A protein against Newcastle disease virus and its application

Abstract

This invention relates to the application of chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs against Newcastle disease virus (NDV), belonging to the field of antiviral biological product technology. This invention provides the application of chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs against NDV. The application described in this invention enables the preparation of a novel biological product (immunostimulant) against NDV, which can inhibit NDV replication, increase IFN-α transcription levels, promote STAT1 upregulation, and regulate the transcriptional expression levels of ISGs; exogenously overexpressed Mertk protein can exert an anti-NDV effect and plays a certain role in innate immunity against NDV.

Description

Technical Field

[0001] This invention relates to the field of antiviral drug technology, specifically to the application of chicken-derived receptor tyrosine kinase protein in the preparation of drugs against Newcastle disease virus. Background Technology

[0002] Newcastle disease primarily infects chickens, pheasants, turkeys, and quails. Chickens are the most susceptible, followed by pheasants, and it is highly contagious to poultry. It is one of the reportable infectious diseases designated by the World Organisation for Animal Health (OIE), and often causes significant losses to the poultry industry. The pathogen is Newcastle disease virus (NDV), which belongs to the Paramyxoviridae family, the Orthomomavirus genus, and is a member of avian orthomomavirus type 1. Its coding genome is a single-stranded, negative-sense, non-segmented, enveloped RNA virus.

[0003] Currently, Newcastle disease control primarily relies on vaccination, mainly including inactivated and live vaccines. The main drawback of inactivated vaccines is their inability to activate cellular immune responses, resulting in insufficient cross-immunity against different viral genotypes and a potential for immunization failure. Furthermore, live vaccines also have limitations; for example, under conditions of weak disease populations or poor rearing environments, live vaccine strains can cause disease in flocks. Therefore, biological products that promote the enhancement of innate immunity remain relatively scarce. Summary of the Invention

[0004] The purpose of this invention is to provide the application of chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs against Newcastle disease virus. The application described in this invention enables the preparation of a novel biological product against Newcastle disease virus, which can inhibit Newcastle disease virus replication, increase IFN-α transcription levels, promote STAT1 upregulation, and regulate the transcriptional expression levels of ISGs.

[0005] This invention provides the application of chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs against Newcastle disease virus, wherein the amino acid sequence of the chicken-derived receptor tyrosine kinase protein is shown in SEQ ID NO.1.

[0006] The present invention also provides the application of chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs that inhibit the replication of Newcastle disease virus, wherein the amino acid sequence of the chicken-derived receptor tyrosine kinase protein is shown in SEQ ID NO.1.

[0007] This invention also provides the application of chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs that enhance IFN-α transcription levels, wherein the amino acid sequence of the chicken-derived receptor tyrosine kinase protein is shown in SEQ ID NO.1.

[0008] The present invention also provides the application of chicken receptor tyrosine kinase protein in the preparation of biological products or drugs that promote the upregulation of STAT1 expression, wherein the amino acid sequence of the chicken receptor tyrosine kinase protein is shown in SEQ ID NO.1.

[0009] The present invention also provides the application of chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs that regulate the transcriptional expression level of ISGs, wherein the amino acid sequence of the chicken-derived receptor tyrosine kinase protein is shown in SEQ ID NO.1.

[0010] Preferably, the ISGs include one or more of IFITM3, ZAP, and Viperin.

[0011] Preferably, the application includes:

[0012] Overexpression of chicken receptor tyrosine kinase protein is used to prepare biological products or drugs that promote the transcriptional expression level of ISGs; inhibition of chicken receptor tyrosine kinase protein expression is used to prepare biological products or drugs that reduce the transcriptional expression level of ISGs.

[0013] The present invention also provides the use of chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs that promote innate immune responses against Newcastle disease virus, wherein the amino acid sequence of the chicken-derived receptor tyrosine kinase protein is shown in SEQ ID NO.1.

[0014] The present invention also provides a recombinant vector for overexpressing chicken receptor tyrosine kinase protein, wherein the recombinant vector uses pCMV-HA as the backbone vector, and the amino acid sequence of the chicken receptor tyrosine kinase protein is shown in SEQ ID NO.1.

[0015] The present invention also provides a host cell overexpressing a chicken receptor tyrosine kinase protein, wherein the host cell type includes Escherichia coli, and the amino acid sequence of the chicken receptor tyrosine kinase protein is shown in SEQ ID NO.1.

