pX, a protein associated with LMH cell immune response, and its related biomaterials and applications

Abstract

This invention discloses pX, a protein associated with LMH cell immune responses, and related biomaterials and applications. The technical problem this application aims to solve is how to increase the transcriptional levels of Toll-like receptor genes and / or immune effector factors in avian cells and / or tissues and / or avian organisms. This invention discloses the applications of proteins, substances that upregulate or enhance or increase the expression of genes encoding said proteins, or substances that upregulate or enhance or increase the activity or content of said proteins in the following ways: A1) to increase the transcriptional levels of Toll-like receptor genes and / or immune effector factor genes in avian organisms; A2) to prepare products that increase the transcriptional levels of Toll-like receptor genes and / or immune effector factor genes in avian organisms. The transcriptional levels of Toll-like receptor genes and / or immune effector factor genes in cultured avian tissues and / or organs and / or avian organisms are significantly increased, which can be used for experimental studies of FADV-4 infection mechanisms.

Description

Technical Field

[0001] This application specifically relates to pX, a protein associated with LMH cell immune responses, and its related biomaterials and applications. Background Technology

[0002] Avian adenovirus can infect chickens, geese, ducks, and wild birds. Avian adenovirus type 4 (AADV4) typically infects young chickens, especially broilers under 5 weeks old, causing hydropericardium syndrome (HHS). Its clinical features include clear or strawberry-colored effusion in the pericardium, pulmonary edema, swollen and discolored liver, and distended kidneys, accompanied by focal necrosis and petechial hemorrhages. In recent years, AADV4 has caused multiple outbreaks in chicken flocks, resulting in severe economic losses for stakeholders. Infection is severe, with a mortality rate as high as 80%, posing a serious threat to the poultry industry.

[0003] Avian adenovirus is a non-enveloped, double-stranded DNA virus. Viral particles are 70–90 nm in diameter, with an icosahedral structure, and each particle consists of 252 capsid particles. Hexon, penton, and fiber are the main structural proteins of the capsid; nucleocapsid proteins also include PX, PVI, PVII, and PVⅢ. Adenovirus non-structural proteins mainly consist of E1A, E1B, E3, E4, 100K, and 52 / 55K. Hexon proteins neutralize surface antigens and are the main proteins used for serotyping. Penton proteins can bind to other intracellular proteins and play an important role in viral entry into cells. Fiber proteins include long fiber spike (fiber-1) and short fiber spike (fiber-2). Fiber-2 protein has good antigenicity and can induce antibodies, effectively resisting adenovirus infection. The role of the PX protein after avian adenovirus enters the cell is unclear. Studies have found that human adenovirus protein X (PX) can regulate the expression of E2 protein and participate in carrying the linear double-stranded DNA genome to the capsid during viral replication. However, the role of chicken adenovirus PX remains largely unknown. Summary of the Invention

[0004] This application provides pX, a protein associated with LMH cell immune responses, and related biomaterials and applications.

[0005] To address the aforementioned issues, this application provides the following applications.

[0006] The protein, the substance that upregulates, enhances, or increases the expression of the gene encoding the protein, or the substance that upregulates, enhances, or increases the activity or content of the protein, in any of the following applications:

[0007] The application is any one of the following:

[0008] A1) is used to increase the transcription level of Toll-like receptor genes in avian cells and / or avian tissues and / or avian animals;

[0009] A2) Products used to prepare products that increase the transcription level of Toll-like receptor genes in avian cells and / or avian tissues and / or avian animals;

[0010] A3) is used to increase the transcription level of immune effector gene transcription in avian cells and / or avian tissues and / or avian animals;

[0011] A4) Products used to prepare products that increase the transcription level of immune effector gene transcription in avian cells and / or avian tissues and / or avian animals;

[0012] The protein is any one of the following:

[0013] B1) The amino acid sequence of the protein is shown in sequence 2;

[0014] B2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in B1), which has more than 80% identity with the protein shown in B1) and has the ability to upregulate or enhance or increase the transcriptional level of Toll-like receptor genes and / or immune effector genes in avians.

[0015] B3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2).

