product had the nucleotide shown in Sequence 1 in the sequence table, which was the IFN-γ2 gene, and the protein encoded by it was rtIFN The amino acid sequence of -γ2 is sequence 2 in the sequence listing. The plasmid containing this PCR product was named pMD18Tsimple-IFN-γ2.

2. Construction of recombinant vector

Use restriction endonucleases BamHI and HindIII to cut pMD18Tsimple-IFN-γ2, recover the 489bp restriction enzyme fragment, and digest the restriction fragment with the vector pET32a ( 69015-3CN) connection, transformed Escherichia coli DH5α, and obtained transformants.

The plasmids of the transformants were extracted, and PCR identification (primers were upstream and downstream primers), BamHI single enzyme digestion identification and double enzyme digestion identification (BamHI and HindIII enzyme digestion) were carried out respectively.

The results are shown in Figure 1B, M1.DL2000DNAMarker; M2.DL15000DNAMarker; 1. PCR identification of recombinant plasmid; 2. Single cut identification of recombinant plasmid; All of them obtained a product of 489bp, indicating that it was a positive plasmid.

The positive was sent for sequencing. As a result, the positive plasmid was a plasmid obtained by inserting the IFN-γ2 gene shown in Sequence 1 in the sequence listing between the BamHI and HindIII restriction sites of the vector pET32a. It expressed protein rtIFN-γ2, named pET32a -rtIFN-γ2.

The transformant containing pET32a-rtIFN-γ2 was named DH5α/pET32a-rtIFN-γ2.

Embodiment 2, application of rainbow trout IFN-γ2 gene

1. Protein expression and expression pattern analysis

The recombinant strain DH5α/pET32a-rtIFN-γ2 was cultured in 5mL LB medium containing 100μg/mLAMP at 37°C overnight with shaking at 220rpm. The next day, the overnight culture was inoculated in 100mL LB medium at a ratio of 1:100, cultured with shaking at 220rpm until the OD value reached 0.6-0.8, and the expression was induced by adding 1mMIPTG. After 4h, the cells were collected by centrifugation at 4000rpm at 4°C.

The bacteria were resuspended in PBS with 1/10 of the initial medium volume, and divided into supernatant and precipitate after ultrasonic lysis and crushing. SDS-PAGE was used to analyze the existing form of the target protein.

The results are shown in Figure 2, M is the protein molecular mass standard; 1 is before the induction of the expression strain; 2 is after the induction of the expression strain; 3 is the ultrasonic supernatant after the induction of the expression strain; 4 is the ultrasonic precipitation after the induction of the expression strain; 5 is the purpose Purification of the protein, it can be seen that the results of SDS-PAGE showed that the induced recombinant bacteria had an obvious expression band at the expected position of 38.4KD, but no such band appeared before induction, indicating that the target protein can be expressed in the prokaryotic expression system Correctly expressed (swimming lane 2), the supernatant and precipitate were collected for SDS-PAGE analysis after lysing the bacteria by ultrasonic, which showed that the target protein was mainly expressed in the form of inclusion bodies (swimming lane 4).

2. Purification and renaturation of target protein

Purification: with an appropriate volume of refolding solution (2M urea, 0.4MTrisBase, 1mM 2-mercaptoethanol, 0.5mM oxidized glutathione, 0.5mM reduced glutathione, 10% glycerol, 10% acetone, pH8.0 ) fully wash the inclusion bodies twice. Add 3ml of denaturing solution (8M urea, 0.1M NaH ₂ PO ₄ , 0.01M Tris-cl, pH 8.0), and dissolve overnight at 4°C.

Refolding: Add the lysate dropwise to 27ml ice-bathed refolding solution, stir while adding, and let stand at 4°C overnight. The next day, centrifuge at 5000rpm at 4°C for 30min, transfer the supernatant to a dialysis bag, place it in a PBS solution containing 3M urea for 12h at 4°C, and then change the PBS solution twice (containing 1M and 0M urea). Stand at 4°C for 12h.

The electrophoresis results after renaturation are shown in lane 5 of Figure 2. After the inclusion bodies were washed and dissolved, the target protein rtIFN-γ2 with high purity was obtained.

The target protein was renatured by the dilution renaturation method, and only a small amount of precipitation was produced during the process. Finally, the concentration of the target protein after renaturation was measured by ultraviolet spectrophotometer to be about 0.207 mg/mL.

The plasmids of other irrelevant proteins also expressed by pET32a were introduced into E. coli DH5α to obtain control recombinant bacteria, and the above-mentioned expression and purification of the target protein was used to obtain a 40KD control protein.