[0016] This invention provides the application of chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs against Newcastle disease virus (NDV). The application described in this invention enables the preparation of a novel biological product or drug against NDV, which can inhibit NDV replication, increase IFN-α transcription levels, promote STAT1 upregulation, and regulate the transcriptional expression levels of ISGs, wherein ISGs include one or more of IFITM3, ZAP, and Viperin; exogenously overexpressed Mertk protein can exert an anti-NDV effect and plays a certain role in innate immunity against NDV. Attached Figure Description

[0017] Figure 1 The figure shows the results of fluorescence quantitative PCR detection of changes in the expression of the Mertk (Mer) gene provided by this invention;

[0018] Figure 2 The results of the fluorescence quantitative PCR detection of IFN-α gene expression changes provided by the present invention are shown in the figure.

[0019] Figure 3 The figure shows the results of the fluorescence quantitative PCR detection of STAT1 gene expression changes provided by the present invention.

[0020] Figure 4 The image shows the results of fluorescence quantitative PCR detection of IFITM3 gene expression changes provided by this invention.

[0021] Figure 5 The image shows the results of quantitative real-time PCR detection of ZAP gene expression changes provided by this invention.

[0022] Figure 6 The image shows the results of the quantitative real-time PCR detection of Veripin gene expression changes provided by this invention.

[0023] Figure 7 The figure shows the comparison of the effects of Mertk overexpression on the expression of Newcastle disease virus NP protein and host cell STAT1 by Western Blot analysis provided by this invention.

[0024] Figure 8 The half-maximum tissue culture infection dose (TCID) provided by this invention 50 The titer of Newcastle disease virus in DF1 cell supernatant was measured. Supernatant from normal DF1 cells and supernatant from Mertk-overexpressing DF1 cells were collected at 12 h and 24 h for TCID testing. 50 The results of detecting and comparing the changes in Newcastle disease virus titers are shown in the graph. Detailed Implementation

[0025] This invention provides the application of a chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs against Newcastle disease virus. The amino acid sequence of the chicken-derived receptor tyrosine kinase protein is shown in SEQ ID NO.1. In this invention, the nucleotide sequence of the gene encoding the chicken-derived receptor tyrosine kinase protein is shown in SEQ ID NO.2. In a specific embodiment of this invention, a recombinant vector overexpressing the chicken-derived receptor tyrosine kinase protein was transfected into chicken fibroblast cell line DF1. The transcription level of Mertk gene mRNA and changes in Mertk protein expression were detected. It was found that both Mertk mRNA and protein expression levels were upregulated. Further analysis of the half-maximal tissue culture infectious dose (TCID50) of the virus in the supernatant of Newcastle disease virus-infected cells was performed.50 The results showed a significant decrease in Newcastle disease virus titer (p<0.05), confirming that exogenously overexpressed Mertk protein can exert an anti-Newcastle disease virus effect.

[0026] This invention also provides the application of chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs that inhibit Newcastle disease virus replication, wherein the amino acid sequence of the chicken-derived receptor tyrosine kinase protein is shown in SEQ ID NO.1. Specific embodiments demonstrate that overexpression of the chicken-derived receptor tyrosine kinase protein can inhibit the replication of Newcastle disease virus.

[0027] This invention also provides the application of a chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs that enhance IFN-α transcription levels, wherein the amino acid sequence of the chicken-derived receptor tyrosine kinase protein is shown in SEQ ID NO.1. Specific embodiments demonstrate that overexpression of the chicken-derived receptor tyrosine kinase protein can enhance IFN-α transcription levels.

[0028] This invention also provides the application of a chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs that promote the upregulation of STAT1 expression, wherein the amino acid sequence of the chicken-derived receptor tyrosine kinase protein is shown in SEQ ID NO.1. Specific embodiments demonstrate that overexpression of the chicken-derived receptor tyrosine kinase protein can promote the upregulation of STAT1 expression.

