27 results about "Fibroblast cell line" patented technology

The present invention relates to a method for constructing an eight-gene-edited xenogeneic organ transplantation donor pig, belonging to the field of animal biotechnology. In the wild-type porcine fetal fibroblast cell line, the GGTA1, β4GalNT2, and CMAH genes are knocked out by using the CRISPR / Cas9 gene editing technology, and the humanized genes of hCD39, hCD46, hCD55, hCD59, and hTBM are transfected. Combining with somatic cell cloning technology, GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene-edited cloned pigs are constructed. Further, through genotype, mRNA, protein expression identification and functional analysis, eight-gene-edited xenogeneic organ transplantation donor pigs are obtained. The present invention solves the technical problems of high production difficulty, low efficiency, and low survival rate of donor pigs for multi-gene-edited xenogeneic organ transplantation, and maximally solves the common problems of immune rejection reaction and complement dysregulation faced during xenogeneic organ transplantation, laying a foundation for more targeted development of donor pigs suitable for different tissue and organ xenotransplantation, and having important value for promoting the clinical transformation of xenogeneic organ transplantation.

The invention discloses an immortalized human pelvic kidney cancer related fibroblast line RPCcaf-X1 and application thereof, and belongs to the field of microbial animal cell lines. The immortalized human pelvic kidney cancer related fibroblast line is named as RPCcaf-X1, and the preservation number of the immortalized human pelvic kidney cancer related fibroblast line is CCTCC (China Center For Type Culture Collection) NO: C2025306. The invention also discloses the application of the immortalized human pelvis and kidney cancer related fibroblast line RPCcaf-X1 in the research of the pathogenic mechanism of the pelvis and kidney cancer. The invention further discloses the application of the immortalized human pelvic kidney cancer related fibroblast line RPCcaf-X1 in tumor microenvironment research. The invention further discloses the application of the immortalized human pelvic kidney cancer related fibroblast line RPCcaf-X1 in drug screening or research and development. The invention further discloses the application of the immortalized human pelvis and kidney cancer related fibroblast line RPCcaf-X1 in screening of potential biomarkers related to pelvis and kidney cancer.

The present invention discloses a porcine fibroblast cell line capable of long-term passage and its application. The present invention uses porcine fibroblast tissue as a source, obtains porcine fibroblasts from different parts through tissue block separation, performs long-term in vitro passage, and induces the cells to break through the Hayflick limit. Further, monoclonal screening is performed through limiting dilution to obtain a single-source porcine fibroblast cell line. After screening, porcine fibroblasts from two parts, ear margin fibroblasts and hind leg fibroblasts, can be long-term passaged in vitro and have the ability to secrete collagen. These cells have been deposited with the China Center for Type Culture Collection under the accession numbers CCTCC NO: C202511 and CCTCC NO: C202512. This cell line has broken through the Hayflick limit and has been passaged to 42 generations. The cells exhibit a healthy, plump, long spindle shape. Long-term passage does not affect proliferation levels, and the cell line can be used in the preparation of cell-cultured meat.

This invention relates to the interdisciplinary field of cell biology, experimental oncology, and translational medicine, specifically to a cancer-associated fibroblast cell line, its construction method, and its applications. This invention fills the research gap in mouse-derived oral squamous cell carcinoma-associated fibroblast cell line models and provides a method for constructing such a cell line. Through the inverted adherent culture and enzymatic digestion methods of this invention, a mouse-derived oral squamous cell carcinoma-associated fibroblast cell line with a clear origin, high purity, and long-term phenotypic and functional stability can be obtained. This allows for the establishment of a cryopreservable and resuscitable mouse oral squamous cell carcinoma-associated fibroblast cell bank, providing technical support for research related to the tumor microenvironment and immunotherapy.

The present application relates to a CRISPR-mediated immortalized sheep fetal skin fibroblast cell line in the field of variation or genetic engineering and application thereof.The construction of the CRISPR-mediated immortalized sheep fetal skin fibroblast cell line of the present application comprises the following steps: step one, constructing a Cas9 site-directed knockout plasmid containing an FGF5 gene target sequence; step two, constructing a homologous repair plasmid Donor-2 kb HA-SV40LT containing an FGF5 gene homologous arm; step three, constructing a donor DNA containing an SV40LT protein expression frame; and step four, preparing a sheep fetal fibroblast cell line stably expressing SV40LT protein.The immortalized SFF-FGF5 / SV40LT monoclonal cell strain created by the present application can become an ideal tool cell line for genome editing researches such as sheep CRISPR library screening, multiple gene editing and mutation gene function exploration.

