CN106892976A - A kind of recombination chicken interferon lambda（rChIFN‑λ）Clonal expression of gene and its preparation method and application - Google Patents

Abstract

The invention belongs to the interferon field in technical field of bioengineering, and in particular to a kind of clonal expression of recombination chicken interferon lambda (rChIFN λ) gene and its preparation method and application.By recombination chicken interferon lambda (rChIFN λ) gene from POLY (I:C) extracted in the template obtained by the CEF cells for stimulating, be cloned into pET32a expression vectors, obtain recombinant plasmid pET32a rChIFN λ；By pET32a rChIFN λ recombinant plasmid transformeds to BL21 competence, Amplification Culture, after inclusion body modification, purifying and renaturation, recombination chicken interferon lambda (rChIFN λ) albumen needed for being obtained.

Description

Cloning and expression of a recombinant chicken interferon λ (rChIFN-λ) gene and its preparation method law and application

technical field

The invention belongs to the field of interferon in the technical field of bioengineering, and in particular relates to the cloning and expression of a recombinant chicken interferon λ (rChIFN-λ) gene and its preparation method and application.

Background technique

IFN-λ is a new type of cytokine discovered in recent years, named as type III interferon, and its family members include IFN-λ1, IFN-λ2, IFN-λ3 and IFN-λ4, but chicken IFN-λ has only one ChIFN -λ, the DNA fragment is 561bp, and the protein size is about 22kDa. Chicken IFN-λ has a pair of specific functional receptor complexes (IFN-λR1/IL-10R2), when chicken IFN-λ binds to the receptor complex, it leads to receptor heterodimerization, which can activate the downstream JAK -STAT signal transduction, thereby exerting a wide range of biological effects, including antiviral effects, immune regulation and antitumor, etc. Studies have shown that IFN-λ exerts a good antiviral effect on the following viruses, such as influenza virus, herpes simplex virus, hepatitis virus and so on.

Newcastle disease is an acute and highly contagious infectious disease of poultry caused by Newcastle Disease (ND) of the Avian Paramyxoviridae family, mainly affecting chickens and turkeys. The disease mainly damages the digestive tract, gastrointestinal tract and nervous system. Newcastle disease not only seriously harms the poultry industry, but also causes huge losses to the world economy. It is listed as a legally notifiable disease by the World Organization for Animal Health and a Class I animal disease by China.

Contents of the invention

The purpose of the present invention is to invent the cloning and expression of a recombinant chicken interferon λ (rChIFN-λ) gene with high antiviral activity and its preparation method and application. The chicken interferon λ of the invention is easy to produce, low in cost, high in activity, and has good therapeutic effects on Newcastle disease and influenza.

The first object of the present invention is to provide the above-mentioned recombinant chicken interferon lambda (rChIFN-λ) protein, the nucleotide sequence of which is shown in SEQ ID NO:1.

Another object of the present invention is to provide the production method of the protein and the preparation method of the prokaryotic expression plasmid pET32a-rChIFNλ.

Another object of the present invention is to provide the application and effect of the protein in inhibiting virus reproduction on cells and clinically treating animal viral diseases.

To achieve the above object, the present invention adopts the following technical solutions:

Step 1: Primer design and synthesis. According to the chicken IFN-λ gene sequence (GenBank no.KF680102.1) provided by Genbank, design a pair of primers IFN-λ-F1, IFN-λ-R1, predict and remove the signal peptide through SignalP, design primers, respectively upstream and downstream Insert two restriction sites of EcoR1 and Hind3, which are IFN-λ-F and IFN-λ-R. The sequence is as follows:

IFN-λ-F1: ATGGTATGCTACGGGGTCAC

IFN-λ-R1: CTAAGTGCAATCCTCGCGCTG

IFN-λ-F: CCGGAATTCCAGGTCACCCCCGAAGAA

IFN-λ-R: CCCAAGCTTCTAAGTGCAATCCTCGCGCTGGGC

Step 2: Gene cloning and identification. Self-made chicken embryo fibroblast (CEF) cells with SPF chicken embryos were stimulated with POLY(I:C) for 2 hours, RNA was extracted, and the template cDNA for amplifying the chicken IFN-λ gene was obtained by inversion, using IFN-λ-F , IFN-λ-R primer amplified the gene fragment of chicken IFN-λ without signal peptide.

Step 3: Cloning the gene into pMD-19T vector. Connect the chicken IFN-λ gene to the pMD-19T vector, connect at 16°C for more than 4 hours, transform it into an ampicillin-containing agar plate, pick a single clone and put it in LB medium to shake the bacteria, the positive clone is identified by bacterial liquid PCR, and the sequence is correct After expanded culture, the plasmid pMD-19T-rChIFN-λ was extracted.