[0029] This invention also provides the application of a chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs that regulate the transcriptional expression levels of ISGs, wherein the amino acid sequence of the chicken-derived receptor tyrosine kinase protein is shown in SEQ ID NO.1. In this invention, the ISGs preferably include one or more of IFITM3, ZAP, and Viperin. In this invention, the application preferably includes: overexpressing the chicken-derived receptor tyrosine kinase protein to prepare biological products or drugs that promote the transcriptional expression levels of ISGs; and inhibiting the expression of the chicken-derived receptor tyrosine kinase protein to prepare biological products or drugs that reduce the transcriptional expression levels of ISGs.

[0030] This invention also provides the application of chicken-derived receptor tyrosine kinase protein in the preparation of biological products or drugs that promote innate immune responses against Newcastle disease virus, the amino acid sequence of which is shown in SEQ ID NO.1. Interferon (IFN), as an innate immune response to viral infection, is an immediate response designed to control and inhibit viral growth and spread, and is mainly classified into types I, II, and III. IFN can activate the expression of interferon-stimulated genes (ISGs) through the Janus-activated kinase (JAK) and signal transducer and activator of transcription (STAT) signaling pathways, making it the most important innate immune response against viruses. This invention amplifies the chicken-derived Mertk gene in vitro, constructs a recombinant Mertk eukaryotic expression plasmid, and transfects it into DF1 cells. The effects of overexpression of Mertk protein on the expression of various interferon-stimulated genes (ISGs) and on NDV replication were examined. The results showed that overexpression of Mertk protein plays a role in innate immunity against NDV. Chicken-derived receptor tyrosine kinase protein, as an immune agonist, can promote STAT1 expression, regulate the transcriptional expression level of ISGs, and enhance IFN-α transcription levels, thereby regulating the innate immune response against Newcastle disease virus.

[0031] In this invention, the biological product preferably includes a vaccine or an immune agonist. In the preparation of a vaccine product for the prevention of Newcastle disease virus, recombinant expression of exogenous Mertk protein can induce a type I interferon immune response in the body, thereby enhancing the immune effect.

[0032] This invention also provides a recombinant vector for overexpressing a chicken receptor tyrosine kinase protein, wherein the recombinant vector uses pCMV-HA as a backbone vector, and the amino acid sequence of the chicken receptor tyrosine kinase protein is shown in SEQ ID NO.1. Specifically, this invention preferably involves polymerase chain reaction (PCR) amplification to obtain the full-length Mertk gene, followed by ligation into the eukaryotic expression vector pCMV-HA to obtain the recombinant plasmid pCMV-HA-Mertk. In this invention, the primers used to construct the recombinant vector are preferably shown in SEQ ID NO.3 and SEQ ID NO.4.

[0033] The present invention also provides a host cell overexpressing a chicken-derived receptor tyrosine kinase protein, wherein the host cell type includes *Escherichia coli*, and the amino acid sequence of the chicken-derived receptor tyrosine kinase protein is shown in SEQ ID NO.1. Preferably, the *Escherichia coli* includes DH-5α.

[0034] The application of the chicken-derived receptor tyrosine kinase protein of the present invention in the preparation of biological products or drugs against Newcastle disease virus is further described in detail below with reference to specific embodiments. The technical solutions of the present invention include, but are not limited to, the following embodiments.

[0035] Example 1

[0036] 1. Construction of recombinant chicken Mertk gene eukaryotic expression vector plasmid

[0037] The corresponding nucleotide sequence of the chicken-derived Mertk gene was downloaded from Genbank. Primers were designed using Primer software. The upstream primer of Mertk was preceded by the restriction endonuclease SalI (gtcgac), and the downstream primer was preceded by the restriction site of KpnI (ggtacc), as shown in Table 1. The primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0038] Table 1. Primer sequences for Mertk

[0039]

[0040] RNA was extracted from chicken fibroblast cell line DF1, and reverse transcription was performed using MLV reverse transcriptase to obtain cellular cDNA, which was then used as a template for PCR amplification of the Mertk gene. The reaction system is shown in Table 2. After mixing the above reaction solutions, the mixture was centrifuged and then placed in a PCR instrument for reaction. The reaction conditions were: 94℃ pre-denaturation for 5 min; 95℃ denaturation for 20 s, 58℃ annealing for 30 s, 72℃ extension for 90 s, for 30 cycles; followed by a final extension at 72℃ for 10 min. A 1% agarose gel was prepared using 1×TAE buffer, and 1 μL of green-blue nucleic acid dye was added. After PCR amplification, the product was detected by gel electrophoresis. The gel purification product was recovered using a gel extraction kit.

