Application of pigeon interferon λ in the preparation of drugs against pigeon pox virus

Abstract

The present invention relates to the field of antiviral interferon, and in particular to the use of pigeon λ interferon in the preparation of drugs against pigeon pox virus. The pigeon λ interferon provided by the present invention has an amino acid sequence as shown in SEQ ID NO.1, and a nucleotide sequence of a gene encoding the pigeon λ interferon as shown in SEQ ID NO.2. The inventors have found that the pigeon λ interferon provided by the present invention can activate the promoters of downstream antiviral genes Mx and ISRE, and significantly increase the expression levels of antiviral genes Mx and OAS. Compared with chicken and duck λ interferons, pigeon λ interferon has stronger antiviral activity and can be used as a candidate drug for preventing and treating pigeon viral diseases such as pigeon pox virus.

Description

Technical Field

[0001] The invention relates to the field of antiviral interferon, in particular to the application of pigeon lambda interferon in the preparation of medicines against pigeon pox virus. Background Art

[0002] Pigeon pox is a highly contagious disease of pigeons caused by the Pigeonpox virus (PGPV). It can infect pigeons of different stages and breeds. Infection of young pigeons can lead to severe mass deaths, causing serious economic losses. Currently, the prevention of pigeon pox virus mainly relies on vaccination, but with the continuous emergence of new strains and variants of pathogens, the protective effect of vaccines has also been limited. Currently, there are only a few veterinary antibiotics that can be used for the treatment of bacterial diseases such as Escherichia coli and Salmonella in pigeons. Once viral infectious diseases such as pigeon pox virus occur, there is often no cure, causing significant economic losses.

[0003] Interferon is a type of cytokine with a wide range of biological functions, including antiviral, anti-tumor, and immune regulation. According to the genome structure, receptors, and biological functions, interferon is divided into type I (including IFN-α, IFN-β, etc.), type II (IFN-γ), and type III (IFN-λ, etc.). At present, interferon has been widely used in human clinical treatment of various viral diseases such as hepatitis B, chronic hepatitis C, and viral myocarditis, and has important clinical application value. Although interferon was first discovered in poultry, pigeons are a special species of poultry, and research on pigeon interferon is still lacking. Current research mainly focuses on pigeon type I interferon (IFN-α). For example, Harvey M Santos and other scholars found that pigeon IFN-α has the function of inhibiting pigeon circovirus (Santos HM, et al. Influence of pigeon interferon alpha (PiIFN-α) on pigeon circovirus (PiCV) replication and cytokine expression in Columba livia [J]. Veterinary Microbiology, 2020); Li Siwen also found that pigeon IFN-α has obvious anti-vesicular stomatitis virus (VSV) ability (Li Siwen. Effect of p53 on the antiviral effect of recombinant pigeon α interferon [D]. Northeast Forestry University); Qian Jing and other scholars expressed pigeon IFN-α through the Pichia yeast expression system and found that it has anti-VSV virus effect (Qian Jing et al. A recombinant pigeon interferon α and its expression engineering strain and preparation method: CN202310095939.); Elena Fringuelli and other scholars cloned pigeon type II interferon (IFN-γ) in pigeons, but did not clarify its antiviral function (Fringuelli E, et al. Cloning and expression of pigeon IFN-γ gene [J]. Research in Veterinary Science, 2010). At present, no research reports on pigeon λ interferon and its antiviral function have been found. In addition, there are no research reports on other avian interferons against pigeon pox virus, which greatly limits the clinical prevention and treatment of pigeon pox virus. Therefore, in order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a pigeon lambda interferon that can resist pigeon pox virus. Summary of the invention

[0004] The purpose of the present invention is to provide an application of pigeon λ interferon in the preparation of a drug against pigeon pox virus, so as to solve the problems existing in the above-mentioned prior art. The present invention provides an application of pigeon λ interferon with an amino acid sequence as shown in SEQ ID NO.2 in the prevention and treatment of pigeon pox. The antiviral activity of the pigeon λ interferon provided by the present invention against pigeon pox virus is 1.26×10 6 UI / mg, which is higher than the antiviral activity of chicken and duck λ interferon against pigeon pox virus.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of pigeon lambda interferon in preparing a drug for resisting pigeon pox virus. The amino acid sequence of the pigeon lambda interferon is shown in SEQ ID NO.2.

