阅读说明：本技术 重组虹鳟i型干扰素、其原核表达和纯化方法及应用 (Recombinant rainbow trout type I interferon, prokaryotic expression and purification method and application thereof ) 是由 魏文燕 李良玉 汪开毓 刘家星 杨马 陈霞 杨倩 唐洪 刘韬 张小丽 陈健 杨 于 2019-10-08 设计创作，主要内容包括：本发明公开了一种重组虹鳟I型干扰素、其原核表达和纯化方法及应用,原核表达和纯化方法包括虹鳟I型干扰素基因的获取、虹鳟I型干扰素基因的筛选、原核表达载体的构建与筛选和重组蛋白的表达与纯化等几个步骤,制备得到的虹鳟I型干扰素具有良好的生物活性,安全、高效,能够用于水产养殖中预防病毒感染性疾病,具有良好的应用前景。(The invention discloses a recombinant rainbow trout type I interferon, a prokaryotic expression and purification method and application thereof, wherein the prokaryotic expression and purification method comprises the steps of obtaining rainbow trout type I interferon genes, screening the rainbow trout type I interferon genes, constructing and screening a prokaryotic expression vector, expressing and purifying recombinant protein and the like, and the prepared rainbow trout type I interferon has good biological activity, is safe and efficient, can be used for preventing virus infectious diseases in aquaculture and has good application prospect.)

1. A prokaryotic expression and purification method of recombinant rainbow trout type I interferon is characterized by comprising the following steps:

s1 acquisition of rainbow trout type I interferon gene: extracting total RNA from the kidney of the rainbow trout infected with the infectious hematopoietic necrosis virus, and synthesizing cDNA by adopting RT-PCR reaction; designing a specific primer for amplifying the rainbow trout type I interferon gene, carrying out PCR amplification on the rainbow trout type I interferon by using the synthesized cDNA as a template and the specific primer, and recovering and purifying an amplification product by using agarose gel to obtain a DNA fragment;

s2 screening of rainbow trout type I interferon gene: connecting the DNA fragment with a pMD19-T vector to obtain a first connecting product, transforming the first connecting product into a DH5 alpha competent cell, and selecting a white single colony from a transformed cell colony for primary PCR identification, wherein the primary PCR identification reaction system is the same as the PCR amplification in the step S1; extracting plasmids from strains identified as positive by primary PCR, and performing primary enzyme digestion reaction identification on the extracted plasmids, wherein the primary enzyme digestion reaction identification comprises identification of a single enzyme digestion system and identification of a double enzyme digestion system, the primary enzyme digestion reaction product is analyzed by agarose gel electrophoresis, the identification is successful when the identification result of the single enzyme digestion system is a 3100-3200 bp strip, the identification is successful when the identification result of the double enzyme digestion system is a 2650-2750 bp strip and a 400-500 bp strip, and sequencing analysis is performed on the plasmids with the successful identification results of the single enzyme digestion system and the double enzyme digestion system;

s3, constructing and screening a prokaryotic expression vector: carrying out enzyme digestion on the plasmid with correct sequencing identification, recovering an rainbow trout I-type interferon gene fragment, connecting the rainbow trout I-type interferon gene fragment with a pET-32a (+) vector to obtain a second connecting product, transforming the second connecting product into BL21 competent cells, and carrying out secondary PCR identification and secondary enzyme digestion reaction identification on the transformed cells; the secondary PCR identification comprises a first identification and a second identification, wherein the first identification reaction system is the same as the PCR amplification in the step S1, and primers in the second identification reaction system adopt T7 and T7-ter; the reaction system of the secondary enzyme digestion reaction identification is the same as the primary enzyme digestion reaction identification, the identification is successful when the identification result of the single enzyme digestion system is a 6300-6400 bp strip, and the identification is successful when the identification result of the double enzyme digestion system is a 5850-5950 bp strip and a 400-500 bp strip;

s4, expression and purification of recombinant protein: carrying out liquid culture on the strains of which the secondary PCR identification is positive and the secondary enzyme digestion reaction is successful, carrying out induction expression by IPTG, centrifugally collecting thalli, carrying out ultrasonic crushing after resuspending the thalli, centrifugally collecting supernatant and precipitate respectively after ultrasonic treatment, washing and dissolving the precipitate, and filtering the supernatant and the dissolved solution of the precipitate respectively; respectively purifying the protein of the supernatant and the dissolved solution of the precipitate by adopting a medium-high pressure chromatography system, and carrying out gradient dialysis renaturation on the purified precipitate at 4-6 ℃, wherein the dialysis gradient is 6mol/L urea solution, 4mol/L urea solution, 2mol/L urea solution, 1mol/L urea solution and PBS buffer solution; and (3) dialyzing and renaturing the purified supernatant at 4-6 ℃ by adopting a PBS buffer solution, and renaturing to obtain the recombinant protein of the rainbow trout type I interferon.