[0041] Table 2 PCR amplification system

[0042]

[0043] 4 μL of the recovered Mertk gene was mixed with 1 μL of pMD19-T vector, and then 5 μL of Solution I solution was added. Ligation was performed at 16°C for 30 min. Subsequently, 5 μL of the ligation product was transformed into *E. coli* DH-5α competent cells. Several single colonies were picked and inoculated into 1 mL of LB medium containing ampicillin. The cells were cultured on a shaker at 37°C for approximately 6 h before bacterial PCR identification. The bacterial PCR reaction system is shown in Table 3.

[0044] Table 3. Bacterial PCR Reaction System

[0045]

[0046] After identification, positive bacterial cultures were selected and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Plasmids were extracted from the correctly sequenced positive bacterial cultures and named pMD-19T-Mertk. The obtained pMD-19T-Mertk fragment was double-digested with Sal I / Kpn I (purchased from Takara Bio Inc.) and ligated into the pCMV-HA vector (purchased from Clontect Inc.), which was then transformed into DH5α (E. coli). Single colonies were picked and identified by PCR and Sal I / Kpn I digestion to obtain positive clones, which were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The positive clone was named pCMV-HA-Mertk, with the nucleotide sequence shown in SEQ ID NO.2 and the amino acid sequence shown in SEQ ID NO.1.

[0047] 2. Effects and applications of Mertk overexpression on Newcastle disease virus replication

[0048] DF1 cells were transferred to six-well plates and cultured overnight in a constant temperature incubator at 37°C and 5% CO2. When the cell density was about 70%, they were used for plasmid transfection and / or NDV infection.

[0049] Add 200 μL of Opti-MEM medium and 6 μL of FuGENE transfection reagent to a 1.5 mL EP tube and incubate for 5 min. Then add 2 μg of recombinant plasmid pCMV-HA-Mertk and incubate for another 20 min. A control with empty vector (pCMV-HA) is also included. After incubation, add the mixture to the medium in a six-well plate and mix well. Incubate at 37°C for 24 h, then infect with NDV for 3 h. After incubation with recombinant plasmid pCMV-HA-Mertk, the Mertk overexpression group (Mertk) is obtained. After incubation with pCMV-HA-Mertk and NDV infection for 3 h, the Mertk overexpression and NDV infection group (Mertk+NDV) is obtained.

[0050] Meanwhile, several control groups were designed. Control group 1 was the NDV-infected group alone (NDV), that is, when the cell density was about 70%, the cells were directly infected with NDV for 3 hours. Control group 2 was the DF1 cell group treated with IFN-α (IFN-α), that is, when the cell density was about 70%, IFN-α was added for treatment. Control group 3 was the IFN-α-infected group plus NDV infection (IFN-α+NDV), that is, when the cell density was about 70%, IFN-α was added for treatment and then NDV infection was carried out for 3 hours. Control group 4 was the Mertk inhibitor UNC2250-treated group (UNC2250), that is, when the cell density was about 70%, Mertk inhibitor was added for treatment for 1 hour. The Mertk inhibitor-infected group plus NDV infection (UNC2250+NDV), that is, when the cell density was about 70%, Mertk inhibitor was added for treatment for 1 hour and then NDV infection was carried out for 3 hours.

[0051] RNA was extracted from a portion of the cells and reverse transcribed into cDNA. The mRNA expression levels of genes such as NDVNP, STAT1, IFITM3, Viperin, and ZAP were detected using quantitative real-time PCR (relative quantification).

[0052] Quantitative real-time PCR results showed that the level of Mertk gene (also known as Mer) mRNA was as follows: Figure 1 As shown, the transcriptional level of Mertk gene in DF1 cells of the Mertk overexpression group was significantly higher than that in the blank cell control group (p<0.05). After Mertk overexpression, the transcriptional level of Mertk in the NDV infection group was significantly upregulated compared with the NDV infection group alone (p<0.05), but significantly lower than the upregulation of the Mertk overexpression group compared with the NDV infection group alone (p<0.05).