[0016] The amino acid sequence of sequence 2 is shown below:

[0017] MPAVLLTGGRAASKRKFSTKQRRKKAVSVPKIRSRSGKRSGVRKRSSISVPVSGTASASERAALQNLAQRLQRGN YTAWRSADPSVAASEAAKAAAASGAAAYVRDLTTGTAAEAVPLTGTGRRRRTGARRSMRGGFFPALIPLIAAAIGAIPGI AGTAVGIASLKEQQRQFNKLYGNK.

[0018] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0019] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0020] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0021] In the aforementioned protein, sequence 2 (SEQ ID No. 2) consists of 179 amino acid residues.

[0022] In the above text, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0023] In the above applications, the Toll-like receptor gene may be one or more of chTLR1b, chTLR2a, chTLR3, chTLR7, and chTLR15;

[0024] and / or;

[0025] The immune effector factors are one or more of interferon and interleukin.

[0026] In the above text, the interferon may be one or more of IFN-α and IFN-β.

[0027] In the above text, the interleukin may be one or more of IL-6, IL-8, IL-15 and IL-1β.

[0028] In the above applications, the protein may be derived from avian adenovirus. Summary of the Invention:

[0030] In the above text, the avian adenovirus may be avian adenovirus type 4, abbreviated as FADV-4. The protein is abbreviated as pX protein or protein pX.

[0031] In this document, the substance regulating the activity and / or content of the protein may be a substance regulating gene expression, and the protein encoded by the gene may be any of the following:

[0032] C1) The amino acid sequence is the protein shown in sequence 2;

[0033] C2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in C1), which has more than 80% identity with the protein shown in C1) and upregulates or enhances or increases the transcriptional levels of avian Toll-like receptor genes and / or immune effector genes.

[0034] To address the above problems, this application provides any of the following applications.

[0035] Applications of related biomaterials in any of the following:

[0036] The biomaterial is any one of the following:

[0037] D1) Nucleic acid molecules encoding any of the proteins described above;

[0038] D2), an expression cassette containing the nucleic acid molecules described in D1);

[0039] D3), ​​a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0040] D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3);

[0041] D5) A transgenic animal cell line containing the nucleic acid molecule described in D1), or a transgenic animal cell line containing the expression cassette described in D2), or a transgenic animal cell line containing the recombinant vector described in D3);

[0042] D6) Transgenic animal tissue containing the nucleic acid molecule described in D1), or transgenic animal tissue containing the expression cassette described in D2), or transgenic animal tissue containing the recombinant vector described in D3);

[0043] D7) Transgenic animal organs containing the nucleic acid molecules described in D1), or transgenic animal organs containing the expression cassette described in D2), or transgenic animal organs containing the recombinant vector described in D3);

[0044] The application is any one of the following:

[0045] A1) Application of biomaterials in improving the transcription level of Toll-like receptor genes in avian cells and / or avian tissues and / or avian animals;

[0046] A2) Application of biomaterials in the preparation of products that enhance the transcriptional level of Toll-like receptor genes in avian cells and / or avian tissues and / or avian animals;

[0047] A3) Application of biomaterials in improving the transcription level of immune effector gene transcription in avian cells and / or avian tissues and / or avian animals;

[0048] A4) Application of biomaterials in the preparation of products that enhance the transcription levels of immune effector gene genes in avian cells and / or avian tissues and / or avian animals.

[0049] In the above text, the substance that upregulates, enhances, or improves the expression of the gene encoding the protein may be the nucleic acid molecule encoding any of the proteins described above (D1), the expression cassette containing the nucleic acid molecule described in D1), or the recombinant vector containing the nucleic acid molecule described in D1) or the recombinant vector containing the expression cassette described in D2).

[0050] In the nucleic acid molecule described in D1), those skilled in the art can easily mutate the nucleotide sequence encoding the protein pX of the present invention using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides that have 80% or more of the same nucleotide sequence as the protein pX isolated in the present invention, as long as they encode protein pX and have the function of protein pX, are all derived from and equivalent to the nucleotide sequence of the present invention.

[0051] The aforementioned 80% or higher identity can be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0052] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0053] In the above-mentioned biological materials, the nucleic acid molecule described in D1) may be the gene encoding the protein. Specifically, the nucleic acid molecule described in D1) may be a DNA molecule whose coding sequence is shown in Sequence 1.