Step 4: Construction of pET32a-rChIFN-λ recombinant plasmid. Respectively cut pMD-19T-rChIFN-λ and pET32a empty vectors with EcoR1 and Hind3 fast-cutting enzymes. After the gel was recovered, they were connected with T4 ligase at room temperature for more than 30 minutes, then transformed into ampicillin-containing agar plates, and single clones were picked and placed on the plate. Shake the bacteria in LB medium. The positive clones were identified by PCR of the bacteria liquid. After the sequencing was correct, the culture was expanded and the recombinant plasmid pET32a-rChIFN-λ was extracted.

The beneficial effects of the present invention are as follows:

The invention can extract protein with higher concentration and activity, which lays a good foundation for subsequent experiments, and has good antiviral activity at the cell level, and has a significant curative effect on Newcastle disease in animals.

Description of drawings

Fig. 1 is the chicken IFN-λ gene agarose nucleic acid electrophoresis image amplified by PCR; wherein, swimming lane M is DNA marker DL2000, swimming lane 1 is a negative control, and swimming lane 2 is the amplification result of chIFN-λ gene.

Figure 2 is the result of SDS-PAGE identification of recombinant chicken interferon lambda (rChIFN-λ) protein expression; wherein, lane M is the low molecular weight protein Marker, lane 1 is pET-32a empty vector inclusion body before denaturation, and lane 2 is pET- 32a After renaturation of inclusion bodies with empty vector, lane 3 is before denaturation of inclusion bodies of recombinant chicken interferon λ (rChIFN-λ), and lane 4 is after renaturation of inclusion bodies of recombinant chicken interferon λ (rChIFN-λ).

Figure 3 is a Western blot identification of recombinant chicken interferon λ (rChIFN-λ) protein expression results; wherein, lane M is a low molecular weight protein Marker, lane 1 is the pET-32a empty vector supernatant control, and lane 2 is pET-32a empty Vector inclusion body control, lane 3 is the supernatant of recombinant chicken interferon λ (rChIFN-λ), and lane 4 is the precipitation of inclusion body of recombinant chicken interferon λ (rChIFN-λ).

detailed description

Below in conjunction with specific embodiment, further illustrate the present invention. But the present invention is not limited thereto.

The construction of embodiment 1 chicken IFN-λ gene prokaryotic expression vector

(1) Design and synthesis of related primers

Primer design is based on the gene sequence of chicken IFN-λ provided by Genbank (GenBank no.KF680102.1), a pair of primers IFN-λ-F1 and IFN-λ-R1 are designed, the signal peptide is predicted and removed by SignalP, and the primers are designed. Two restriction sites, EcoR1 and Hind3, are inserted in the upstream and downstream respectively, which are IFN-λ-F and IFN-λ-R. The sequence is as follows:

IFN-λ-F1: ATGGTATGCTACGGGGTCAC

IFN-λ-R1: CTAAGTGCAATCCTCGCGCTG

IFN-λ-F: CCGGAATTCCAGGTCACCCCCGAAGAA

IFN-λ-R: CCCAAGCTTCTAAGTGCAATCCTCGCGCTGGGC

(2) Gene cloning and identification

Take 10-day-old SPF chicken embryos, use tweezers to remove the head, limbs and viscera, shred the chicken, trypsinize it for 4 minutes, filter it, and make CEF cells. Discard the supernatant, take the cell sample to extract RNA, obtain the template cDNA for amplifying the chicken IFN-λ gene by inversion, first use IFN-λ-F1, IFN-λ-R1 to amplify the chicken IFN-λ gene, and then use IFN-λ-F and IFN-λ-R primers were used to amplify the chicken IFN-λ gene without the signal peptide fragment. The reaction system is as follows: 25 μl Premix ExTaq enzyme, 2.5 μl template DNA (cDNA), 0.5 μl upstream primer, 0.5 μl downstream primer, and make up to 50 μl with distilled water. The reaction program was as follows: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 1 min, annealing at 55°C for 30 s, extension at 72°C for 40 s, and extension at 72°C for 7 min, including 35 cycles from steps 2 to 4. The size of the nucleic acid electrophoresis band is about 492bp.