2. The prokaryotic expression and purification method of recombinant rainbow trout type I interferon according to claim 1, wherein the specific primers in step S1 comprise an upstream primer and a downstream primer, the sequence of the upstream primer is shown in SEQ ID No.1, and the sequence of the downstream primer is shown in SEQ ID No. 2.

3. The method for prokaryotic expression and purification of recombinant rainbow trout type I interferon according to claim 1, wherein in step S4, 2mol/L urea solution is used for washing the precipitate, and 8mol/L urea solution is used for dissolving the precipitate.

4. The prokaryotic expression and purification method of recombinant rainbow trout type I interferon according to claim 1, wherein in the step S4, IPTG concentration is 0.2-0.3 mmol/L and induction time is 4-4.5 h is used for induction expression.

5. The prokaryotic expression and purification method of recombinant rainbow trout type I interferon according to claim 1, wherein in the step S4, the power of the ultrasonication is 200W, the work time is 2-3S, the interval is 1S, each time lasts for 20-22 min, and the process is repeated for 2-4 times.

6. A recombinant rainbow trout type I interferon produced by the method of any one of claims 1 to 5.

7. The recombinant rainbow trout type I interferon of claim 6, for use in a medicament for preventing viral infectious diseases in aquaculture animals.

8. The use of claim 7, wherein the aquaculture animal is rainbow trout.

9. The use of claim 8, wherein the recombinant rainbow trout type I interferon is used at a concentration of 0.2-0.5 mg/ml.

Technical Field

The invention relates to the field of bioengineering genes, in particular to recombinant rainbow trout type I interferon, a prokaryotic expression and purification method and application thereof.

Background

At present, as global demand for aquatic products far exceeds sustainable fishing of marine/freshwater species, aquaculture must meet the increasing demand for aquatic product consumption by people. Aquaculture has become the fastest growing food production sector in the world, currently accounting for over 30% of the world's food fish, and this trend is projected to increase to 50% by the year 2030. According to statistics, the fishery industry provides 17% of animal protein for the world, and the export income created for developing countries exceeds the sum of the export income of meat, tobacco, rice and sugar. Indeed, research into the nutrition, reproduction and disease prevention of fish has led to a significant increase in annual aquaculture production. To maintain this growth rate, the aquaculture industry must reduce the significant impact of disease on fish farming and the resulting economic losses.

Fish pathogens, particularly RNA viruses, can have a devastating economic impact on the aquaculture industry every year. Infectious Hematopoietic Necrosis Virus (IHNV) is responsible for a large part of the world's salmon farming industry. Depending on the fish species, viral strain and environmental conditions, an outbreak of IHNV may result in an 80-100% loss in salmon farms. After the outbreak of the disease, the surviving fish may become a virus carrier without any clinical symptoms. Rainbow trout, a salmon belonging to the salmonidae pelagia genus of salmonidae order, is known as "ginseng in water", has the characteristics of much meat, stabbing softness and less fishy smell, and is a popular edible fish. IHNV is a fish disease which is mainly prevented and treated in rainbow trout cultivation, and because the control strategy of adopting antibiotics and chemotherapy is ineffective to viruses, the prevention of virus infectious diseases needs to be carried out by an anti-immune preparation, but the commercial development of the fish disease is limited by the requirements of low dosage, low cost, easy use and safety of the immune preparation brought by aquaculture.

Interferons (IFNs) are a class of glycoproteins with high species specificity that have antiviral, cytostatic, immunoregulatory, and antitumor effects. Interferons are classified into two types, I and ii, according to factors such as production cells, receptors and activities of interferons, I-type IFN has a broad spectrum of antiviral activity and acts at the early stage of viral infection, and I-type interferon is also known as a pleiotropic cytokine that links innate and acquired immunity by acting on highly specialized immune cells. For example, they initiate the maturation of dendritic cells and direct the differentiation of T helper cells to the Th1 phenotype. It has been reported that typical adjuvants such as Freund's adjuvant act by inducing endogenous type I IFN that mediates Th1 immune responses, suggesting that type I IFN itself is a powerful immunostimulant.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a prokaryotic expression and purification method of recombinant rainbow trout type I interferon, and the prepared rainbow trout type I interferon has good biological activity and can be used for preventing virus infectious diseases in aquaculture.