3. Application of target protein rtIFN-γ2 in inhibiting IHNV activity

1) Antiviral experiment

Cultivate CHSE-214 cells (ATCCCRL-1681) in a 96-well cell culture plate to a monolayer, add 100 μl of 4-fold diluted purified rtIFN-γ2 protein to the 96-well plate, and store in 15°C CO ₂ Culture in the incubator for 12 hours, discard the culture medium and add 100 TCID ₅₀ IHNV (recorded in the following literature: Xu Liming, Liu Hongbai and Lu Tongyan, Infectious hematopoietic necrosis virus-Sn strain matrix protein gene cloning and bioinformatics analysis. Acta Aquatica Sinica, 2013(09): pp. 1409-1415. Xu Liming, Liu Hongbai, Xu Jin, Lu Tongyan, Establishment of a high-efficiency RT-PCR detection method for infectious hematopoietic necrosis virus. Acta Hydrobiology: Page 1; public available Obtained from the Heilongjiang Fisheries Research Institute of the Chinese Academy of Fishery Sciences), at the same time set up a control group without interferon, a control protein group, and a control group without virus, continue to culture and observe and record the pathological changes of the cells every day, and finally wash with PBS Add 0.1% crystal violet to each well to stain the cells for 10 min to assist in judging cell death. Referring to the virus TCID ₅₀ assay method, the lesion-inhibiting wells were replaced by the lesion wells in the toxicity assay, and the anti-IHNV activity of rtIFN-γ2 on CHSE-214 cells was calculated by the Reed-Meunch method.

The results of the anti-virus experiment are shown in Figure 3, A. the control group without interferon; B. the control protein group; C. the interferon treatment group, D. the virus-free control group. It shows that the cells in the control protein group grow well, the cells in the positive control group (Group A and Group B) are completely pathological, and the CHSE-214 cells treated with the target protein rtIFN-γ2 have no pathological changes; the IHNV inhibition rate of the control group without virus is 100% , the IHNV inhibition rate of the control group without interferon was 0%, and the IHNV inhibition rate of the control protein group was 0%. The calculated target protein rtIFN-γ2 inhibits IHNV activity on CHSE-214 cells at about 6.63×10 ⁶ U/mg.

The above antiviral experiments were carried out with different concentrations of rtIFN-γ2, and the results are shown in Table 1:

Table 1 shows the results of IHNV inhibition rate of different concentrations of rtIFN-γ2

rtIFN-γ2 (μg/mL) IHNV inhibition rate (100%) 20.70 100 5.18 93.75 1.29 87.5 0.32 81.25 0.081 70 0.020 62.5 0.0051 53.57142857 0.0013 46.875

2) Plaque reduction experiment

Cultivate CHSE-214 cells in a 6-well cell culture plate to a single layer, add cell culture solution containing 1:20 times diluted recombinant rtIFN-γ2 protein to each well, culture in a CO ₂ incubator at 15°C for 12 hours, discard After the culture solution, 10 ³ , 10 ⁴ , and 10 ⁵ -fold diluted IHNV were added respectively, and the culture was continued for 1 hour, and a control group without interferon was established at the same time. Discard the supernatant and add 1:1 mixed 2×MEM culture solution and 1.5% low-melting point agarose to each well, culture in a CO ₂ incubator at 15°C, and observe daily until obvious plaques can be observed under the microscope , add neutral red staining solution with a final concentration of 0.05%, incubate in the dark for 1 hour, and observe the formation of plaques.

The results of the plaque reduction experiment are shown in Figure 4, showing that when CHSE-214 cells were inoculated with 10 ³ -fold diluted IHNV, too many plaques in the control group could not be accurately counted, while the plaques in the recombinant rtIFN-γ2 treatment group were significantly reduced (specifically about 40), when 10 ⁴ -fold diluted IHNV was inoculated into CHSE-214 cells, plaques could be reduced to 1/10 (specifically 8) after rtIFN-γ2 treatment, and when 10 ⁵ -fold diluted IHNV was inoculated with CHSE-214 cells, rtIFN -γ2 treatment produced no plaques (specifically 0).

The above results confirm that the prepared target protein has better anti-IHNV activity.

Claims (10)

1.rtIFN-γ 2 albumen or its encoding gene or the recombinant vector containing its encoding gene suppress the application in infectious hematopoietic necrosis's viral product in preparation;

The aminoacid sequence of described rtIFN-γ 2 albumen is the sequence 2 in sequence table.

2. application according to claim 1, is characterized in that: the described virus that suppresses is for suppressing the activity of virus; Described rtIFN-γ 2 dietary protein origin is in rainbow trout.

3. application according to claim 1 and 2, is characterized in that: the nucleotides sequence of described rtIFN-γ 2 encoding gene is classified as sequence 1 in sequence table.

4. application according to claim 1 and 2, is characterized in that: described recombinant vector is the recombinant vector obtained by described rtIFN-γ 2 encoding gene insertion expression vector, and described expression vector is specially pET32a.

5. application according to claim 1 and 2, is characterized in that: described product is test kit or vaccine.

6. suppress a recombinant vector for infectious hematopoietic necrosis's virus, for rtIFN-γ 2 encoding gene being inserted the recombinant vector that expression vector obtains;

The nucleotides sequence of described rtIFN-γ 2 encoding gene is classified as sequence 1 in sequence table.

7. recombinant vector according to claim 6, is characterized in that: described expression vector is pET32a.

8. suppress a recombinant bacterium for infectious hematopoietic necrosis's virus, for the recombinant vector described in claim 6 or 7 being imported host cell, the recombinant bacterium obtained.

9. recombinant bacterium according to claim 8, is characterized in that: described host cell is escherichia coli.

10. infectious hematopoietic necrosis's vaccine, its active component is the recombinant bacterium shown in rtIFN-γ 2 albumen or its encoding gene or the recombinant vector containing its encoding gene or claim 8.