(3) Cloning the gene into the pMD-19T vector

The rChIFN-λ gene was connected to the pMD-19T vector, and the reaction system was as follows: 5.0 μl Ligation Solution Ⅰ, 0.5 μl pMD 19-T vector, 4.5 μl chIFN-λ+αPCR recovery product. Connect at 16°C for more than 4 hours, use DH5α to transform it into an ampicillin-containing agar plate, place it overnight at 37°C, pick a single clone and put it in the ampicillin-containing LB medium for 6-8 hours. The system is as follows: 7.5 μl Premix rTaq enzyme, 6.1 μl ddH ₂ O, 0.2 μl upstream primer IFN-λ-F, 0.2 μl downstream primer IFN-α-R, 1.0 μl bacterial solution, the reaction procedure is as follows: pre-denaturation at 94°C for 4 minutes, Denaturation at 94°C for 1 min, annealing at 55°C for 30 s, extension at 72°C for 40 s, and extension at 72°C for 7 min, including 30 cycles from the 2nd to the 4th steps. The positive samples were selected and sent for sequencing. After the sequencing was correct, they were expanded and cultivated, and the plasmid pMD-19T-rChIFN-λ was extracted.

(4) Construction of pET32a-rChIFN-λ recombinant plasmid

Respectively digest pMD-19T-rChIFN-λ and pET32a empty vectors with EcoR1 and Hind3 enzymes, and the reaction system is as follows: 1.5 μl EcoR1 enzyme, 1.5 μl Hind3 enzyme, 5 μl 10×Buffer, 2-5 μg plasmid, ddH ₂ Make up to 50 μl with O, and bathe in water at 37°C for 30 minutes. After gel recovery, use T4 ligase to digest the product according to the ratio of pMD-19T-rChIFN-λ: pET32a empty vector = 3:1, add 8 μl, 1 μl T4 ligase, 1 μl T4 ligation buffer, connect at room temperature for more than 30 minutes, and transform into On the ampicillin agar plate, pick the monoclonal bacteria and put them into the ampicillin-containing LB medium to shake the bacteria. The positive clones were identified by the PCR of the bacteria liquid, and sent for sequencing. After the sequencing was correct, they were expanded and cultured, and the recombinant plasmid pET32a-rChIFN-λ+α was extracted.

Example 2 protein expression and identification

The recombinant plasmid pET32a-rChIFN-λ was transformed with BL21 competently. After the monoclonal bacteria were expanded and cultivated, they were inoculated with ampicillin LB medium at a ratio of 1:100 and cultured on a shaker at 37°C until the OD600nm value was about 0.6. Then, IPTG was added to induce expression Afterwards, centrifuge the bacterial solution, discard the supernatant, resuspend in PBS, and wash by centrifugation for 2 times. Lysis with an ultrasonic breaker at 200W for 15 minutes, centrifuged at 4°C to separate the precipitate, washed 2 times with pre-cooled PBS, and washed with Lysis Equilibration Buffer containing 8M urea. Dissolve the inclusion bodies, incubate at room temperature for 60 min, centrifuge at 4°C to remove insoluble impurities, filter the supernatant with a 0.45 μm filter, combine it with a Ni column, and elute with different concentrations of imidazole eluent until the OD280 value is 0, and finally use 250mmol/ Elution Buffer of L imidazole was eluted, and the filtrate was collected. Put the filtrate into a dialysis bag treated with EDTA, carry out gradient dialysis with 8M, 6M, 4M, 2M, 0M, and PBS respectively, with an interval of 12 hours each time, and finally identify it by SDS-PAGE.

Example 3 In vitro antiviral activity identification

Plate DF-1 cells on a 96-well cell plate, wait until they grow to 80%-90%, add 100 μl of rChIFN-λ to each well in a 4-fold gradient dilution, from 4 ^-1 -4 ^-10, respectively, for each gradient 24 replicates, incubated at 37°C for 12h, respectively added 100TCID50VSV, NDV, AIV virus, 100μl per well, incubated at 37°C for 1h, discarded the virus liquid, washed twice with PBS, replaced with 2% serum DMEM, put At 37°C for 48h, the virus content was detected. The results showed that rChIFN-λ had a good protective ability on DF-1 cells, and had good VSV and AIV antiviral activities on DF-1 cells in vitro, which were 1.87*10 ³ IU/mg and 7.8*10 ³ IU/mg respectively. mg.