The purpose of the invention is realized by the following technical scheme:

a prokaryotic expression and purification method of recombinant rainbow trout type I interferon comprises the following steps:

s1, obtaining an I-type rainbow trout interferon gene: total RNA was extracted from the kidney of rainbow trout infected with infectious hematopoietic necrosis virus, and cDNA was synthesized by RT-PCR reaction. Designing a specific primer for amplifying the gene of the rainbow trout type I interferon, taking the synthesized cDNA as a template, carrying out PCR amplification on the rainbow trout type I interferon by adopting the specific primer, and recovering and purifying an amplification product by using agarose gel to obtain a DNA fragment. Further, the specific primers comprise an upstream primer and a downstream primer, wherein the sequence of the upstream primer is shown as SEQ ID No.1, and the sequence of the downstream primer is shown as SEQ ID No. 2.

S2, screening of rainbow trout type I interferon genes: and (3) connecting the DNA fragment with a pMD19-T vector to obtain a first connecting product, transforming the first connecting product into DH5 alpha competent cells, and selecting a white single colony from a transformed cell colony for primary PCR identification, wherein the primary PCR identification reaction system is the same as the PCR amplification in the step S1. Extracting plasmids from strains identified as positive by primary PCR, and performing primary enzyme digestion reaction identification on the extracted plasmids, wherein the primary enzyme digestion reaction identification comprises identification of a single enzyme digestion system and a double enzyme digestion system, the primary enzyme digestion reaction product is analyzed by agarose gel electrophoresis, the identification is successful when the identification result of the single enzyme digestion system is a 3100-3200 bp strip, the identification is successful when the identification result of the double enzyme digestion system is a 2650-2750 bp strip and a 400-500 bp strip, and sequencing analysis is performed on the plasmids with the successful identification results of the single enzyme digestion system and the double enzyme digestion system.

S3, constructing and screening a prokaryotic expression vector: carrying out enzyme digestion on the plasmid with correct sequencing identification, recovering the rainbow trout I-type interferon gene fragment, connecting the rainbow trout I-type interferon gene fragment with a pET-32a (+) vector to obtain a second connecting product, transforming the second connecting product into BL21 competent cells, and carrying out secondary PCR identification and secondary enzyme digestion reaction identification on the transformed cells. The secondary PCR identification comprises a first identification reaction system and a second identification reaction system, wherein the first identification reaction system is the same as the PCR amplification in the step S1, and primers in the second identification reaction system adopt T7 and T7-ter. The reaction system of the secondary enzyme digestion reaction identification is the same as the primary enzyme digestion reaction identification, the identification is successful when the identification result of the single enzyme digestion system is a band of 6300-6400 bp, and the identification is successful when the identification result of the double enzyme digestion system is a band of 5850-5950 bp and a band of 400-500 bp.

S4, expression and purification of recombinant protein: and (3) carrying out liquid culture on the strains which are positive in secondary PCR identification and successful in secondary enzyme digestion reaction identification, and carrying out IPTG induced expression, preferably, the IPTG concentration is 0.2-0.3 mmol/L and the induction time is 4-4.5 h. And centrifugally collecting the thalli, carrying out ultrasonic crushing after the thalli are resuspended, preferably, the power of ultrasonic crushing is 200W, the work lasts for 2-3 s at an interval of 1s, each time lasts for 20-22 min, and the operation is repeated for 2-4 times. And after ultrasonic treatment, respectively collecting supernatant and precipitate by centrifuging again, washing and dissolving the precipitate, and respectively filtering supernatant and dissolved precipitate. Preferably, the precipitate is washed with a 2mol/L urea solution, and the precipitate is dissolved with an 8mol/L urea solution. Respectively purifying the supernatant and the dissolved solution of the precipitate by using a medium-high pressure chromatography system, and carrying out gradient dialysis renaturation at 4-6 ℃ on the purified precipitate, wherein the dialysis gradient is 6mol/L urea solution, 4mol/L urea solution, 2mol/L urea solution, 1mol/L urea solution and PBS buffer solution; and (3) dialyzing and renaturing the purified supernatant at 4-6 ℃ by adopting a PBS buffer solution, and renaturing to obtain the recombinant protein of the rainbow trout type I interferon.