[0053] IFN-α gene mRNA level as Figure 2 As shown, the levels of IFN-α treated with NDV infection and NDV infection after Mertk overexpression were significantly higher than those in the blank cell control group and the NDV infection group alone (p<0.05). Furthermore, the Mertk overexpression group induced a higher level of IFN-α transcription compared to the IFN-α treatment group. This indicates that Mertk overexpression has a stronger ability to induce type I IFN production. The significant increase in IFN-α transcription levels due to Mertk overexpression (p<0.05) plays a crucial role in promoting the activation of downstream ISGs of type I IFN, thereby inhibiting viral replication.

[0054] STAT1 gene mRNA level as Figure 3As shown, the IFN-α treatment group, the Mertk overexpression group, and the Mertk overexpression followed by NDV infection group were all significantly higher than the blank cell group, the NDV infection group alone, the IFN-α treatment followed by NDV infection group, the Mertk inhibitor UNC2250 treatment group, and the Mertk inhibitor treatment followed by NDV infection group (p<0.05). This indicates that the overexpression of Mertk can effectively upregulate the transcription level of the STAT1 gene, and its upregulation ability is better than that of the positive control IFN-α treatment group. This further demonstrates that overexpression of Mertk can effectively promote the upregulation of STAT1, an important transduction gene in the IFN-α signaling pathway.

[0055] IFITM3 gene mRNA level as follows Figure 4 As shown, the Mertk overexpression group and the Mertk overexpression plus NDV infection group were significantly higher than the IFN-α treatment group (p<0.05) and the NDV infection group alone (p<0.05). Furthermore, the Mertk inhibitor UNC2250 treatment group and the Mertk inhibitor treatment plus NDV infection group were approximately 3-fold and 4-fold lower than the NDV infection group alone and the IFN-α treatment group, respectively. This indicates that Mertk overexpression significantly increases the transcriptional expression level of the interferon-stimulated gene (ISG) IFITM3 (p<0.05), while inhibiting Mertk expression decreases the transcriptional expression level of the IFITM3 gene.

[0056] The mRNA levels of the other two ISGs are as follows: Figure 5 (ZAP gene) and Figure 6 As shown in the image (Viperin gene), similar to the IFITM3 gene, the Mertk overexpression group and the Mertk overexpression plus NDV infection group were significantly higher than the IFN-α treatment group and the NDV infection alone group (p<0.05). The Mertk inhibitor UNC2250 treatment group and the Mertk inhibitor treatment plus NDV infection group were lower than the NDV infection alone group and the IFN-α treatment group. This indicates that overexpression of Mertk can upregulate the transcriptional levels of multiple ISGs, such as IFITM3, ZAP, and Viperin, significantly upregulating their expression (p<0.05).

[0057] For the other portion of cells, the culture medium was discarded, and the cells were collected with 2×SDS loading buffer. The cells were then heated at 100°C for 10 min to denature the proteins. Protein samples were separated by polyacrylamide gel electrophoresis (80V, adjusted to 120V after 30 min), and then transferred to an NC membrane (250mA, 90 min). The membrane was then blocked with 5% skim milk powder for 2 h. The blocking solution was discarded, and the membrane was washed three times with TBST buffer for 10 min each time. The corresponding tag anti-STAT1 antibody, anti-phosphorylated STAT1 antibody, or NDV NP protein antibody (1:1000 dilution) was added, and the membrane was incubated overnight at 4°C on a shaker. The membrane was washed three more times with TBST buffer for 10 min each time. The corresponding goat anti-rabbit or goat anti-mouse secondary antibody (1:5000 dilution) was added, and the membrane was incubated at room temperature for 1 h. The membrane was then washed three more times with TBST buffer for 10 min each time. Finally, the membrane was exposed to chemiluminescence solution.

[0058] like Figure 7 As shown in the Western blot results, the expression levels of STAT1 and phosphorylated STAT1 in DF1 cells overexpressing Mertk were significantly higher than those in DF1 cells without Mertk recombinant plasmid transfection (p<0.05). In normal DF1 cells, NDV expression increased significantly over time, with NP protein expression reaching a high level at 12 h. However, in the Mertk overexpression group, NP protein expression was absent at 12 h, significantly lower than in the normal DF1 cell group (p<0.05), and only showed weak expression at 18 h. This indicates that overexpression of Mertk protein can increase the expression level of phosphorylated STAT1, which is beneficial for type I IFN signal transduction and promotes the function of downstream ISGs, thereby inhibiting NDV replication in cells.