The invention discloses a rumen fibroblast line as well as a construction method, a culture method and application thereof, the rumen fibroblast line G8 is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: C2025332, the rumen fibroblast line G8 has a typical fibroblast-like form and can be stably passaged, and the rumen fibroblast line G8 can be used as a rumen fibroblast cell line. The cell culture medium also has the characteristic of highly expressing fibroblast marker genes and the function of tolerating and utilizing rumen core metabolite butyric acid, and the proliferation rate and metabolic activity of the cell culture medium under the condition that 15mmol / L butyric acid is used as a main energy source are obviously superior to those of glucose culture; according to the construction method of the rumen fibroblast line, the steps of function screening and molecular identification are introduced, so that the identity authenticity of the constructed cell line can be ensured; the culture method of the rumen fibroblast line is simple and effective, culture medium materials are easy to obtain, and culture conditions are easy to realize; the rumen fibroblast line can be used for rumen in-vitro research, and is beneficial to clarification of a mechanism for regulating rumen interstitial metabolism and tissue repair by butyric acid.

An sgRNA targeting a porcine PLIN1 gene and use thereof. The sgRNA action site is located on the second exon and the fourth exon of the porcine PLIN1 gene. Provided is a method for knocking out a PLIN1 gene in a porcine fetal fibroblast cell line by using a CRISPR / CAS9 system. The method comprises designing and synthesizing an sgRNA targeting a porcine PLIN1 gene according to a porcine PLIN1 gene sequence, then constructing a CRISPR / Cas9 targeting vector containing the sgRNA, and transferring same into a porcine fetal fibroblast cell to obtain a PLIN1 gene-knocked-out porcine fetal fibroblast cell. The cell can be used for the preparation of a PLIN1 gene-edited porcine, and is not only of great significance for porcine breeding, but also of great reference value for revealing the occurrence mechanism of human hereditary lipodystrophy and seeking a treatment method.

The invention relates to a CRISPR (clustered regularly interspaced short palindromic repeats)-mediated immortalized sheep fetal skin fibroblast line and application thereof in the field of variation or genetic engineering. The construction of the CRISPR-mediated immortalized sheep fetal skin fibroblast line comprises the following steps: step 1, constructing a Cas9 site-directed knockout plasmid containing an FGF5 gene target sequence; step 2, constructing a homologous repair plasmid Donor-2 kb HA-SV40LT containing a homologous arm of the FGF5 gene; step 3, constructing a donor DNA (Deoxyribose Nucleic Acid) containing an SV40LT protein expression cassette; and step 4, preparing the sheep fetal fibroblast line capable of stably expressing the SV40LT protein at a fixed point. The immortalized SFF-FGF5 / SV40LT monoclonal cell strain created by the invention can become an ideal tool cell line for genome editing research such as sheep CRISPR library screening, multiple gene editing, mutant gene function exploration and the like.

The invention discloses a human immortalized fallopian tube fibroblast strain as well as a construction method and application thereof, and belongs to the technical field of cell immortalization construction. The preparation method comprises the following steps: firstly, separating, culturing and purifying fibroblasts from human fallopian tube tissues by virtue of an enzyme digestion method, identifying markers of the fibroblasts, then introducing a simian virus 40 large T antigen gene into primary fibroblasts by virtue of a lentiviral vector, and screening by virtue of puromycin to obtain a cell strain capable of stably expressing SV40T. Finally, the human immortalized fallopian tube fibroblast which can be stably passaged is constructed. The cell strain has the characteristics of strong multiplication capacity and high hereditary stability, and the cells are not obviously aged along with the increase of the number of generations of the cells, which indicates that the human immortalized fallopian tube fibroblast line is successfully established. According to the invention, the problem that the in-vitro culture life of primary fibroblasts is limited is solved, and a reliable in-vitro cell model is provided for research in the fields of fallopian tube related diseases, ovarian cancer origin, tumor microenvironment and the like.