The invention also provides a recombinant rainbow trout type I interferon, which is prepared by the method.

The invention also provides application of the recombinant rainbow trout type I interferon in preparing a medicament for preventing virus infectious diseases of aquaculture animals. Preferably, the aquaculture animal is rainbow trout, and the application concentration of the recombinant rainbow trout type I interferon is 0.2-0.5 mg/ml.

The invention has the beneficial effects that:

1) the recombinant rainbow trout type I interferon can be prepared into a product with good biological activity and high protein product titer through prokaryotic expression. The invention obtains the gene fragment of the target gene by designing a specific primer, cultures and induces the expression of the selected expression vector after a plurality of times of screening and identification, and strictly controls the conditions of induction, extraction and renaturation to obtain the recombinant protein product which can be used for preventing virus infectious diseases in aquaculture.

2) The prokaryotic expression and purification method greatly controls the preparation cost of the recombinant rainbow trout type I interferon, effectively improves the antiviral bioactivity of the recombinant rainbow trout type I interferon, and provides the greatest possibility for the later-stage industrial production.

3) The recombinant rainbow trout type I interferon has the characteristics of good medicinal effect and high safety, effectively improves the acute mortality rate of infectious hematopoietic necrosis, is favorable for improving the current situation of the major threat of virus infectious diseases to aquaculture, and can be used for preventing virus infectious diseases in aquaculture, especially infectious hematopoietic necrosis.

Drawings

FIG. 1 is a diagram showing an electrophoretic analysis of PCR amplification products according to a first embodiment of the present invention;

FIG. 2 shows the result of the primary enzyme digestion reaction of the first embodiment of the present invention;

FIG. 3 shows the results of two types of secondary PCR assays according to the first embodiment of the present invention;

FIG. 4 shows the result of the secondary enzyme digestion reaction according to the first embodiment of the present invention;

FIG. 5 shows the result of verifying the expression product according to the first embodiment of the present invention;

FIG. 6 is a graph of the in vitro anti-IHNV protective effect of recombinant IFNa;

FIG. 7 is a graph showing the survival of recombinant IFNa.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of protection of the present invention.

The following experimental materials and all reagents used in the present invention can be purchased commercially, and the prokaryotic expression and purification method of recombinant rainbow trout type I interferon of the present invention will be described below with reference to the drawings and examples of the specification.

Strains and plasmids:

cloning vector19-T Simple Vector was purchased from Bao bioengineering (Dalian) Inc.; the prokaryotic expression vector pET-32a (+) was purchased from EnxWeijie Jie corporation (Invitrogen, CA, US). Coli (Escherichia coli) DH5 alpha and BL21(DE3) competent cells were purchased from Tiangen Biochemical technology, Inc. (Beijing).

The main reagents are as follows:

and (3) bacterial culture: tryptone (Tryptone), Yeast extract (Yeast extract) were purchased from OXOID, UK, and ampicillin (Amp, 100mg/ml) was purchased from Biotechnology (Shanghai) GmbH.

Cloning genes: total RNA extraction reagent RNAiso Plus, Prime Script^TMRT reagent Kit (Perfect Real Time) was purchased from Takara, Inc. (Beijing) of medical technology, Baozi, Inc.; 2 × TaqMaster Mix was purchased from Biotechnology Ltd of New Scout of Beijing; the agarose gel recovery kit is purchased from Tiangen Biotechnology (Beijing) Ltd; both primer synthesis and gene sequencing are available from Biotechnology engineering (Shanghai) Inc.; plasmid DNA miniprep kits were purchased from Biotechnology engineering (Shanghai) Inc.

Nucleic acid electrophoresis: biowest regular AGAROSE G-10 (Spanish AGAROSE) was purchased from Kyoho technologies, Inc.; nucleic acid dye Gold view was purchased from Beijing Solaibao science and technology, Inc.; DNA marker was purchased from Takara Bio Inc.; 6 × loading buffer is available from Tiangen Biochemical technology (Beijing) Ltd.