[0059] like Figure 8 As shown, the median tissue culture infectious dose (TCID) in the supernatant of cells infected with Newcastle disease virus was further detected. 50 It was found that at 12h and 24h time points, the NDV titer in the supernatant of DF1 cells overexpressing Mertk protein and reinfected with Newcastle disease virus was significantly lower than that in the supernatant of normal DF1 cells infected with NDV (p<0.05). This indicates that overexpression of Mertk protein can inhibit NDV replication in cells and exert antiviral activity.

[0060] In summary, the recombinant chicken-derived Mertk protein with anti-Newcastle disease virus provided by this invention can exert anti-Newcastle disease virus effects at the cellular level, which is helpful for the development of novel anti-Newcastle disease virus biological products or drugs.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences <120> A protein against Newcastle disease virus and its application <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 834 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Gly Gly Arg Cys Ala Leu Leu Cys Ala Leu Leu Cys Ala Leu 1 5 10 15 Pro Leu Pro Arg Cys Gly Ala Ala Glu Phe Gly Asp Ala Gly Gly His 20 25 30 Gly Gly Leu Arg Ser Leu Ser Ala Gly Ala Pro Leu Ala Arg Pro Leu 35 40 45 Trp Ala Lys His His Arg Pro Lys Arg Gly Leu Thr Ser Gly Arg Trp 50 55 60 Pro Pro Gln Gly Ala Lys Pro Ser Ala Thr Ser Val Gly Gln Leu Lys 65 70 75 80 Phe Asn Pro Thr Val Gly His Val Val Ile Asn Glu Leu Lys Asp Val 85 90 95 Thr Phe Asn Cys Ser Ile Lys Val Pro Gln Leu Leu Val Arg Pro Asp 100 105 110 Ser Pro Gly Ile Ser Leu Trp Lys Asp Gly Arg Glu Leu His Thr Leu 115 120 125 Asp Arg Ile Ala Thr Ser His Phe Glu Ile Leu Asp Glu Glu Glu Val 130 135 140 Ala Met Thr Ser Thr Phe Ser Ile Arg Ala Ala Gln Arg Ser Asp Asn 145 150 155 160 Gly Ser Tyr Val Cys Lys Leu Asn Ile Ser Gly Ile Glu Ile Ala Ser 165 170 175 Asp Pro Ile Leu Val Gln Leu Glu Gly Leu Pro His Phe Ile Gln Gln 180 185 190 Pro Glu Lys Leu Asn Val Thr Arg Asn Ser Pro Phe Asn Leu Thr Cys 195 200 205 Gln Ala Val Gly Pro Pro Glu Pro Val Glu Ile Tyr Trp Phe Arg Asn 210 215 220 Asn Val Gln Leu Asn Met Lys Pro Tyr Ile Ser Pro Ser Val Leu Thr 225 230 235 240 Val Pro Gly Leu Asn Glu Thr Ala Leu Phe Ser Cys Glu Ala His Asn 245 250 255 Ser Lys Gly Leu Thr Ala Ser Asn Pro Gly Gln Val Asn Val Lys Gly 260 265 270 Ile Pro Ser Ala Pro Lys Ala Val His Val Leu Lys Arg Met Ala His 275 280 285 Ser Ile Val Ile Ser Trp Val Pro Gly Phe Asp Ala Phe Ser Ala Leu 290 295 300 Asn Ser Cys Ser Val Gln Val Lys Glu Ala Val Pro Gln Ser Asn Val 305 310 315 320 Ser Leu Leu Leu Phe Asn Thr Ser Val Pro Pro His Val Tyr Arg Ile 325 330 335 Gln Gln Leu Trp Pro Met Ala Asp Tyr Asn Ile Ser Val Ser Cys Lys 340 345 350 Asn Glu Val Gly Trp Ser Ala Phe Ser Pro Trp Ile Thr Ala Ser Thr 355 360 365 Thr Glu Gly Ala Pro Thr Thr