[0054] The nucleotide sequence of sequence 1 is shown below:

[0055] .

[0056] In the aforementioned biological materials, the expression cassette described in D2) refers to DNA capable of expressing the gene in a host cell. This DNA may include not only a promoter to initiate gene transcription but also a terminator to terminate gene transcription. Furthermore, the expression cassette may also include an enhancer sequence. The promoter may be PEF1α (human elongation factor 1alpha promoter), or simply human elongation factor 1α promoter.

[0057] In D3) above, a recombinant expression vector containing the gene expression cassette can be constructed using an animal expression vector. Specifically, it can be a pEF1α-HA Vector.

[0058] It can also be an expression cassette cloning vector containing the aforementioned nucleic acid molecule or a nucleic acid molecule. The vector can be pMD18-T. The microorganism mentioned in D4) above can be Escherichia coli. The Escherichia coli can be DH5α.

[0059] The transgenic animal tissue described in D5) above or the transgenic animal organ described in D6) above can be prepared from the animal cell line described in D5) by biological means.

[0060] In the above applications, the animal cells described in D5)-D7 can be any of the following:

[0061] E1) Aves cells

[0062] E2) Cells of Galliformes animals;

[0063] E3) Pheasant cells;

[0064] E4) Cells of animals belonging to the genus *Gnaphalium*.

[0065] In the above text, the animal cells mentioned in D5)-D7 can be chicken liver cancer cells, abbreviated as LMH cells.

[0066] In the above applications, the substance that upregulates, enhances, or improves the expression of the protein-coding gene can be the aforementioned biological material.

[0067] To address the aforementioned problems, this application provides a method for enhancing gene transcription in avian cells.

[0068] The method for enhancing gene transcription in avian cells provided in this application includes upregulating or enhancing or increasing the expression of the gene encoding any of the proteins described above, or the activity or content of the proteins, in avian cells to upregulate or enhance or increase the transcription of Toll-like receptor genes and / or immune effector factor genes in avian cells.

[0069] In the above text, the gene transcription is the transcription of Toll-like receptor genes and / or immune effector factor genes. Specific types are as described above.

[0070] To address the aforementioned issues, this application provides a method for preparing avian cells with high levels of transcription of Toll-like receptor genes and / or immune effector genes.

[0071] The method for preparing avian cells with high transcription levels of Toll-like receptor genes and / or immune effector genes includes upregulating or enhancing or increasing the expression level of the encoding gene of any of the aforementioned proteins or the activity and / or content of the proteins in the target avian cells, thereby obtaining avian cells with high transcription levels of Toll-like receptor genes and / or immune effector genes, wherein the transcription levels of Toll-like receptor genes and / or immune effector genes in the avian cells with high transcription levels of Toll-like receptor genes and / or immune effector genes are higher than those in the target avian cells.

[0072] In the above method, the upregulation, enhancement, or increase of the expression level of the coding gene of any of the proteins mentioned above in the target plant, or the activity or content of the protein, is to introduce the above-mentioned biological material into the target avian cell.

[0073] The biomaterials mentioned above are as follows:

[0074] D1) The nucleic acid molecule that encodes the above-mentioned protein.

[0075] D2) Expression cassettes containing the nucleic acid molecules described in D1).

[0076] D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2). The recombinant vector may be as described above, specifically a pEF1α-HA vector.

[0077] The importation described above can be achieved through liposome transfection technology. Specifically, it can be:

[0078] D10), an artificial biomembrane containing a recombinant vector of the nucleic acid molecule described in D1), or an artificial biomembrane containing a recombinant vector of the expression cassette described in D2).

[0079] Artificial biomembranes, also known as liposomes, are lipid bilayers formed when phospholipids are dispersed in water. They can be used in liposome transfection technology.

[0080] The nucleic acid molecule encoding the above protein can be:

[0081] E1) The nucleic acid sequence is the DNA shown in Sequence 1;

[0082] E2) DNA obtained by substituting and / or deleting and / or adding nucleotide residues to the DNA described in E1), which has more than 80% identity with the DNA shown in E1) and has upregulated or enhanced or increased transcription levels of avian Toll-like receptor genes and / or immune effector genes.