Protein expression: restriction enzymes EcoRI and XhoI were purchased from Thermo Fisher, Germany; the kit for quickly preparing the pre-dyed protein marker and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) denatured acrylamide gel is purchased from Biotechnology engineering (Shanghai) GmbH; IPTG (Isopropyl β -D-Thiogalactopyranoside) was obtained from Beijing Sorbo technology Ltd; protein Loading Buffer (4X) was purchased from Boston Biotech Limited.

Protein purification: Bio-Scale Mini Nuvia IMAC Ni-Charged (1X 5ml) was purchased from Bio-Rad, USA; imidazole (Imidazole) is commercially available from bio-engineering (shanghai) gmbh.

Cell culture penicillin-streptomycin solution (100 ×) and 0.25% trypsin were purchased from Sigma company, usa; MEM nutrient solution was purchased from Hyclone, USA; fetal Bovine Serum (FBS) was purchased from Zeta Life corporation; PBS buffer was purchased from Biotechnology engineering (Shanghai) Inc.

The main apparatus is as follows:

5424R type high speed refrigerated centrifuge (Eppendorf, Germany);

905SS-ULTS type ultra-low temperature refrigerator (Thermo company, Germany);

Milli-Q Advantage A10 ultra-pure water meter (Milli-Q, USA);

c1000 Thermal Cycler PCR amplification instrument (Bio-Rad, USA);

agarose nucleic acid electrophoresis apparatus & horizontal electrophoresis tank (Bio-Rad, USA);

Mini-Protean cell type gel imaging System (Bio-Rad, USA);

power Pac 100 protein electrophoresis apparatus (U.S. Bio-Rad corporation);

medium and high pressure chromatography systems (Bio-Rad, USA);

TS100 inverted biomicroscope (Nikon corporation);

micro-loading guns (Eppendorf, Germany);

JY92-IIN ultrasonic crusher (Ningbo Xinzhi Biotech GmbH);

an AL204 type electronic balance (shanghai precision instruments ltd);

nucleic acid protein instrument (Bio-Rad company)

The main reagent formula is as follows:

LB liquid medium: 5g of yeast extract, 10g of tryptone and 10g of NaCl, adding ddH2O to dissolve and fix the volume to 1000mL, adjusting the pH value to 7.0-7.3, and sterilizing at 121 ℃ for 30 min.

LB solid medium (Amp-containing): adding 2.5% agar powder into LB liquid culture medium, sterilizing at 121 deg.C for 30min, cooling to about 50 deg.C at room temperature, adding Amp (100mg/mL) with final concentration of 100 μ g/mL, and pouring into plate.

10 × TBE electrophoresis buffer (stock solution): adding 108g of Tris, 7.44g of Na2.EDTA.2H2O and 55g of boric acid, adding deionized water to fully dissolve, adjusting the pH value to 8.3 and metering to 1000 mL.

1.0% agarose gel: 1.0g agarose, adding 90mL deionized water and 10mL 10 × TBE, microwave oven heating to melt, and making gel.

Binding Buffer(1L)PH 8.0：500mM NaH₂PO₄.2H₂O; 300mM NaCl; 5mM imidazole, 8M urea was added for purification of the precipitate.

Elution Buffer(1L)PH 8.0：500mM NaH₂PO₄.2H₂O; 300mM NaCl; 400mM imidazole, 8M urea was added for purification of the precipitate.

The invention provides a technical scheme that: a prokaryotic expression and purification method of recombinant rainbow trout type I interferon comprises the following steps:

s1, obtaining an I-type rainbow trout interferon gene: total RNA was extracted from the kidney of rainbow trout infected with infectious hematopoietic necrosis virus, and cDNA was synthesized by RT-PCR reaction. Designing a specific primer for amplifying the gene of the rainbow trout type I interferon, taking the synthesized cDNA as a template, carrying out PCR amplification on the rainbow trout type I interferon by adopting the specific primer, and recovering and purifying an amplification product by using agarose gel to obtain a DNA fragment.

The specific primers comprise an upstream primer and a downstream primer, wherein the sequence of the upstream primer is shown as SEQ ID No.1, and the sequence of the downstream primer is shown as SEQ ID No. 2.