[0007] Preferably, the nucleotide sequence of the gene encoding the pigeon λ interferon is shown in SEQ ID NO.1.

[0008] Preferably, the drug achieves the effect of resisting pigeon pox virus by increasing the expression levels of the antiviral genes Mx and OAS.

[0009] The present invention provides a medicine for preventing and / or treating pigeon pox, wherein the effective component of the medicine comprises pigeon lambda interferon; the amino acid sequence of the pigeon lambda interferon is shown in SEQ ID NO.2.

[0010] Preferably, the nucleotide sequence of the gene encoding the pigeon λ interferon is shown in SEQ ID NO.1.

[0011] Preferably, the drug achieves the effect of resisting pigeon pox virus by increasing the expression levels of the antiviral genes Mx and OAS.

[0012] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0013] The present invention discloses the following technical effects:

[0014] The present invention provides an application of pigeon λ interferon in the preparation of a drug against pigeon pox virus. The present invention analyzes the sequence characteristics of various avian λ interferons, clones the pigeon λ interferon gene from Shiqi pigeon for the first time using specific primers, and successfully obtains the pigeon λ interferon by prokaryotic expression. It is found that the pigeon λ interferon has the function of activating the promoter of downstream antiviral genes Mx and ISRE, and inducing the expression of downstream antiviral genes Mx and OAS. In terms of antiviral ability, the antiviral activity of the pigeon λ interferon provided by the present invention against pigeon pox virus reaches 1.26×10 6 UI / mg, which is higher than the antiviral activity of chicken and duck lambda interferon against pigeon pox virus. It can be seen that the pigeon lambda interferon provided by the present invention can be used as a candidate drug for preventing and treating pigeon pox virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is a high-fidelity clone of pigeon interferon λ; lanes 1, 2, and 3 are electrophoretic images of pigeon interferon λ sequences cloned from pigeon spleen cDNA using specific primers, with a fragment length of about 561 bp;

[0017] Figure 2 This is the phylogenetic tree of pigeon λ interferon;

[0018] Figure 3 This is a colony PCR detection diagram of pigeon λ interferon prokaryotic expression engineering bacteria; wherein, lane 1 is a marker, and the remaining lanes are PCR detection diagrams of single colonies;

[0019] Figure 4 The figure is a Western-Blot image of pigeon λ interferon protein; wherein, lane 1 is the inclusion body solution after urea denaturation, lane 2 is the inclusion body refolding solution, lane 3 is the product after the refolding solution is purified by nickel column, and lane 4 is the sample collected after the purified product is dialyzed to remove imidazole;

[0020] Figure 5 It is a bar chart showing the ability of pigeon λ interferon to activate the antiviral factor Mx and ISRE promoter; wherein A is the activation level of pigeon λ interferon on ISRE promoter, and B is the activation level of pigeon λ interferon on MX promoter;

[0021] Figure 6 It is a bar chart showing the expression of the pigeon interferon-activated antiviral gene Mx gene and OAS gene, where A is the expression level of the Mx gene and B is the expression level of the OAS gene;

[0022] Figure 7 The microscopic observation of the ability of pigeon interferon to inhibit the replication of pigeon pox virus; AF is the dilution of pigeon interferon 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 The effect against pigeon pox virus indicates that the pigeon interferon λ can provide effective protection; G is the dilution of pigeon interferon λ by 10 -7Anti-pox virus effect (cannot provide effective protection); H is the positive control with only virus added; I is the negative control without any treatment. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] 1. Obtaining pigeon λ interferon nucleic acid fragments

[0030] Use 100 μL 10 6 EID 50The 2-week-old pigeons were infected with a dose of H9N2 subtype avian influenza virus. On the third day after infection, the pigeons were dissected and the spleens of the pigeons were aseptically taken for RNA extraction and reverse transcription to obtain the pigeon spleen cDNA. According to the gene sequences of λ interferon of pigeons, chickens, ducks and other poultry published by NCBI, the specific primers P1 and P2 of the gene were designed using the primer design software Oligo7 (as shown in Table 1 below), and sent to Bio-Industry for synthesis. The spleen cDNA obtained above was used as a template, and the gene of pigeon λ interferon was amplified with high fidelity using P1 and P2.