[0083] To address the aforementioned issues, this application provides methods for cultivating avian tissues and / or avian organs and / or avian animals.

[0084] The method for cultivating avian tissues and / or organs and / or animals provided herein includes preparing avian cells with high levels of Toll-like receptor gene and / or immune effector gene transcription using the methods described above, and then using the avian cells with high levels of Toll-like receptor gene and / or immune effector gene transcription to cultivate avian tissues and / or organs and / or animals with high levels of Toll-like receptor gene and / or immune effector gene transcription.

[0085] In this application, the bird cells described in the application or method can be any of the following:

[0086] R1) Galliformes;

[0087] R2) Pheasant family;

[0088] R3) Genus *Gyrococcus*;

[0089] R4) Chicken liver cancer cells.

[0090] The chicken liver cancer cells mentioned may be LMH cells.

[0091] Previous studies in this research group revealed significant changes in the mRNA transcription levels of Toll-like receptors and effector cytokines after infection of LMH cells and SPF chickens with virulent FADV-4 strains, suggesting a close relationship between FADV-4 infection and the recognition of host innate immune pattern receptors and the production of their effector cytokines. However, whether the FADV-4PX gene participates in regulating the host's innate immune response remains unclear and requires further investigation. This study used the FADV-4PX gene as the research subject, constructed a pEF1α-HA-px recombinant expression plasmid, transfected LMH cells, and used real-time quantitative PCR to detect the mRNA transcription levels of 10 Toll-like receptors and 6 effector cytokines to analyze and explore the interaction between the FADV-4PX gene and the host's innate immunity. This provides data for further elucidating the pathogenic mechanism and immune response mechanism of FADV-4 infection.

[0092] Beneficial effects

[0093] This invention discloses the protein pX associated with the immune response in LMH cells, its related biomaterials, and its applications. This application constructs an overexpression vector pEF1α-HA-PX encoding the pX protein gene. The overexpression vector was transfected into LMH cells to obtain pEF1α-HA-PX-transfected LMH cells. Detection revealed that, compared to pEF1α-HA-transfected LMH cells as a control, the pX protein content in pEF1α-HA-PX-transfected LMH cells was significantly increased. Furthermore, the transcription levels of chTLR1b, chTLR2a, chTLR3, chTLR7, chTLR15, chTLR2a, IFN-α, IFN-β, IL-6, IL-8, IL-15, and IL-1β genes were significantly increased in pEF1α-HA-PX-transfected LMH cells. The results indicate that pX protein expression in LMH cells activates the LMH cell immune response. LMH cells transfected with pEF1α-HA-PX can be used to study the mechanism of LMH cell response to FADV-4 invasion. Attached Figure Description

[0094] Figure 1 Electrophoresis results of PX gene PCR amplification products; M: Trans 2K DNA Marker; 1-4: pX gene amplification products.

[0095] Figure 2 PCR identification of pEF1α-HA-PX recombinant plasmid; M: Trans 2K DNA Marker; 1: pEF1α-HA-PX PCR positive product; 2-3: negative products.

[0096] Figure 3 Western blot validation results for recombinant PX protein; M: protein marker (11-180 kDa); 1: pEF1α-HA-PX plasmid; 2: PEF1α-HA empty plasmid.

[0097] Figure 4 The results of indirect immunofluorescence verification of recombinant PX protein (100×); A: PEF1α-HA empty plasmid; B: pEF1α-HA-PX plasmid.

[0098] Figure 5 The effect of FADV-4pX protein on the expression level of Toll-like receptor mRNA in LMH cells.

[0099] Figure 6 The effect of FADV-4pX protein on the expression levels of LMH cell effector factor mRNA. Detailed Implementation

[0100] 1. Experimental Materials

[0101] 1.1 Viruses and Cells

[0102] The PEF1α-HA vectors were all preserved by the Key Laboratory of Biotechnology, Veterinary Research Institute of Guangxi Zhuang Autonomous Region, and are described in the following literature: Ren Hongyu, Xie Zhixun, Xie Liji, Wang Sheng, Huang Jiaoling, Fan Qing, Luo Sisi, Zhang Yanfang, Zeng Tingting, Zhang Mingxiu, Xie Zhiqin, Deng Xianwen. Expression of avian reovirus σA protein in HEK293T cells [J]. Heilongjiang Animal Husbandry and Veterinary Medicine, 2019(14):59-62.