The total RNA is extracted from the kidney of the rainbow trout infected with the infectious hematopoietic necrosis virus by the following specific operations: 20-30 mg of IHNV infected rainbow trout kidney is extracted, immediately ground into powder under the condition of 40-80 ml of liquid nitrogen, and poured into 0.5-1 ml of RNAioso Plus. And then centrifuging the RNAioso Plus dissolved with the rainbow trout kidney tissues for 5-6 min at 4-6 ℃ at 12000r/min, transferring the supernatant into a first RNAase-free EP tube, adding 1/5 volume of chloroform into the supernatant, manually shaking up and down for 45-50 s, uniformly mixing, and standing at room temperature for 5-7 min. Centrifuging at 12000r/min at 4-6 ℃ for 15-16 min, sucking 0.4-0.5 ml of supernatant liquid into a second RNAase-free EP tube, adding isopropanol with the same volume to obtain a mixed liquid, reversing and uniformly mixing, and standing at room temperature for 10 min. Centrifuging the mixed solution added with the isopropanol at 12000r/min for 10-15 min at 4-6 ℃, removing supernatant, retaining precipitate, and adding 1-1.2 ml of pre-cooled 75% ethanol. Centrifuging at 12000r/min for 5min at 4-6 ℃, pouring out the ethanol solution in the step S130, and standing at room temperature for drying. Finally, 30ul ddH was added to the EP tube₂O (RNAase-free) sufficiently dissolves RNA, and the RNA is stored at-80 ℃ for further use.

The reverse transcription reaction system for synthesizing cDNA by adopting RT-PCR reaction is：5x Primer Scrip Buffer 2μl，Primer Scrip RT Enzyme mix 0.5μl，Oligo dT Primer 0.5μl，Random 6mers 2μl， RNAfree ddH₂O2. mu.l, RNA 3. mu.l; the parameters for maintaining the reverse transcription reaction were: 15min at 37 ℃; the reaction parameters for inactivation of the anti-transcriptase are: 5s at 85 ℃; storing at-20 to-22 ℃ for later use.

The reaction system for carrying out PCR amplification on the rainbow trout type I interferon is as follows: 10pmol/ul of the upstream primer 1.0. mu.l, 10pmol/ul of the downstream primer 1.0. mu.l, template 2. mu.l, 2 XTAQA Master Mix 10. mu.l, H₂O6 mu l; the reaction conditions are as follows: at 94 ℃, 5min, and the cycle number of 1 time; the cycle times (94 ℃, 30 s; 57 ℃, 30 s; 72 ℃, 45-60 s) are 35 times; at 72 deg.C for 10min, and the cycle number is 1.

S2, screening of rainbow trout type I interferon genes: connecting the DNA fragment with a pMD19-T vector to obtain a first connecting product, transforming the first connecting product into DH5 alpha competent cells, selecting a white single colony from transformed cell colonies for primary PCR identification, and adopting a primary PCR identification reaction system to be the same as the PCR amplification reaction system. Extracting plasmids from strains identified as positive by primary PCR, and performing primary enzyme digestion reaction identification on the extracted plasmids, wherein the primary enzyme digestion reaction identification comprises identification of a single enzyme digestion system and a double enzyme digestion system, the primary enzyme digestion reaction product is analyzed by agarose gel electrophoresis, identification is successful when the identification result of the single enzyme digestion system is a 3100-3200 bp strip, identification is successful when the identification result of the double enzyme digestion system is a 2650-2750 bp strip and a 400-500 bp strip, and sequencing analysis is performed on the plasmids with the successful identification results of the single enzyme digestion system and the double enzyme digestion system.

Wherein, the connection reaction system for connecting the DNA fragment and the pMD19-T vector is as follows: 4-5 ul of DNA fragments are obtained,1-2 ul of 19-T Simple Vector and 5-6 ul of Solution I, and reacting for 12-16 h at 15.5-16.5 ℃.

The specific process for transformation of the first ligation product into DH5 α competent cells was as follows: adding 10-13 ul of the first connection product into 50-60 ul of Escherichia Coli (Escherichia Coli) DH5 alpha competent cells, uniformly blowing and stirring the cells, incubating the cells on ice for 30-32 min on the premise of keeping the integrity of the competent cells, heating the cells for 90-95 sec by a 42 ℃ constant-temperature incubator, standing the cells on ice for 5-6 min, adding 0.9-1.2 ml of LB liquid culture medium, culturing the cells for 2-2.5 h at 36-37 ℃ under the condition of oscillation frequency of 150-160 r/min, uniformly spreading 200-250 ul of bacterial liquid on an Amp-LB agar plate, and culturing the cells for 12-12.5 h at 36-37 ℃ to obtain bacterial colonies.

