Porcine bone marrow macrophage, construction method therefor, and use thereof

By introducing pTERT, pCDK4 (R24C) and pCCND1 genes into pig bone marrow macrophages and introducing Cre genes into this cell line, immortalized cell lines without viral oncogene and screening tags were constructed, solving the problems of passage instability and exogenous gene risks of pig macrophage lines in the prior art, and achieving efficient gene deletion vaccine construction and vaccine safety improvement.

WO2025065908A1 Publication Date: 2025-04-03NANJING AGRICULTURAL UNIVERSITY
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Patent Information

Application Number
PCT/CN2023/138714
Authority / Receiving Office
WO · WO
Patent Type
Applications
Current Assignee / Owner
Priority Date
2023-09-25
Filing Date
2023-12-14
Publication Date
2025-04-03

AI Technical Summary

Technical Problem

It is difficult to establish a mature pig macrophage line with stable passage in the prior art, and there is a risk of exogenous genes and screening markers in vaccine production, affecting the safety and approval process of the vaccine.

Method used

Immortalized porcine bone marrow macrophage IPBM 27w7 was constructed by electroporation, and the Cre gene was introduced into this cell line to obtain a cell line that stably expresses Cre in order to remove screening markers in gene deletion recombinant viruses.

Benefits of technology

Immortalized pig bone marrow macrophage line without viral oncogene, viral vector gene and screening tags have the characteristics of stable passage and high-level expression of macrophage-specific surface molecules, simplifying the construction process of gene deletion vaccines and reducing the risk of vaccines.

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Abstract

The present invention relates to the technical field of biology, in particular to a porcine bone marrow macrophage, a construction method therefor, and the use thereof. The method comprises a method of performing electroporation on a primary porcine bone marrow macrophage to introduce pTERT, pCDK4(R24C) and pCCND1 genes into same so as to immortalize the porcine bone marrow macrophage; a method of continuously constructing, on the basis of the cell, a porcine bone marrow macrophage cell line stably expressing the Cre gene; and a method of removing a screening marker gene on a gene-deleted recombinant virus strain by means of using the cell line. The methods comprise: by means of electroporation, separately introducing into the primary porcine bone marrow macrophage transposon vectors and transposase vectors which respectively contain pTERT, pCDK4(R24C) and pCCND1, and screening out an immortalized porcine bone marrow macrophage; and continuing to perform electroporation on the immortalized porcine bone marrow macrophage to introduce the Cre gene so as to enable same to stably express a Cre protein. The present invention can be used for removing screening marker genes during the process of constructing gene-deleted recombinant virus strains.
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