[0103] The FAdV-4 isolate GX2019-010 (also known as FAdV-4-GX2019-010 strain) is described in the following literature: Luan Yongjiao, Xie Zhixun, Wang Sheng, et al. Dynamic distribution and shedding patterns of avian adenovirus type 4 in various tissues of SPF chickens infected with serum type 4 avian adenovirus [J]. Chinese Journal of Veterinary Medicine, 2021, 41(03):463-468.

[0104] Chicken liver cancer cells (LMH) are described in the following literature: Shi Yongli, Xie Zhixun, Luo Sisi, et al. Dynamic changes in the transcriptional level of Toll-like receptor mRNA in LMH cells infected with serum avian adenovirus type 4 [J]. Chinese Journal of Veterinary Science, 2022, 52(01):85-92. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0105] 1.2 Main Reagents

[0106] 2×Transtaq-T PCR Super Mix, viral DNA / RNA purification kit, and E. coli DH5α competent cells were all purchased from Beijing TransGen Biotech Co., Ltd.; pMD18-T vector, T4 DNA ligase, EcoRI and KpnI restriction endonucleases were all purchased from Takara Bio Engineering (Dalian) Co., Ltd.; DMEM / F12 cell culture medium, fetal bovine serum (FBS), transfection kit, HA-labeled mouse monoclonal antibody, and HRP-labeled goat anti-mouse IgG (H+L) were purchased from Thermo Fisher Scientific, Inc.; Universal Genomic DNA Kit was purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; Gene JET RNAPurification Kit and 2×SYBR Green Master Mix were purchased from Ingenium Biotech, Inc.; PrimeScript RT Master Mix was purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.

[0107] 1.3 Main Instruments

[0108] Gel Imaging Analyzer (Model Gel Doc) TMXR+ was purchased from Bio-Rad, USA; NanoDrop2000 ultra-micro spectrophotometer and Quant Studio 5 real-time fluorescence quantitative PCR instrument were purchased from Thermo Fisher Scientific, USA; and ECLIPSETi2-U inverted fluorescence microscope was purchased from Nikon, Japan.

[0109] 2 Methods

[0110] 2.1 Primer Design and Synthesis

[0111] Based on the sequenced pX gene coding region isolated from FAdV-4-GX2019-010 strain (accession number MW439040) by the Biotechnology Laboratory of the Guangxi Zhuang Autonomous Region Veterinary Research Institute, PCR primers were designed using Oligo 7.37 software. The upstream primer was ccggaattcGGATGCCCGCCGTGCTTTTGAC (underlined indicates EcoRI restriction site), and the downstream primer was cccggtaccTCACTTGTTGCCATACAACTTATTG (underlined indicates KpnI restriction site). The primers were synthesized by BGI Genomics Co., Ltd. in Shenzhen and were used for PCR amplification of the pX gene coding region sequence.

[0112] 2.2 PCR amplification of the PX gene coding region sequence

[0113] FAdV-4- samples were obtained by inoculating LMH cells with the FAdV-4-GX2019-010 strain. DNA was extracted from the FAdV-4- samples according to the viral DNA / RNA purification kit instructions. The coding region sequence of the FAdV-4- gene was amplified using the extracted DNA as a template. The amplification system (50 μL) consisted of: 25 μL of 2×TransTaq-T PCR Super Mix, 2 μL each of the forward and reverse primers from step 2.1 (primer concentration 10 μmol / L), 3 μL of DNA template, and 20 μL of nuclease-free water. The amplification program was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 56℃ annealing for 1 min, 72℃ extension for 1 min, for a total of 34 cycles; and a final extension at 72℃ for 5 min. The PCR amplification products were detected by 1.5% agarose gel electrophoresis.

[0114] Electrophoresis results of PCR amplification products are shown in the figure. Figure 1 .Depend on Figure 1As can be seen, using the cDNA obtained by reverse transcription as a template, the PCR amplification product separated by agarose gel showed a band of approximately 540 bp, consistent with the expected size. Sequence analysis showed that the cloned PX gene ORF was 540 bp long (Sequence 1), encoding 179 amino acids (Sequence 2), and had 100% nucleotide sequence similarity to the PX gene of avian adenovirus type 4 strain 2018 uploaded by our laboratory in August, as reported in GenBank (accession number MN577984.1).