The invention provides a sheep embryo fibroblast line and application thereof, and belongs to the technical field of cell biology. The name of the sheep embryo fibroblast line is sheep embryo fibroblast OEF, the sheep embryo fibroblast line is preserved in China Center for Type Culture Collection, the address is Wuhan University, Wuhan, China, the preservation date is January 26, 2022, and the preservation number is CCTCC NO: C202212. The sheep embryo fibroblast line can be used for separation and culture of bovine and sheep pox viridae viruses such as LSDV, GTPV, SPPV and ORFV, has the characteristics of short proliferation time and high virus titer, and can avoid the risk that other viruses are polluted by viruses generated by primary cell separation.

The invention belongs to the field of cell biology, and relates to a shelduck fibroblast cell line which is finally obtained by performing primary culture, screening and optimized subculture on shelduck embryos serving as materials. The fibroblasts cultured by the method are slender, fusiform, triangular and irregular, and can be continuously subcultured for at least 82 generations; the shelduck fibroblast cell line is used for culturing waterfowl viruses such as duck plague virus, novel duck reovirus, duck tembusu virus, duck parvovirus and the like for the first time; compared with the existing duck-derived fibroblast culture, the duck-derived fibroblast culture medium has the advantages of high virus content and wide application range. The shelduck fibroblast cell line provided by the invention can provide a large amount of high-quality materials for scientific researches of cell engineering, molecular biology, immunology and the like; the method is used for culturing, separating and identifying poultry viruses, provides raw materials for development and production of poultry attenuated vaccines and inactivated vaccines, can shorten the production period and reduce the production cost, and has certain practical significance for large-scale production of vaccines.

The invention relates to a method for constructing a porcine endogenous retrovirus inactivated xenotransplantation donor pig, and belongs to the technical field of xenotransplantation. And the copy number of the PERVs and the genotype of the PERV-pol are determined through ddPCR, Sanger sequencing and second-generation PCR deep sequencing. The method comprises the following steps: designing and synthesizing a porcine PERV-pol gene targeting sequence based on a CRISPR / Cas9 RNP system, co-transfecting the porcine PERV-pol gene targeting sequence and spCas9 protein into a porcine fetal fibroblast line, screening to obtain positive cells, carrying out somatic cell cloning and embryo transplantation, taking out a fetus during pregnancy, identifying the PERV-pol knockout condition, and if the PERV-pol knockout condition is completely carried out, carrying out somatic cell cloning and embryo transplantation; and if the PERV-pol gene is not completely knocked out, secondarily designing and synthesizing an sgRNA targeting sequence, transfecting the sgRNA targeting sequence into the fetal fibroblast cell line, screening to obtain a cell line in which the PERV-pol gene is completely knocked out, and carrying out somatic cell cloning and embryo transplantation to obtain the PERVs inactivated xenotransplantation donor pig. The problem that the PERVs inactivation efficiency is low is solved, and the PERVs-inactivated xenotransplantation donor pig can be obtained through one round or multiple rounds.

The present invention relates to extracellular vesicles derived from hematopoietic stem cells (HSCs) or natural killer cells (NK cells) differentiated from induced pluripotent stem cells (iPSCs), a method for producing same, and use thereof. Exosomes derived from HSCs differentiated from iPSCs, according to the present invention, promote the growth of human vascular endothelial cells, and exhibited the effect of promoting wound healing in a wound healing assay performed in human fibroblast cell lines. It was also confirmed that exosomes derived from NK cells differentiated from iPSCs include specific miRNA. The exosomes not only exhibited anti-inflammatory activity in macrophages, but also inhibited the gene expression of fibrosis marker proteins (a-SMA, COL1A1) in hepatic stellate cells. Since the exosomes derived from HSCs or NK cells differentiated from iPSCs, according to the present invention, are obtained from single iPSCs, there is no inconvenience of collecting tissues or blood from donors. In addition, the exosomes have uniform properties and can be mass-produced. Therefore, the extracellular vesicles according to the present invention can be used to treat various diseases such as infectious diseases, fibrosis, cancer, neurological diseases, autoimmune diseases, and skin diseases.