Gln Pro Leu Asn Val Thr Val Ser Leu 370 375 380 Asn Glu Ser Ser Ser Phe Leu Glu Ile Arg Trp Val Lys Pro Pro Leu 385 390 395 400 Glu Arg Thr His Gly Glu Leu Gln Gly Tyr His Ile Trp His Thr Trp 405 410 415 Gln Asp Ser Lys Gly Leu Gln Asn Ile Ser Leu Glu Ala Gln Pro Asn 420 425 430 Ala Thr Val Ala Ile Leu Pro Val Val Ala Thr Asn Ala Thr Cys Ser 435 440 445 Val Arg Val Ala Ala Val Thr Lys Gly Gly Val Gly Pro Phe Ser Ser 450 455 460 Pro Val Glu Val Phe Val Pro Ala Ser Gly Leu Ile Thr Ser Ser Pro 465 470 475 480 Ser Ser Thr Pro Ala Ser Gly Asn Thr Asp Ser Phe Ile Val Ala Leu 485 490 495 Gly Phe Val Cys Gly Thr Val Ala Val Gly Leu Ile Leu Cys Leu Ser 500 505 510 Val Val Ile Gln Lys Arg Cys Met Glu Thr Lys Tyr Gly Asn Ala Phe 515 520 525 Ser Arg Asn Asp Ser Glu Leu Val Val Asn Tyr Thr Ala Lys Lys Ser 530 535 540 Tyr Cys Arg Arg Ala Val Glu Leu Thr Leu Gly Ser Leu Gly Val Ser 545 550 555 560 Ser Glu Leu Gln Gln Lys Leu Gln Asp Val Val Ile Asp Arg Asn Ala 565 570 575 Leu Ser Leu Gly Lys Val Leu Gly Glu Gly Glu Phe Gly Ser Val Met 580 585 590 Glu Gly Arg Leu Ser Gln Pro Glu Gly Thr Pro Gln Lys Val Ala Val 595 600 605 Lys Thr Met Lys Leu Asp Asn Phe Ser His Arg Glu Ile Glu Glu Phe 610 615 620 Leu Ser Glu Ala Ala Cys Met Lys Asp Phe Asp His Pro Asn Val Ile 625 630 635 640 Lys Leu Leu Gly Val Cys Ile Glu Leu Ser Ser Gln Gln Ile Pro Lys 645 650 655 Pro Met Val Val Leu Pro Phe Met Lys Tyr Gly Asp Leu His Ser Phe 660 665 670 Leu Leu Arg Ser Arg Leu Glu Met Ala Pro Gln Phe Val Pro Leu Gln 675 680 685 Met Leu Leu Lys Phe Met Val Asp Ile Ala Leu Gly Met Glu Tyr Leu 690 695 700 Ser Ser Arg Gln Phe Leu His Arg Asp Leu Ala Ala Arg Asn Cys Met 705 710 715 720 Leu Arg Asp Asp Met Thr Val Cys Val Ala Asp Phe Gly Leu Ser Lys 725 730 735 Lys Ile Tyr Ser Gly Asp Tyr Tyr Arg Gln Gly Arg Ile Ala Lys Met 740 745 750 Pro Val Lys Trp Ile Ala Ile Glu Ser Leu Ala Asp Arg Val Tyr Thr 755 760 765 Thr Lys Ser Asp Val Trp Ala Phe Gly Val Thr Met Trp Glu Ile Ala 770 775 780 Thr Arg Gly Met Thr Pro Tyr Pro Gly Val Gln Asn His Glu Ile Tyr 785 790 795 800 Glu Tyr Leu Phe His Gly Gln Arg Leu Lys Lys Pro Glu Asn Cys Leu 805 810 815 Asp Glu Leu Ala Asp Arg Gln Gly Ala His Arg Ile Thr His Thr Gly 820 825 830 Trp Leu <210> 2 <211> 2505 <212> DNA <213> Artificial Sequence <400> 2 atgggcggcg ggcgctgcgc gctgctctgc gcgctgctct gcgctctgcc cctcccgcgc 60 tgcggggccg ctgagtttgg tgatgctggt ggacatggag gcctgaggtc cctatctgca 120 ggagccccgc tggcaaggcc cctgtgggca aagcaccacc gccccaaacg aggcctcacc 180 agtggccgct ggcctcccca gggcgcaaag ccttctgcca cctccgtggg acagctgaaa 240 tttaacccca cagtgggaca cgttgtgata aatgagctca aagatgtcac atttaactgc 300 tccatcaaag tacctcagct gctagtccgg ccagactccc ctggcatttc cctgtggaag 360 gatggcaggg agctgcacac gctggaccgc atcgccacca gccactttga gatccttgat 420 gaggaggagg tagccatgac ctctacattc agcatccgtg ctgctcagcg ctcggataac 480 ggctcctacg tctgcaaact caacatctct