[0031] Table 1 Primer list

[0032] Primer name Primer sequence (5'-3') SEQ ID NO. P1 ATGTTGTGCCTCAGCTTCAC 3 P2 CTAAGTGCATTGCTCCCGGC 4

[0033] The PCR system included: PrimeSTAR Max premix 25 μL, P1 1 μL, P2 1 μL, cDNA template 3 μL, and sterile distilled water 20 μL; the PCR reaction conditions were: 98°C 10 s, 55°C 5 s, and 72°C 20 s; for a total of 35 cycles.

[0034] After the reaction was completed, the reaction was separated by agarose gel electrophoresis. Figure 1 As shown, a band of about 561 bp can be observed on the electrophoresis diagram. After the band is cut, the electrophoresis product is recovered using a gel recovery kit, and the recovered product is sent to the company for sequencing. The sequencing results show that the correct pigeon λ interferon gene sequence is obtained. The nucleic acid sequence of pigeon λ interferon is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2.

[0035] SEQ ID NO.1:

[0036] ;

[0037] SEQ ID NO.2:

[0038] MLCLSFTSLLVLVLGVSLGAAFPQGALKKSCSLSKYKFIMRHEMKAARKMTEHFENIKLQSDRKCNTRLFHRKWKIADLSVPDRVILVEAELNLTIAMLELPTVPSFDEARQRPLAFLTQVREDLRGCMTMEATSHQPSGRLRHWLQNLQTAKNTETTGCLEASAILNIFQVLEDLRCTARREQCT.

[0039] Then, DNAStar MegAlign software was used to perform molecular evolution analysis on the interferon-λ sequences of different species of birds and mammals. Figure 2 As shown, it indicates that the pigeon λ interferon gene belongs to the avian λ interferon branch.

[0040] 2. Construction of prokaryotic expression engineering bacteria for pigeon λ interferon

[0041] Two restriction sites, BamH1 and XhoI, were introduced at both ends of specific primers P1 and P2, respectively, and primers P3 and P4 were designed and synthesized (as shown in Table 2). P3 and P4 primers were used to perform PCR amplification using the above-obtained pigeon λ interferon product as a template. The pigeon λ interferon gene was directionally inserted into the pET-28a prokaryotic expression vector by performing double restriction digestion, ligation, transformation, and positive clone screening on the recovered product and the expression vector pET-28b to obtain a recombinant positive plasmid. The recombinant positive plasmid was identified by PCR (as shown in Table 2). Figure 3 The pigeon λ interferon prokaryotic expression plasmid pET-28b-PiIFN-λ was successfully constructed, and the pET-28b-PiIFN-λ prokaryotic expression plasmid obtained above was transformed into the prokaryotic expression strain BL21 (DE3). The pigeon λ interferon prokaryotic expression engineering bacteria pET-28b-PiIFN-λ / BL21 (DE3) were obtained through positive clone screening and sequencing steps. The specific steps are as follows:

[0042] PCR amplification was performed using primers P3 and P4 with the pigeon λ interferon product obtained above as a template, wherein the PCR system included: PrimeSTAR Max premix 25 μL, P3 1 μL, P4 1 μL, cDNA template 1 μL, and sterile distilled water 22 μL; the PCR reaction conditions were: 98°C 10 s, 55°C 5 s, and 72°C 20 s; for a total of 35 cycles.

[0043] After the reaction is completed, the results are separated by agarose gel electrophoresis. Figure 3As shown, a band of 561bp can be observed on the electrophoresis diagram. After the band is cut, the electrophoresis product is recovered using a gel recovery kit, and the recovered product is double-digested with BamH1 and XhoI, and then the DNA is recovered using a gel recovery kit; similarly, the pET28a plasmid vector is double-digested with BamH1 and XhoI, recovered by agarose electrophoresis and connected with the recovered target DNA fragment. The connection reaction conditions are: 1μL T4 ligase, 1μL 10× buffer, 4μL enzyme-digested vector pET28a, 4μL purified enzyme-digested fragment product, and the above mixture is reacted at 37℃ for 1h. After 1 hour, the above ligation product was transformed into Escherichia coli DH5α, competent cells, coated with kanamycin resistance plates, cultured at 37°C overnight, and a single colony was picked as a template. P3 and P4 primers (see Table 2) were used for amplification. The product was detected by agarose electrophoresis, and a specific band appeared at about 500bp in the positive clone. To further confirm its sequence, the positive clone strain was extracted from the plasmid, and the plasmid was sent to the company for sequencing. The plasmid with correct sequencing represented that the pigeon λ interferon prokaryotic expression vector pET-28b-PiIFN-λ was constructed. The above-obtained prokaryotic expression plasmid was transformed into the prokaryotic expression strain BL21 (DE3), and the pigeon λ interferon prokaryotic expression engineering bacteria pET-28b-PiIFN-λ / BL21 (DE3) were obtained through positive clone screening and sequencing steps.