The invention relates to the technical field of molecular biology and genetic engineering, and discloses sgRNA targeting a chicken CD30 gene and a combination and application thereof. The sgRNA and the sgRNA combination provided by the invention perform targeted knockout on the seventh exon region (transmembrane region) of the chicken CD30 gene, and the sgRNA knockout target of the chicken CD30 is transfected into a chicken fibroblast line DF-1 to prepare the CD30 knockout DF-1 cell, namely the CD30 / -DF-1 cell, and compared with a control group cell through detection, the CD30 expression quantity in the CD30 / -DF-1 cell is extremely low, and the result shows that the CD30 is successfully edited. The establishment of the CD30- / -DF-1 cell line provides an experimental material for deeply researching the functions of CD30.

The invention relates to a CRISPR (clustered regularly interspaced short palindromic repeats)-mediated red fluorescence labeled sheep fibroblast line and application thereof in the field of variation or genetic engineering. The construction and the application of the CRISPR-mediated red fluorescence labeled sheep fibroblast line disclosed by the invention comprise the following steps: step 1, constructing a Cas9 site-directed knockout plasmid containing an MSTN gene target sequence; step 2, constructing a plasmid Donor-1 kb HA-mCherry containing a homologous arm of the MSTN (myostatin) gene; step 3, constructing a donor DNA containing a fluorescent protein mCherry expression cassette; and step 4, co-transfecting the donor DNA containing the fluorescent protein mCherry expression cassette and the fixed-point knockout plasmid in the step 1 into the sheep fibroblast cell line, so as to obtain the sheep fibroblast cell line marked by red fluorescence.

The invention relates to an immortalized muscovy duck embryo fibroblast line and an establishment method and application thereof.The immortalized muscovy duck embryo fibroblast line is immortalized muscovy duck embryo fibroblasts iMDEF / SV40-LT, and the establishment method of the cell line comprises the steps that cloned SV40LT genes are inserted into lentivirus plasmids through homologous recombination, and the lentivirus plasmids are subjected to homologous recombination to obtain the immortalized muscovy duck embryo fibroblast line. According to the present invention, the muscovy duck embryo fibroblasts are transfected by using lentivirus, such that the muscovy duck embryo fibroblasts stably express SV40LT, and puromycin screening is performed to obtain the immortalized muscovy duck embryo fibroblasts, the immortalized muscovy duck embryo fibroblasts (iMDEF / SV40-LT) are successfully constructed by using the SV40LT gene, the immortalized muscovy duck embryo fibroblasts stably grow for more than 50 generations, the fibroblast morphology is maintained, and the muscovy duck embryo fibroblasts can be obtained; the method is free of endogenous viruses, good in safety and free of teratogenesis and senescence signs, and can be used for virus culture and the like.

The copy number of the ten-gene edited porcine endogenous retroviruses (PERVs) in a whole genome is determined by ddPCR, and the PERV-Pol genotype is determined through second-generation whole genome deep sequencing. The method comprises the following steps: constructing a porcine PERV-Pol gene knockout targeting vector, co-transfecting the vector into a ten-gene edited porcine fetal fibroblast line, screening to obtain a PERV-Pol knockout fibroblast line, carrying out somatic cell nuclear transfer and embryo transfer, taking out a fetus during pregnancy, and identifying PERV-Pol gene knockout and PERVs inactivation conditions of the fetus. Furthermore, the continuous cloning technology is used for batch production of the PERVs completely inactivated eleven-gene edited xenotransplantation donor pigs. The technical problems that the PERV-Pol gene, especially the multi-copy gene, is high in editing difficulty, low in efficiency and long in period are solved, and the method has important significance on overcoming the problems of biosafety and immunological rejection in the xenogeneic organ transplantation process.

The invention provides a proliferation culture medium for long-term stable passage of bovine-derived cells, a method for promoting long-term stable passage of bovine-derived cells and application of the proliferation culture medium. The proliferation culture medium comprises the following components: a complete culture medium, cycloastragenol and metformin hydrochloride. Researches show that the proliferation culture medium can make bovine embryo fibroblasts (BFFs) break through the proliferation limit in the in-vitro subculture process, a continuous cell line is formed, and production of cell culture meat products with excellent quality is facilitated. The culture medium provided by the invention can support continuous passage of BFFs to 145 generations, and the cell type of the bovine embryo fibroblast cell line subjected to passage to 145 generations is unchanged, so that the culture medium has the advantage of long-term stable passage. Through induction of the proliferation culture medium, the BFFs can break through aging limitation, long-term stable passage (more than or equal to 100 generations) is realized, the waveform protein positive rate of more than 90% is maintained, the requirement of mass production of cell culture meat on the cell number is met, and the proliferation culture medium has a very wide application prospect.

