CN-121975804-A - SgRNA of targeted chicken CD30 gene and combination and application thereof

Abstract

The invention relates to the technical fields of molecular biology and genetic engineering, and discloses sgRNA of a targeted chicken CD30 gene, and a combination and application thereof. The sgRNA and sgRNA combined targeting knockout chicken CD30 gene seventh exon region (transmembrane region) provided by the invention is transfected into chicken fibroblast line DF-1 aiming at the sgRNA knockout target of chicken CD30 to prepare CD30 knockout DF-1 cells, namely CD30 ‑/‑ DF-1 cells, and compared with cells in a detection group and a control group, the result shows that the CD30 editing is successful, and the expression level of the CD30 in the CD30 ‑/‑ DF-1 cells is extremely low. The establishment of the CD30 ‑/‑ DF-1 cell line provides experimental material for the intensive study of CD30 function.

Inventors

• LIAN LING
• Du Yushuang
• LI JUAN
• ZHANG JIANNAN

Assignees

• 中国农业大学

Dates

Publication Date

20260505

Application Date

20260106

Claims (9)

1. 1. An sgRNA targeting the chicken CD30 gene, characterized in that it is sgRNA1 or sgRNA2; The nucleotide sequence of the sgRNA1 acting site is 5'-AGAGACCAAGAGAAGGCAGA-3'; The nucleotide sequence of the sgRNA2 acting site is 5'-AGGTCTTGTTTTCTGGGCAG-3'.
2. 2. A sgRNA combination targeting the chicken CD30 gene, characterized by consisting of sgRNA1 and sgRNA 2; the sgrnas 1 and 2 are identical to the sgrnas 1 and 2 described in claim 1.
3. 3. A nucleic acid construct comprising a nucleic acid sequence encoding the sgRNA of claim 1 or the combination of sgrnas of claim 2.
4. 4. The nucleic acid construct of claim 3, further comprising a nucleic acid sequence encoding a Cas protein.
5. 5. A biological material comprising the sgRNA of claim 1 or the combination of sgrnas of claim 2, or the nucleic acid construct of claim 3 or 4; the biological material is an expression cassette, a transposon, a plasmid vector, a viral vector, engineering bacteria or a transgenic cell line.
6. 6. A host cell comprising the sgRNA of claim 1 or the combination of sgrnas of claim 2, or comprising the nucleic acid construct of claim 3 or 4; The host cell is a chicken-derived cell, preferably a chicken fibroblast line DF-1 cell.
7. 7. A method for editing a chicken CD30 gene in vitro, comprising introducing the sgrnas of claim 1 or the combination of the sgrnas of claim 2, or the nucleic acid construct of claim 3 or 4 into a cell; The editing results in a decrease in the expression level or functional activity of the CD30 protein encoded by the chicken CD30 gene.
8. 8. Use of the sgRNA of claim 1 or the combination of sgrnas of claim 2, or the nucleic acid construct of claim 3 or 4, or the biological material of claim 5 or the host cell of claim 6 for the preparation of a cell model or an animal model for studying chicken CD30 gene function.
9. 9. A kit for editing chicken CD30 gene, characterized in that it comprises the sgRNA of claim 1 or the combination of sgrnas of claim 2, or the nucleic acid construct of claim 3 or 4.