[0115] 2.3 Construction of pEF1α-HA-PX expression vector

[0116] The target fragment was recovered from the gel according to the EZNAGel Extraction Kit instructions. The recovered gel product was ligated with the pMD18-T vector at 16℃ overnight. The ligation product was transformed into DH5α competent cells, and positive clones were screened. Samples were identified by double digestion with EcoRI and KpnI. Samples with correct digestion identification were sent to BGI Genomics Co., Ltd. for sequencing. Plasmids (recombinant cloning vector named pMD18-T-pX) were extracted from the correctly sequenced strains according to the PlasmidMini Kit instructions and stored at -20℃ for later use. The pEF1α-HA vector and the recombinant cloning vector pMD18-T-pX were simultaneously digested with EcoRI and KpnI restriction endonucleases at 37℃ for 4 h. The target fragment was recovered from the gel and ligated with both at 16℃ overnight. The ligation product was transformed into DH5α competent cells, and single colonies were picked for PCR verification (using GATTACGCTCTTATGGCCATG as the upstream primer and TTATCATGTCTGGATCCCCGC as the downstream primer). PCR results are shown Figure 2 .Depend on Figure 2 It can be seen that the recombinant expression plasmid was amplified to obtain a 640bp fragment, indicating that the PX target fragment has been correctly inserted into the pEF1α-HA vector.

[0117] The correct recombinant expression plasmid with the correct PCR band size was then sequenced. Sequencing by BGI Genomics showed that the inserted PX gene size, location, and frame of reference were all correct, indicating successful construction of the recombinant expression plasmid. The recombinant expression plasmid, verified by both enzyme digestion and sequencing, was named pEF1α-HA-pX (also known as pEF1α-HA-pX plasmid, pEF1α-HA-PX recombinant plasmid, pEF1α-HA-pX expression plasmid, pEF1α-HA-pX vector, pEF1α-HA-pX recombinant expression vector, or pEF1α-HA-pX recombinant vector).

[0118] pEF1α-HA-pX is obtained by replacing the DNA fragment with the nucleotide sequence of sequence 1 between the recognition sites of the restriction endonucleases EcoRI and KpnI in the pEF1α-HA vector, while keeping the other nucleotide sequences of the pEF1α-HA vector unchanged.

[0119] 2.4pEF1α-HA-PX recombinant plasmid transfected LMH cells

[0120] Press 1×10 6 LMH cells were seeded at a density of cells / mL in 6-well cell culture plates and allowed to grow into a monolayer. The culture medium (DMEM / F-12 (1:1) medium, catalog number: 8121748) was then discarded. Endotoxin-free plasmids pEF1α-HA-PX (experimental group) and pEF1α-HA (control group) were transfected into LMH cells using the Lipfectamine 3000 Transfection Kit (2 μg / well). After incubation for 40 min, 2 mL of culture medium (DMEM / F-12 (1:1) medium, catalog number: 8121748) was added, and the cells were cultured for another 48 h to obtain pEF1α-HA-PX transfected LMH cells. Cell samples were collected according to experimental requirements.

[0121] Western blot and indirect immunofluorescence assays of 2.6pEF1α-HA-PX protein

[0122] 2.6.1 Western blot assay method: Protein sample acquisition steps: (1) Remove the culture medium, collect the cells in an EP tube, and wash once with PBS; (2) Add RIPA lysis buffer (purchased from Sangon Biotech (Shanghai) Co., Ltd., product number: C50005) mixed in the ratio (A solution: B solution: C solution = 1 mL: 1 μL: 5 μL), and mix well; (3) Place on ice for 10 min and then sonicate to disrupt the cells; (4) Centrifuge at 12000 rpm and 4℃ for 5 min, and take the supernatant for WB assay. After SDS-PAGE analysis, recombinant protein samples were transferred to PVDF membranes and blocked with 4% skim milk at room temperature for 4 h. Then, HA-labeled mouse monoclonal antibody diluted 1:2000 was added and incubated overnight at 4°C. The next day, the membranes were washed 4 times with PBST, and HRP-labeled goat anti-mouse secondary antibody diluted 1:2000 was added and incubated at room temperature for 1 h. The membranes were washed 4 times with PBST and then developed and photographed using DAB-enhanced reagent.