Example 4 In vivo antiviral activity identification

The antiviral activity of rChIFN-λ was detected in 2-week-old SPF chickens. Give 2-week-old SPF chickens intravenous injection of 100 μgrChIFN-λ, 4 hours later, inject the same amount intravenously again, and then inject Newcastle disease virus after an interval of 2 hours. After 1, 3, 5, 7, 9, and 11 days, throat swabs and cloacal swabs were collected, and ordinary chicken embryos were inoculated to detect detoxification; the chickens were observed daily for food intake and mental status; and death was recorded. The results showed that the protection rate of the rChIFN-λ group to SPF chickens could reach 33%, while the survival rate of the challenged control group was only 8%. Compared with the rChIFN-λ group, detoxification can be delayed for 4-9 days, and the amount of detoxification is significantly reduced; the SPF chickens in the rChIFN-λ group eat normally and are in good spirits.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

SEQUENCE LISTING

South China Agricultural University

Cloning and expression of a recombinant chicken interferon lambda (rChIFN-λ) gene and its preparation method and application

(1) Chicken IFN-λ gene

ATGGTATGCT ACGGGGTCAC AATTATTTTG GTGGGGACCC TGGGGTCCCT CCTGGTGGGT 60

GCCTTCCCCC AGGTCACCCCC GAAGAAGAGC TGCAGCCTCT CCAAGTACCA GTTCCCTGCA 120

CCTTTGGAGT TGAAGGCAGT GTGGAGGATG AAGGAGCAGT TTGAAGACAT CATGCTGTTA 180

ACAAACAGAA AATGCAACAC CAGACTCTTC CATCGGAAGT GGGACATAGC TGAGCTGTCG 240

GTACCTGACC GAATCACCCT GGTGGAGGCT GAGCTGGACC TCACCATCAC CGTGCTCACA 300

AACCCCCACAA CCCAGAGACT GGCAGAGACG TGCCAACAGC CCCTGGCCTT CCTTACCCAA 360

GTCCAGGAGG ACCTGCGAGA CTGCTTGGCC CTCGAGGCAC CTTCACATCA GCCCTCTGGG 420

AAACTGAGGC ACTGGCTGCA GAAGCTGGAG ACAGCCAAGA AGAAGGAGAC CGCCGGCTGC 480

CTGGAGGCCT CAGCCATCCT CCACATCTTC CAAGTACTGA ACGACCTGCG GTGGCAGCCC 540

AGCGCGAGGATTGCACTTAG 560

(2) IFN-λ-F1

ATGGTATGCT ACGGGGTCAC 20

(3) IFN-λ-R1

CTAAGTGCAA TCCTCGCGCT G 21

(4) IFN-λ-F

CCGGAATTCC AGGTCACCCCC GAAGAA 26

(5) IFN-λ-R

CCCAAGCTTC TAAGTGCAAT CCTCGCGCTG GGC 33

Claims (5)

1. a highly antiviral activity recombinant chicken interferon λ (rChIFN-λ), characterized in that: its nucleotide sequence is as shown in SEQ ID NO: 1. 2. the preparation method of a kind of high antiviral activity recombinant chicken interferon λ (rChIFN-λ) according to claim 1 is characterized in that, is prepared by the following steps: a. According to the chicken IFN-λ gene sequence (GenBank no.KF680102.1) provided by Genbank, design primers without the signal peptide, use CEF to self-amplify the required template, cDNA, and amplify the rChIFN-λ gene fragment; b. Cloning the gene into the pMD-19T vector to construct the cloning vector pMD-19T-rChIFN-λ. Sequencing showed that the homology with the chicken IFN-λ gene sequence provided by Genbank was 100%; c. Digest the pMD-19T-rChIFN-λ and pET32a empty vectors with EcoR1 and Hind3 fast enzymes, clone the target gene into the pET32a expression vector, transform and express the host E.coli DH5α, and obtain the recombinant plasmid pET32a-rChIFN-λ; d. Transform the recombinant plasmid pET32a-rChIFN-λ with BL21 competently, expand the culture, add IPTG to induce expression, take inclusion bodies after ultrasonic disruption, collect recombinant chicken interferon λ (rChIFN-λ) after denaturation, purification and renaturation )protein. 3. according to the preparation method of a kind of high antiviral activity recombinant chicken interferon λ (rChIFN-λ) described in claim 2, it is characterized in that, in step d, IPTG induction concentration is 1.0mmol/L, and induction temperature is 37 ℃ , the induction time is 8h. 4. a kind of highly antiviral activity recombinant chicken interferon λ (rChIFN-λ) described in claim 1 suppresses the pathogenic effect of vesicular stomatitis virus (VSV) and influenza virus (H5N6) on DF-1 in preparation application in medicine. 5. a kind of high antiviral activity recombinant chicken interferon λ (rChIFN-λ) described in claim 1 reduces and delays Newcastle disease virus (GM) lethal action in chicken body in preparation, suppresses the application in the drug of detoxification amount.