This invention discloses an anti-Vibrio polypeptide, Lvvibriocin-GK, and its applications, the amino acid sequence of which is shown in SEQ ID NO. 01. This invention is a positively charged cationic, highly effective anti-Vibrio polypeptide that exhibits significant antibacterial effects against common aquatic pathogens such as Vibrio. Furthermore, Lvvibriocin-GK showed no cytotoxic effects on zebrafish embryonic fibroblast cell line (ZF4) and human kidney epithelial cell line (HEK-293T); its antibacterial activity is unaffected by high temperature, salt ion concentration, or pH changes, demonstrating good environmental stability.

The present invention discloses a head and neck squamous cell carcinoma-related fibroblast and organoid co-culture system and its construction method and application, which belongs to the field of tumor biology. The construction method of the co-culture system is to enzymatically digest the head and neck squamous cell carcinoma tissue, culture it into a fibroblast cell line and organoid tissue respectively, and then use organoid culture medium for co-culture. The organoid cells obtained by co-culture of the present invention have a 3-5-fold increase in stemness and a 5-7-fold increase in drug resistance. Moreover, the method is simple to operate, takes little time, and can complete cell separation and organoid culture within 1 hour. The present invention provides a basis for establishing an organoid library, conducting drug screening, and promoting clinical transformation.

This invention belongs to the field of animal cell culture technology, specifically relating to chicken embryo fibroblast cell lines, their culture media, acclimatization methods, and applications. This application provides a chicken embryo fibroblast cell line derived from… Gallus gallus The chicken embryo fibroblast suspension cell line CEF-P17 3D is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C202661 and deposit date of March 31, 2026. This application also provides a culture medium for culturing the chicken embryo fibroblast cell line and a method for acclimatizing the chicken embryo fibroblast cell line, which shortens the acclimatization period, simplifies the operation, and is suitable for large-scale culture. This application also provides the application of the culture medium for the chicken embryo fibroblast cell line. Furthermore, this application also provides the application of the chicken embryo fibroblast cell line in the preparation of cultured meat.

The present invention relates to a CRISPR-mediated red fluorescently labeled sheep fibroblast cell line and its application in the field of mutation or genetic engineering. The construction and application of the CRISPR-mediated red fluorescently labeled sheep fibroblast cell line of the present invention include the following steps: step 1, constructing a Cas9 site-directed knockout plasmid containing the MSTN gene target sequence; step 2, constructing a plasmid Donor-1 kb HA-mCherry containing the MSTN gene homology arm; step 3, constructing a donor DNA containing a fluorescent protein mCherry expression cassette; step 4, co-transfecting the donor DNA containing the fluorescent protein mCherry expression cassette and the site-directed knockout plasmid described in step 1 into the sheep fibroblast cell line to obtain a red fluorescently labeled sheep fibroblast cell line.

The copy number of the eight-gene edited PERVs in a genome is measured through ddPCR, and the PERV-Pol genotype is determined by utilizing second-generation whole genome deep sequencing. The method comprises the following steps: constructing a porcine PERV-Pol gene knockout targeting vector, jointly transfecting the vector into an eight-gene edited porcine fetal fibroblast line, screening to obtain a PERV-Pol knockout positive fibroblast line, carrying out somatic cell nuclear transfer and embryo transfer, taking out a fetus after pregnancy, and identifying PERV-Pol gene knockout and PERVs inactivation conditions of the fetus. And designing and constructing a targeting sgRNA expression vector again according to the knockout condition, and transfecting the targeting sgRNA expression vector into the PERV-Pol gene knockout fetal fibroblast line until the nine-gene edited pig fibroblast line with completely inactivated PERVs is obtained through screening for somatic cell nuclear transplantation, thereby finally obtaining the nine-gene edited xenotransplantation donor pig with completely inactivated PERVs. According to the invention, the technical problems of high difficulty and low efficiency of editing the endogenous retrovirus, especially the multi-copy gene, are solved, and the problems of biological safety and immunological rejection in the xenogenic organ transplantation process are solved.