[0123] 2.6.2 Indirect Immunofluorescence Method: 48 h after transfection of cells with recombinant plasmids, the culture medium was discarded. 500 μL of 4% paraformaldehyde was added to each well, and staining was performed for 15 min. After discarding the paraformaldehyde, 500 μL of permeabilization buffer (Triton X-100) was added, and staining was performed for 15 min. After discarding the permeabilization buffer, 500 μL of blocking buffer (5% BSA) was added, and staining was performed for 15 min. The blocking buffer was discarded, and 500 μL of HA-labeled mouse monoclonal antibody (1:500) was added to each well, and incubated overnight at 4°C. The primary antibody was discarded, and the cells were washed three times with PBS. 500 μL of FITC-labeled rabbit anti-mouse secondary antibody (1:1000) was added to each well, and incubated at room temperature for 1 h. The secondary antibody was discarded, and the cells were washed three times with PBS. The results were observed using an inverted fluorescence microscope.

[0124] The verification results are shown below. Figure 3 and Figure 4 .Depend on Figure 3 It was found that when pEF1α-HA-PX (experimental group) and pEF1α-HA (control group) were transfected into LMH cells, the protein lysed from cells transfected with the pEF1α-HA-PX recombinant plasmid specifically reacted with the mouse-derived HA-labeled monoclonal antibody, showing a specific band at 27 kDa, while the control group did not show a corresponding band at this location. Figure 4 It was observed that cells transfected with the pEF1α-HA-PX recombinant plasmid exhibited abundant green fluorescence, while the control group showed no green fluorescence. This indicates that the pEF1α-HA-PX recombinant protein was correctly expressed in LMH cells.

[0125] 2.5 Real-time quantitative PCR for the determination of cell samples

[0126] RNA was extracted from LMH cells transfected with pEF1α-HA-PX according to the Gene JET RNAPurification Kit instructions. The concentration was measured using a micro-spectrophotometer. One-step reverse transcription was performed. The reverse transcription reaction system (total volume 20 μL) consisted of: 4 μL of 5×PrimeScript RT Master Mix, 1 μg of RNA, and nuclease-free water to a final volume of 20 μL. The reverse transcription program was: 37℃ for 15 min, 85℃ for 5 s. The obtained cDNA was diluted 10-fold and used as a template for relative real-time quantitative PCR. The reaction system (total volume 20 μL) consisted of: 10 μL of 2×SYBR Green Mix, 1 μL each of 10 μmol / L forward and reverse primers, 2 μL of cDNA template, and 6 μL of RNase-free water. Reaction procedure: Activation at 50℃ for 2 min, pre-denaturation at 95℃ for 2 min, denaturation at 95℃ for 15 s, annealing at 60℃ for 1 min, extension at 60℃ for 1 min, for a total of 40 cycles. Each sample was repeated 3 times. The transcriptional levels of chTLR1a, chTLR1b, chTLR2a, chTLR2b, chTLR3, chTLR4, chTLR7, chTLR9, chTLR15, chTLR21, IFN-α, INF-β, IL-1β, IL-6, IL-8, and IL-15 were detected, with β-actin as the internal reference gene. Primer information is shown in Table 1.

[0127] Statistical analysis of the data: By substituting the Ct values ​​of the target gene β-actin obtained from the experimental group and the control group into 2... -ΔΔCt Relative transcription levels were calculated using a formula. Student t-tests were performed on the data using IBM SPSS Statistics 2.0 software. P < 0.05 indicated a significant difference, and P < 0.01 indicated an extremely significant difference. Graphs were generated using Prism8 software.