The specific procedure for extracting plasmids from strains identified as positive by primary PCR was as follows: inoculating the colony identified as positive by PCR into 10-15 mL of Amp-LB liquid culture medium, and culturing for 16-18 h at 36-37 ℃ under the condition of oscillation frequency of 150-160 r/min; taking 4mL of bacterial liquid, centrifuging at 12000 Xg for 2-3 min at normal temperature, discarding the supernatant, and extracting pMD19-T-IFNa plasmid according to the steps in the manual of the small drawing reagent kit of the biological plasmid.

In the primary enzyme digestion reaction, the reaction system of the single enzyme digestion system is specifically as follows: EcoRI 0.5. mu.l, pMD19-T-IFNa 5. mu.l, 10 XGreen Buffer 1.0. mu.l, ddH₂O3.5 μ l, the reaction system of the double enzyme digestion system is specifically as follows: EcoRI 0.5. mu.l, XhoI 0.5. mu.l, pMD19-T-IFNa 5. mu.l, 10 XGreen Buffer 1. mu.l, ddH₂O3. mu.l. Preparing reaction liquid of a single enzyme digestion system and reaction liquid of a double enzyme digestion system, and respectively incubating for 1-1.5 h at 37 ℃, wherein the reaction conditions of the single enzyme digestion system and the double enzyme digestion system are as follows: and (3) standing and reacting at 37-37.5 ℃ for 60-62 min. And analyzing the primary enzyme digestion reaction product by adopting 1-1.5% agarose gel electrophoresis.

S3, constructing and screening a prokaryotic expression vector: carrying out enzyme digestion on the plasmid with correct sequencing identification, recovering the rainbow trout I-type interferon gene fragment, connecting the rainbow trout I-type interferon gene fragment with a pET-32a (+) vector to obtain a second connecting product, transforming the second connecting product into BL21 competent cells, and carrying out secondary PCR identification and secondary enzyme digestion reaction identification on the transformed cells. The secondary PCR identification comprises first identification and second identification, wherein the first identification reaction system is the same as the PCR amplification in the step S1, and primers in the second identification reaction system adopt T7 and T7-ter. The reaction system of the secondary enzyme digestion reaction identification is the same as the primary enzyme digestion reaction identification, the identification is successful when the identification result of the single enzyme digestion system is a 6300-6400 bp strip, and the identification is successful when the identification result of the double enzyme digestion system is a 5850-5950 bp strip and a 400-500 bp strip.

Wherein, the analysis of the sequencing identification result is obtained by carrying out multiple sequence alignment with rainbow trout IFNa1 protein (accession number CAM28541.1) and IFNa3 protein (accession number AAV39394.1) in NCBI, and the homology of the comparison result is more than 90 percent, which indicates that the clone is correct.

The specific operation of the recovered rainbow trout type I interferon gene segment is as follows: carrying out double enzyme digestion on the plasmid pMD19-T-IFNa with correct sequencing, wherein the double enzyme digestion reaction system is as follows: EcoRI 5. mu.l, XhoI 5. mu.l, pMD19-T-IFNa 25. mu.l, 10 XGreen Buffer 5.0. mu.l, ddH₂O10. mu.l. The reaction conditions are the same as the double enzyme digestion system in the step S2, and after the reaction, 1-1.5% agarose gel electrophoresis is adopted to recover the rainbow trout type I interferon gene fragment. Before connecting the rainbow trout type I interferon gene fragment with a pET-32a (+) vector, carrying out double enzyme digestion on the pET-32a (+) under the same reaction conditions, wherein the reaction system is as follows: EcoRI 5. mu.l, XhoI 5. mu.l, pET-32a (+) 25. mu.l, 10 XGreen Buffer 5.0. mu.l, ddH₂Mu.l of O, and recovering the linear fragment of pET-32a (+) after the reaction.

The reaction system for connecting the rainbow trout type I interferon gene fragment and the pET-32a (+) vector is as follows: 3-3.5 uL of the recovered rainbow trout type I interferon gene fragment, 2-2.5 uL of pET-32a (+) linear fragment, 5-6 uL of Solution I, and reacting for 16-17 h at 15.5-16.5 ℃.