The invention provides a preparation method and application of an in-vivo CAR-T cell for treating interstitial lung disease, and provides a T cell of a chimeric antigen receptor which is modified by genetic engineering and is used for expressing targeted fibroblast activating protein. A targeted fibroblast activation protein (FAP) CAR-T cell therapy model is constructed by targeting CD5 entrapped mRNA nano-liposome (LNP) transfection and lentiviral vector, and the method comprises the following steps: firstly, verifying the difference of the killing ability of FAP CAR-T constructed by CD5 LNP-mRNA and lentivirus in a 293T cell which stably co-expresses FAP, Luciferase and mCherry, and then verifying the difference of the killing ability of FAP CAR-T constructed by LNP-mRNA and lentivirus in the 293T cell which stably co-expresses FAP, Luciferase and mCherry; further verifying the effectiveness of the CAR-T cells constructed by transfecting the CD5 LNP-mRNA on cell lines of human fibroblasts (CDD19Lu, LL29 and LL97A) and primary fibroblasts of human and mice in vitro, and further verifying the effectiveness and safety of the CD5 LNP-FAP CAR-T in treatment of pulmonary fibrosis through in-vivo experiments in animals. A novel and effective anti-fibrosis treatment thought is provided for patients with fibrosis interstitial lung diseases.

This application provides a method for establishing and applying mouse gastric squamous cell carcinoma cell lines and gastric cancer-related fibroblast cell lines. The culture medium for culturing these cell lines comprises L-WRN cell culture supernatant, and 100×N-2 additive (0.5 vol%–1.5 vol%), 50× vitamin A-free B-27 additive (1.5 vol%–2.5 vol%), N-acetylcysteine ​​amide (1 mM–1.5 mM), nicotinamide (8 mM–12 mM), FGF-10 (80 ng / mL–120 ng / mL), EGF (40 ng / mL–60 ng / mL), A83-01 (0.8 μM–1.2 μM), Y-27632 dihydrochloride (8 μM–12 μM), Glutamax additive (0.5 vol%–1.5 vol%), and Matrigel. The culture medium provided in this application contains 5 vol%–7 vol% Hepes buffer (8 mM–12 mM) and gastrin (0.8 nM–1.2 nM). This medium can significantly improve the success rate of in vitro culture of primary cell lines. The mouse gastric squamous cell carcinoma cell lines and gastric cancer-related fibroblast cell lines obtained from this medium are genetically stable and can be used to construct ectopic or orthotopic implantation models in immune-active mice.

The present application relates to a method for constructing a pig endogenous retrovirus inactivated xenotransplant donor pig, and belongs to the technical field of xenogenic organ transplantation. The copy number and PERV-pol genotype of PERVs are determined by ddPCR, Sanger sequencing and second-generation sequencing. Based on the RNP system of CRISPR / Cas9, the pig PERV-pol gene targeting sequence is designed and synthesized, and is co-transfected into a pig fetal fibroblast cell line with spCas9 protein, positive cells are screened, somatic cell cloning and embryo transfer are carried out, and the PERV-pol knockout condition of the fetus is identified when pregnancy occurs. If not completely knocked out, the sgRNA targeting sequence needs to be designed and synthesized again, and the cell line with completely knocked out PERV-pol gene is screened. If completely knocked out, somatic cell cloning and embryo transfer are directly carried out; and the xenotransplant donor pig with inactivated PERVs is obtained.

The invention belongs to the technical field of genetic engineering of animal molecular breeding, and particularly relates to application of chicken FNDC3A in resisting H9N2 subtype avian influenza virus (AIV) infection. It is found that after H9N2 AIV infection, the expression quantity of the FNDC3A gene in a chick embryo fibroblast line (DF-1) is remarkably reduced. The replication of the H9N2 AIV in host cells can be remarkably inhibited by silencing the FNDC3A through an RNA interference technology, which indicates that the FNDC3A gene is a susceptible gene of the H9N2 AIV, and the resistance of a host to the H9N2 AIV can be remarkably improved by knocking down or knocking out the FNDC3A gene. The FNDC3A gene provided by the invention can be used as a target for resisting replication of the H9N2 subtype avian influenza virus, can be used as a disease-resistant molecular marker, is used for carrying out assistant breeding on new varieties of the H9N2 subtype avian influenza virus, and has important application value in genetic assistant disease-resistant breeding.