[0128] 2.5.1 Effect of FADV-4PX protein on the transcriptional level of Toll-like receptor mRNA in LMH cells

[0129] Test results are shown Figure 5The results showed that 48 h after transfection of LMH cells with the pEF1α-HA-pX recombinant plasmid, the expression levels of the 10 detected chTLR mRNAs were upregulated to varying degrees compared with the control group. The receptors with significantly upregulated expression were chTLR1b, chTLR2a, chTLR3, chTLR7, and chTLR15. chTLR2a showed the largest upregulation (6.70-fold, P<0.05), followed by chTLR1b (4.08-fold), chTLR3 (3.09-fold), chTLR7 (1.64-fold), and chTLR15 (1.15-fold). chTLR1a, chTLR2b, chTLR4, chTLR5, and chTLR21 were upregulated by 3.78, 5.57, 1.95, 2.24, and 1.19-fold, respectively, with no significant difference compared to the control group.

[0130] 2.5.2 Effects of FADV-4PX protein on the mRNA transcription levels of LMH cell effector factors

[0131] Compared with the control group, PX protein promoted the overall expression of interferon (IFN-α and IFN-β) and interleukins (IL-6, IL-8, IL-15, IL-1β) mRNA transcription levels, which were 2.90-fold, 2.77-fold, 2.54-fold, 3.3-fold, 1.87-fold, and 3.10-fold higher than those in the control group, respectively. Among them, the mRNA transcription levels of IFN-β, IL-8, and IL-15 showed significant differences compared with the control group (P<0.05).

[0132] Table 1 Primer sequence information

[0133]

[0134]

[0135] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for enhancing gene transcription in avian cells for non-disease treatment purposes, characterized in that, The method includes upregulating or enhancing or increasing the expression level of protein-coding genes or the activity or content of said proteins in the target avian cells to upregulate or enhance or increase the transcription of Toll-like receptor genes and / or immune effector gene genes in avian cells. The process of upregulating, enhancing, or increasing the expression level of protein-coding genes or the activity or content of the protein in the target avian cells involves introducing biological material into the target avian cells. The protein is any one of the following: B1) The amino acid sequence is the protein shown in sequence 2; B2) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1); The biomaterial is any one of the following: D1) The nucleic acid molecule encoding the protein; D2), an expression cassette containing the nucleic acid molecules described in D1); D3), ​​a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3); The avian cells described are LMH cells; The Toll-like receptor gene is one or more of chTLR1b, chTLR2a, chTLR3, chTLR7, and chTLR15; The immune effector factors are interferon and / or interleukin; The interferon is IFN-β; The interleukin is IL-8 and / or IL-15.

2. A method for preparing avian cells with high transcription levels of Toll-like receptor genes and / or immune effector gene genes for non-disease therapeutic purposes, characterized in that, This includes upregulating or enhancing or increasing the expression level of protein-coding genes or the activity and / or content of said proteins in target avian cells, to obtain avian cells with high transcription levels of Toll-like receptor genes and / or immune effector genes, wherein the transcription levels of Toll-like receptor genes and / or immune effector genes in the avian cells with high transcription levels of Toll-like receptor genes and / or immune effector genes are higher than those in the target avian cells. The process of upregulating, enhancing, or increasing the expression level of protein-coding genes or the activity or content of the protein in the target avian cells involves introducing biological material into the target avian cells. The protein is any one of the following: B1) The amino acid sequence of the protein is shown in sequence 2; B2) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1); The biomaterial is any one of the following: D1) The nucleic acid molecule encoding the protein; D2), an expression cassette containing the nucleic acid molecules described in D1); D3), ​​a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3); The avian cells described are LMH cells; The Toll-like receptor gene is one or more of chTLR1b, chTLR2a, chTLR3, chTLR7, and chTLR15; The immune effector factors are interferon and / or interleukin; The interferon is IFN-β; The interleukin is IL-8 and / or IL-15.

3. A method for cultivating avian tissues and / or organs and / or avian animals for non-disease treatment purposes, characterized in that, The method includes preparing avian cells with high Toll-like receptor gene and / or immune effector gene transcription levels using the method of claim 2, and then using the avian cells with high Toll-like receptor gene and / or immune effector gene transcription levels to cultivate avian tissues and / or avian organs and / or avians with high Toll-like receptor gene and / or immune effector gene transcription levels. The avian cells described are LMH cells; The Toll-like receptor gene is one or more of chTLR1b, chTLR2a, chTLR3, chTLR7, and chTLR15; The immune effector factors are one or more of interferon and interleukin; The interferon is IFN-β; The interleukin is IL-8 and / or IL-15.

Images