The specific method for transforming the second ligation product into BL21 competent cells was the same as the method for transforming the first ligation product into DH5 alpha competent cells. In this step, BL21(DE3) strain into which pET-32a (+) empty plasmid was transferred was also set as a control, and the transformation step of pET-32a (+) empty plasmid was identical to that of pET-32a (+) -IFNa.

In the secondary PCR identification, primers T7(TAATACGACTCACTATAGGG) and T7-ter (TGCTAGTTATTGCTCAGCGGG) are adopted, and the reaction conditions are as follows: sequentially reacting for one cycle at 94 ℃ for 5 min; reacting for 35-37 cycles (94 ℃, 30 s; 55 ℃, 30 s; 72 ℃, 1min), and finally reacting for one cycle at 72 ℃ for 10 min. Wherein, a cell with a band of 650-750 bp appears after identification, and is a no-load control strain successfully transferred into pET-32a (+) empty plasmid. The identified cell with a band of 1150-1250 bp is pET-32a (+) -IFNa positive strain. And (4) performing glycerol conservation on the strains which are positive in secondary PCR identification and successful in secondary enzyme digestion reaction, and storing at the temperature of-20 to-22 ℃ for later use.

S4, expression and purification of recombinant protein: and (3) carrying out liquid culture on the strains which are positive in secondary PCR identification and successful in secondary enzyme digestion reaction identification, and carrying out IPTG induced expression, wherein the IPTG concentration is 0.2-0.3 mmol/L and the induction time is 4-4.5 h. And centrifugally collecting thalli, carrying out ultrasonic crushing after resuspending the thalli, wherein the power of ultrasonic crushing is 200W, the work lasts for 2-3 s, the interval is 1s, each time lasts for 20-22 min, and the process is repeated for 2-4 times. And centrifuging again after ultrasonic treatment to collect supernatant and precipitate respectively, washing the precipitate, dissolving the precipitate, washing the precipitate by using 2mol/L urea solution, dissolving the precipitate by using 8mol/L urea solution, filtering the supernatant and the dissolved solution of the precipitate by using filter membranes with the pore diameter of 0.45 mu m respectively, and filtering. Respectively purifying the protein of the supernatant and the dissolved solution of the precipitate by adopting a medium-high pressure chromatography system, and carrying out gradient dialysis renaturation at 4-6 ℃ on the purified precipitate, wherein the dialysis gradient is 6mol/L urea solution, 4mol/L urea solution, 2mol/L urea solution, 1mol/L urea solution and PBS buffer solution; and (3) dialyzing and renaturing the purified supernatant at 4-6 ℃ by adopting a PBS buffer solution, wherein the renaturation time of the purified precipitate and the supernatant is 6-8 h, and obtaining the recombinant protein of the rainbow trout type I interferon after renaturation.

The liquid culture of the strains which are positive in secondary PCR identification and successful in secondary enzyme digestion reaction specifically comprises the following steps: the strain is inoculated into 10-15 ml of Amp-LB liquid culture medium, and shake culture is carried out at 150-160 r/min and 37 ℃ until OD value is 0.6-0.65. After induction of expression, BL21(DE3) strain transformed with empty pET-32a plasmid was used as a control to eliminate background expression.

After the primary expression is completed, the expression product is verified: taking 1-1.5 ml of bacterial liquid, centrifuging at 8000 Xg for 2-3 min, removing supernatant, collecting the bacterial, re-suspending the bacterial by using 40-50 ul of PBS, adding 10-12.5 ul of 5 Xloading buffer, uniformly mixing, heating at 100-106 ℃ for 10-12 min, centrifuging for 30-40 sec, taking 10ul of sample, carrying out SDS-PAGE analysis, taking out gel after electrophoresis, putting the gel into Coomassie brilliant blue staining solution, staining for 3-3.5 h, finally putting the gel into clear water, boiling for 15-20 min, photographing and observing, comparing with a control strain, wherein the PAGE result of a target strain has one more strip than that of the control strain, and when the position size is basically consistent with the expected size of the target protein, the successful expression of the target protein is shown.

After the recombinant rainbow trout type I interferon is renatured, the recombinant rainbow trout type I interferon is checked by 10% SDS-PAGE, the concentration of the recombinant rainbow trout type I interferon is detected by a nucleic acid protein analyzer, and the recombinant rainbow trout type I interferon is stored at the temperature of minus 80 ℃ for later use.