[0044] Table 2 Primer list

[0045] name Primer sequence (5'-3') SEQ ID NO. P3 ATGGATCCATGTTGTGCCTCAGCTTCAC (containing BamH1 restriction site) 5 P4 ACCTCGAGCTAAGTGCATTGCTCCCGGC (with XhoI restriction site) 6

[0046] 3. Prokaryotic expression and protein purification of pigeon λ interferon

[0047] Specific steps of prokaryotic expression: the above-obtained pigeon λ interferon prokaryotic expression engineering bacteria pET-28b-PiIFN-λ / BL21 were inoculated into liquid LB culture medium and cultured at 37°C with shaking. Then, the induction time of the bacteria (2h, 4h, 6h or 8h) and the concentration of the inducer IPTG (0.2mmol / L, 0.4mmol / L, 0.6mmol / L or 1mmol / L) were explored respectively. The results showed that when the OD value was 0.8, the final concentration of IPTG in the culture was 1mmol / L, and the culture time was 6h, the expression amount of interferon protein was the highest. The prokaryotic expression conditions were subsequently used for batch protein expression.

[0048] The protein purification is as follows:

[0049] (1) Obtaining inclusion bodies: 1 L of induced pigeon interferon λ prokaryotic expression engineering bacteria pET-28b-PiIFN-λ / BL21 was centrifuged at 10,000 r / min to collect the cells, and the cells and PBS were resuspended in a mass volume ratio of 1 g:5 mL. In an ice bath, ultrasonic power of 350 W, 5 s:5 s (working time: interval time) was used for 40 min to fully disrupt the cells. Then, the cells were centrifuged at 4°C and 10,000 r / min for 15 min to collect the precipitate (the precipitate is the crude inclusion body).

[0050] (2) Denaturation of inclusion bodies: The crude inclusion bodies obtained in step (1) were mixed with a denaturation solution containing 8M urea at a mass volume ratio of 1 g:5 mL. The mixture was stirred magnetically at room temperature for 6 h, then centrifuged at 12,000 r / min, 4°C, for 20 min, and the supernatant of the denaturation solution was collected.

[0051] (3) Inclusion body renaturation: The inclusion body was renatured by gradient dilution method. The supernatant of the denaturation solution was added to the pre-cooled renaturation solution (containing 0.3 mol / L L-arginine) by dropper in three times to make the volume ratio of the supernatant of the denaturation solution to the renaturation solution reach 1:20. After completion, the solution was transferred to a 4°C refrigerator and allowed to renature for 24 h.

[0052] (4) Protein purification: After renaturation, the renaturation solution was dialyzed against PBS buffer several times to remove L-arginine. The His-tagged target protein was purified according to the GE nickel column purification instructions to obtain the purified pigeon λ interferon protein. Finally, the imidazole solution of pigeon λ interferon was replaced with PBS solution through a dialysis step. Finally, a western-blot experiment was used to further confirm whether the correct target protein was obtained. The results are shown in Figure 2. Figure 4 As shown, the obtained pigeon λ interferon protein is about 21 kDa, which is consistent with the expected size.

[0053] 4. The ability of pigeon interferon-λ to activate downstream antiviral signaling pathways

[0054] 1. Pigeon λ interferon activates downstream Mx and ISRE promoters

[0055] The ability of the purified pigeon λ interferon protein obtained above to activate the Mx and ISRE promoters was analyzed using the dual luciferase reporter gene method. The specific steps are as follows:

[0056] DF-1 cells (a continuous chicken fibroblast cell line) were seeded in 24-well plates overnight until 85% confluence, and then three sets of plasmids were transfected using FuGENE HD transfection reagent: 100 ng of pGL3-Mx-Luc reporter gene plasmid (by inserting the promoter sequence of the Mx gene (AGTTTCGTTTCTAGTTTCGTTTCTAGTTTCGTTTCTAGTTTCGTTTCTAGTTTCGTTTCT, SEQ ID NO.15) was inserted into the pGL3-Basic vector (pGL3-Basic vector manufacturer: Promega (Beijing) Biotechnology Co., Ltd., catalog number: E6881). And 10ng of pRL-TK (manufacturer: Promega (Beijing) Biotechnology Co., Ltd., catalog number: E2231), 100ng of pGL3-ISRE-Luc reporter gene plasmid (by inserting the ISRE promoter sequence (TAGAAAATGAAACCAGGGAAACCGAAACTGAGTTTCACTTTCCTAGTTTCACTTTCCCTAGAAACTGAAAGTGGAAAGTGAAACCTGGGAAAATGAAACTCGGGAAAATGAAACTGGGGAAAACGAAACTGAGG AAATAGAAACTTAGGAAAAGGAAACTGGAGAAACCGAAACTATGGTTTCAGTTTTCCTGGTTTCATTTTCTACAGTTTCTGTTTCCTCAGTTTCGGTTTCCCGAGTTTCATTTCTTCAGGTTTCGTTTCTGCCTGTTTCAGTTTCTAAGGTTTCACTTTCCAGAGTTTCATTTTCCCCAGTTTCATTTTCCCCGCTTTCGTTTCCTCCAGTTTCGTTTTCCCAAGTTTCTATTTCCTCAGTTTCCTTTTCCTTATGTTTCGGTTTCTCTGGTTTCGTTTCCTC, SEQ ID NO.16) was inserted into the pGL3-Basic vector to construct

[0057] 24h after transfection, the transfected DF1 cells were treated with 1000ng / mL pigeon λ interferon, and 12h after treatment, the dual luciferase reporter system of Promega (Beijing) Biotechnology Co., Ltd. was used for detection. The luminescence detection of the supernatant of the lysate was performed using the PromegaGloMax 96 microplate luminescence detector. The results are shown in Figure 5As shown, pigeon λ interferon can significantly activate the ISRE promoter and the promoter of the MX gene. This shows that pigeon λ interferon has a significant ability to activate the downstream antiviral protein Mx and ISRE promoter.

[0058] 2. Pigeon λ interferon induces the expression of downstream antiviral genes

[0059] Fluorescence quantitative PCR was used to study the effect of pigeon interferon λ on the expression of downstream antiviral genes. The specific steps are as follows:

[0060] Take well-growing DF-1 cells, digest and culture them according to the conventional method, then add 1 mL to a 12-well cell culture plate and culture overnight to form a monolayer cell with a density of more than 85%. Add 100 ng / mL and 1000 ng / mL of purified pigeon λ interferon protein to the cells, respectively, and record them as pigeon λ interferon treatment groups. At the same time, set up a negative control group without pigeon λ interferon protein. There are three replicates in each experimental group and negative control group. After 24 hours, collect cells, extract RNA, reverse transcribe, and use DF1 cell-specific primers (Table 3) to quantitatively detect downstream pigeon antiviral genes Mx1 and OAS by fluorescent quantitative PCR. The primers are shown in Table 3 below. The results are shown in Table 3. Figure 6 As shown, pigeon λ interferon protein can significantly increase the expression of Mx and OAS genes, and has a certain dose-dependency. This shows that pigeon λ interferon has the function of activating the expression of downstream antiviral genes.

[0061] Table 3 Primer list

[0062] name Primer sequence (5'-3') SEQ ID NO. qMx-F GTTTCGGACATGGGGAGTAA 7 qX GCATACGATTTCTTCAACTTT 8 qOAS-F GCAGAAGAACTTTGTGAAGTGGC 9 qOAS-R TCGGCTTCAACATCTCCTTGTACC 10 qGAPDH-F ATCAAGAGGGTAGTGAAGGCTGCT 11 qGAPDH-R TCAAAGGTGGAGGAATGGCTGTCA 12

[0063] 5. Antiviral Application of Pigeon λ Interferon

[0064] In vivo antiviral activity

[0065] The antiviral activity of recombinant pigeon interferon λ was detected by cytopathic inhibition assay, and the specific steps are as follows:

[0066] DF-1 cells were inoculated in a 96-well plate overnight until the cells were more than 85% confluent. The test samples of pigeon interferon (protein concentration was about 1 mg / mL) were added to the cells for 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 and 10 -8 100 μL of 100 TCID was added to each well of the experimental group and the negative control group. 50 / mL of pigeon pox virus, observe the pathological changes of cells under a microscope every 6 hours and make records. The content of pigeon λ interferon that can inhibit 50% of the cytopathic effect is defined as one activity unit. The interferon titer is calculated using the Reed-Muench method. The same method is used to detect chicken λ interferon (the amino acid sequence of chicken λ interferon is shown in SEQ ID NO.13, specifically: MVCYGVTIILVGTLGSLLVGAFPQVTPKKSCSLSKYQFPAPLELKAVWRMKEQFEDIMLLTNRKCNTRLFHRKWDIAELSVPDRITLVEAELDLTITVLTNPTTQRLAETCQQPLAFLTQVQEDLRDCLALEAPSHQPSGKLRHWLQKLETAKKKETAGCLEASAILHIFQVLNDLRCAAQREDCT), duck λ interferon (the amino acid sequence of duck λ interferon is shown in SEQ ID NO.13, specifically: MVCYGVTIILVGTLGSLLVGAFPQVTPKKSCSLSKYQFPAPLELKAVWRMKEQFEDIMLLTNRKCNTRLFHRKWDIAELSVPDRITLVEAELDLTITVLTNPTTQRLAETCQQPLAFLTQVQEDLRDCLALEAPSHQPSGKLRHWLQKLETAKKKETAGCLEASAILHIFQVLNDLRCAAQREDCT) and duck λ interferon (the amino acid sequence of duck λ interferon is shown in SEQ ID NO.14, specifically: MLCPVFAAVLVVGLGPLLAGAFPQAALKKSCRLSQYGSPAYSELAEVLKFKKYYENITSKDPKCSTRLFNRKWTPNELSVPDRLLLVEAELDLTIAVLAHPTVDKLAEKSQQPLAFFTQAREDLRGCVAAEAPSHQPSGKLRHWLQKLETAKKTETASCLEYSTIVHLFQVLHDLGCVAIPERCT) anti-pigeon pox virus activity. The experimental results are as follows Figure 7 As shown in Table 4-Table 5. The results showed that: using the cytopathic inhibition method to detect the inhibitory effect of pigeon λ interferon on pigeon pox virus, pigeon λ interferon was diluted 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 ( Figure 7 AF in the solution still has anti-pigeon pox virus effect and can protect cell survival, while pigeon λ interferon has an anti-pigeon pox virus effect after being diluted by 10 -7 After 100 times, it has no protective effect and the cells are basically dead ( Figure 7 (G in Figure 7 H in the figure is the positive control with only virus added, and most of the cells have died. Figure 7 I in the figure is the negative control without any treatment. The cells survived. The specific protection effect of each dilution is shown in Table 4. The interferon titer was further calculated using the Reed-Muench method. The specific activity against pigeon pox virus was about 1.26×10 6UI / mg. Moreover, the anti-pigeon pox virus effects of pigeon, chicken, and duck interferons are shown in Table 5. Pigeon interferon has a stronger anti-pigeon pox virus effect than chicken and duck interferons. The titer of pigeon interferon against pigeon pox virus is 1.26×10 6 UI / mg.

[0067] Table 4 Protective effect of pigeon interferon λ

[0068] Pigeon interferon alpha dilution multiple No CPE holes CPE hole number Cumulative number of holes without CPE Cumulative CPE hole count Percentage of holes without CPE <![CDATA[10 -1 ]]> 8 0 44 0 100% <![CDATA[10 -2 ]]> 8 0 36 0 100% <![CDATA[10 -3 ]]> 8 0 28 0 100% <![CDATA[10 -4 ]]> 8 0 20 0 100% <![CDATA[10 -5 ]]> 7 1 12 1 92.3% <![CDATA[10 -6 ]]> 5 3 5 4 55.6% <![CDATA[10 -7 ]]> 0 8 0 12 0% <![CDATA[10 -8 ]]> 0 8 0 20 0%

[0069] Table 5 Comparison of the effects of interferon lambda from pigeons, chickens and ducks against pigeon pox virus

[0070] name Anti-pigeon pox virus titer Pigeon alpha interferon <![CDATA[1.26×10 6 IU / mg]]> Chicken IFN-λ <![CDATA[2.62×10 5 IU / mg]]> duck interferon alpha <![CDATA[3.58×10 5 IU / mg]]>

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of pigeon lambda interferon in the preparation of a drug against pigeon pox virus, characterized in that: The amino acid sequence of the pigeon λ interferon is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the pigeon λ interferon is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The drug achieves the effect of resisting pigeon pox virus by increasing the expression levels of antiviral genes Mx and OAS.

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