ggcattgaga ttgcatctga tcccatcttg 540 gtacagctgg aagggctccc acacttcatt caacagcctg agaagctgaa tgtcaccagg 600 aacagcccct tcaacctcac gtgccaagct gtgggcccac cagagcctgt ggaaatctac 660 tggtttcgta acaatgtcca actcaacatg aagccctaca tctccccatc agttctgact 720 gtcccaggtc tcaatgaaac agcactgttc agttgtgagg ctcacaacag caaagggctg 780 actgcttcca accccgggca ggtcaacgtg aaaggaatac catctgcacc aaaagctgtg 840 catgtcctga agagaatggc ccacagcatt gtgatctcct gggtgccagg cttcgatgcg 900 ttctctgcct tgaacagctg cagtgtgcag gtcaaggaag ccgttccaca aagcaatgtc 960 tcacttctgc tctttaacac gtcggtgcct ccccacgtgt atcgcatcca gcagctgtgg 1020 cccatggcag actataacat cagtgtttcc tgcaagaatg aagtcggttg gtcggcattt 1080 agcccctgga taacagccag taccacggaa ggagctccaa ctacccagcc actgaatgtc 1140 acagtgtcac tcaacgaatc cagctccttc ctggaaatcc gatgggtgaa gccacccctt 1200 gagagcac acggggagct gcagggatat catatctggc acacgtggca ggactccaag 1260 ggggctcaga acatctcctt ggaagcccag cctaatgcca cagtggccat cctgcctgtg 1320 gtggccacca atgccacgtg ctcagtgcgt gtggctgctg tcaccaaggg aggcgtgggg 1380 cccttcagca gcccagtgga ggtctttgtt cctgccagtg ggctaataac ctcatctccc 1440 tcttctgacac cagcatctgg gaacacagac tcctttatag tagcactggg cttcgtctgt 1500 ggtacggttg ctgttgggct gatcctctgc ttgtctgtgg tcatccagaa aagatgcatg 1560 gaaacaaagt atgggaatgc cttcagcaga aatgattcag agctggtggt aaactacaca 1620 gccaagaagt cctactgccg gagagccgtc gaactgacat tgggtagcct gggagtcagc 1680 agcgagctcc agcagaagct gcaggacgtt gtcattgaca gaaatgccct cagcctgggg 1740 aaggtcctgg gagaggggga gttcgggtca gtgatggagg gacgtctcag ccagccagaa 1800 ggcaccccac agaaggtggc tgtcaagacc atgaagttgg ataacttttc ccatagagag 1860 atagaagaat tcctcagtga agcagcatgc atgaaggact ttgaccaccc caatgtcatc 1920 aagctcctag gtgtgtgcat cgagctgagc tctcagcaga tccccaagcc catggtggtt 1980 ctcccattca tgaaatatgg tgacctgcac agcttcctgc ttcgctcccg gctggagatg 2040 gccccccagt tcgtgcccct gcagatgctg ctgaagttca tggtggatat tgccctggga 2100 atggagtacc tgagcagtcg gcagtttctt cacagggatt tggcggctcg gaactgcatg 2160 ttacgggatg acatgacggt gtgtgtggca gactttgggc tgtccaagaa gatctacagc 2220 ggcgattact accgtcaggg ccgaatagca aaaatgccag tgaagtggat tgcgatagag 2280 tccctggctg accgtgtcta caccaccaag agtgatgtgt gggcatttgg cgttaccatg 2340 tgggagatag cgaccagagg gatgactccg tacccagggg tgcagaacca cgagatttat 2400 gagtatctat tccacgggca gcggctcaaa aagcctgaga actgcttaga tgaactggct 2460 gacaggcagg gagctcacag gatcacacac acgggttggc tctga 2505 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <400> 3 cggtcgacat gggcggcggg cgctgcgcgc tg 32 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <400> 4 cgggtacctc agagccaacc cgtgtgtgtg at 32

Claims

1. The application of chicken-derived receptor tyrosine kinase protein in the preparation of drugs against Newcastle disease virus, wherein the amino acid sequence of the chicken-derived receptor tyrosine kinase protein is shown in SEQ ID NO.1.

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