Description

SgRNA of targeted chicken CD30 gene and combination and application thereof Technical Field The invention relates to the technical fields of molecular biology and genetic engineering, in particular to sgRNA of a targeted chicken CD30 gene, and a combination and application thereof. Background Chicken Marek's Disease (MD) is an infectious and neoplastic disease of poultry caused by Marek's Disease Virus (MDV), a family of herpesviridae, which has important economic and scientific value, and which causes economic losses of 10-20 billion dollars worldwide each year, and is used as an important model for researching virus-induced lymphomas. MD can cause proliferation of lymphoid cells in chickens, and produces T cell lymphomas, chicken Marek's disease lymphomas are natural animal models of lymphomas with high expression of CD30 genes, and CD30 high expression is a marker of lymphocyte tumor transformation. CD30 is a member 8 of the tumor necrosis factor receptor superfamily, namely TNFRSF8, whose encoded protein is a transmembrane glycoprotein, and is involved in immunomodulation and cell signaling, playing a key role in immunomodulation and tumorigenesis. CD30 is expressed primarily in activated T cells, B cells and Natural Killer (NK) cells, and activates downstream signaling pathways, such as NF- κ B, MAPK pathway, through the ligand CD30L (CD 153, TNFSF 8), thereby regulating cell proliferation, survival or apoptosis. While CD30 in normal lymphocytes can promote cell proliferation and induce cell death, expression of CD30 in malignant lymphoma cells exhibits unidirectional regulation of proliferation, which may be closely related to cell canceration. The protooncogene Meq of chicken marek's virus (MDV) is the most important tumorigenic gene of MDV, and forms homo-or heterodimers with other transcription factors, such as c-JUN, c-FOS, etc., playing a key role in MDV-induced T cell lymphogenesis. The Meq and the CD30 expression are positively correlated, and cells with high expression of the CD30 in lymphoma can simultaneously and highly express the Meq gene, so that after MDV infection of MD-resistant chickens, CD 30-specific antibodies can be generated, which suggests that the CD30 possibly participates in the immune escape process of tumors and promotes virus latency and tumorigenesis. To further clarify the role of CD30 in the development of chicken marek's disease, it is highly desirable to establish a highly efficient CD30 gene knockout cell model, thereby studying the role of CD30 in chicken marek's disease development, tumor transformation. Disclosure of Invention The invention aims to provide sgRNA of targeted chicken CD30 gene and a combination and application thereof. The CD30 gene is a tumor necrosis factor receptor superfamily member, is an important target point in lymphoma treatment, is highly expressed in classical Hodgkin lymphoma cells, and is a core index of pathological diagnosis. CD30 plays an important role in the biological function of normal and malignant lymphocytes, but its specific mechanism in lymphopoiesis has not been fully understood. Chicken Marek's Disease (MD) is an infectious, neoplastic disease of poultry and is used as an important model for studying virus-induced lymphoma production. Chicken Marek's disease lymphoma is a natural animal model of lymphoma with high expression of CD30 gene, marek's disease virus protooncogene Meq can significantly promote CD30 transcription level, and high expression of CD30 plays an important role in maintaining growth and proliferation of tumor cells. Therefore, the invention provides an sgRNA combination for targeted knockout of a seventh exon region (transmembrane region) of a chicken CD30 gene, wherein the nucleotide sequence of the sgRNA is shown as SEQ ID NO. 1 and SEQ ID NO. 2. The sgRNA knockout target of chicken CD30 is transfected into chicken fibroblast line DF-1 to prepare CD30 knockout DF-1 cells, namely CD30 -/- DF-1 cells, and the CD30 expression level in the CD30 -/- DF-1 cells is extremely low compared with that in the control group cells through detection, so that the result shows that the CD30 editing is successful. The establishment of the CD30 -/- DF-1 cell line provides experimental material for the intensive study of CD30 function. To achieve the object of the present invention, in a first aspect, the present invention provides an sgRNA targeting the chicken CD30 gene, which is sgRNA1 or sgRNA2; The nucleotide sequence of the acting site of the sgRNA1 is 5 '-AGAGACCAAGAGAAGGCAGA (SEQ ID NO: 1) - (AGG) -3', wherein AGG is a PAM sequence. The nucleotide sequence of the sgRNA2 acting site is 5 '-AGGTCTTGTTTTCTGGGCAG (SEQ ID NO: 2) - (TGG) -3', wherein TGG is PAM sequence. In a second aspect, the invention provides a sgRNA combination targeting the chicken CD30 gene, consisting of said sgRNA1 and sgRNA 2. In a third aspect, the invention provides a nucleic acid construct comprising a nucleic acid sequence encoding said sg