Immortalized porcine macrophage cell strain as well as construction method and application thereof

A technology of macrophage and construction method, applied in the field of immortalized porcine macrophage cell line and its construction and application, to achieve the effect of avoiding the problem of vaccine quality difference, reliable and stable vaccine quality

Active Publication Date: 2021-10-22
JINYUBAOLING BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the immortalized porcine macrophages suitable for cultivating PRRSV disclosed in the above-mentioned literature 1 and literature 2 are not suitable for culturing ASFV, and there is no reliable, stable and continuously passaged immortalized cells suitable for culturing ASFV in vitro in the prior art. A report on pig macrophages

Method used

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  • Immortalized porcine macrophage cell strain as well as construction method and application thereof
  • Immortalized porcine macrophage cell strain as well as construction method and application thereof
  • Immortalized porcine macrophage cell strain as well as construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Construction of Immortalized Porcine Macrophage Cell Line

[0035] In this example, two kinds of immortalized genes (SV40LT gene and hTERT gene) were co-transfected into primary porcine bone marrow-derived macrophages by means of genetic engineering technology to obtain immortalized porcine macrophage cell lines, which specifically included the following steps.

[0036] 1.1. Isolation and culture of primary porcine bone marrow cells

[0037] (1) Take healthy pigs aged 30-45 days (purchased from Grassland Lixin Green Breeding Park Co., Ltd., Baotou City, Inner Mongolia). Bag packaging, low temperature storage;

[0038] (2) The periosteum was removed from the bone in the operating room, and the outer surface of the bone was rinsed and disinfected with PBS containing antibiotics (penicillin 100 U / mL, streptomycin 0.1 mg / mL, amphotericin B 0.25 μg / ml) in a sterile operating table , cut the ribs into small pieces of 3-4cm, cut off the leg bones from the middle, ...

Embodiment 2

[0061] Example 2: Screening of Immortalized Porcine Macrophage Cell Lines Suitable for Cultivating ASFV and Can Obtain High Virus Titers

[0062] In this embodiment, on the basis of the 8 immortalized porcine macrophage cell lines (pBMDM-1 to pBMDM-8) obtained in Example 1, an immortalized porcine macrophage that is suitable for culturing ASFV and can obtain high virus titers The cell line specifically includes the following steps.

[0063] 2.1. Construction of African swine fever virus deletion strain

[0064] (1) The basic strain used to construct the African swine fever virus deletion strain in this step is a Guangdong strain isolated in the field by the Poultry Disease Teaching and Research Office of the Veterinary College of South China Agricultural University. It was identified as African swine fever virus by sequencing and named ASFV GZ2018, The GenBank accession number of the strain's genome data is MT496893. The ASFV GZ2018 strain was transported to the biosafety le...

Embodiment 3

[0073] Example 3: Observation of cell morphology and analysis of cell surface markers of the immortalized porcine macrophage cell line pBMDM-JY05

[0074] In this example, the immortalized porcine macrophage cell line pBMDM-JY05, which is suitable for cultivating ASFV and can obtain high virus titer, was screened in Example 2 to observe cell morphology and analyze cell surface markers, specifically including the following steps.

[0075] 3.1. Observation of cell morphology

[0076] The immortalized porcine macrophage cell line pBMDM-JY05 obtained in Example 2 was inoculated into a T25 cell bottle, and when the confluence reached more than 80%, the cell morphology was observed microscopically, and the results were as follows: figure 1 Shown in A frame in middle; Simultaneously primary porcine bone marrow-derived macrophage (obtained in step 1.1 in embodiment 1) with 5 * 10 6 Cells / ml density inoculated into T25 cell flask, 10ml / flask, placed at 37±0.5°C, 5% CO 2 Cultivate ove...

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Abstract

The invention discloses an immortalized porcine macrophage cell strain as well as a construction method and an application thereof, and belongs to the technical field of bioengineering. The immortalized porcine macrophage cell strain provided by the invention is obtained by co-transfecting immortalized genes SV40LT and hTERT into primary porcine bone marrow-derived macrophages in a lentivirus infection manner, the provided immortalized porcine macrophage cell strain is suitable for culturing African swine fever virus, can obtain high virus titer, is reliable and stable, can be continuously passed, and can be effectively applied to immunological research in the aspect of resisting African swine fever virus infection and production of African swine fever virus vaccines.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and relates to an immortalized porcine macrophage cell line and its construction method and application, in particular to an immortalized porcine macrophage cell line suitable for cultivating African swine fever virus and its construction method and application , particularly relates to a reliable, stable and continuously passaged immortalized porcine macrophage cell line suitable for cultivating African swine fever virus and capable of obtaining high virus titer and its construction method and its anti-African swine fever virus infection in pigs Immunological research and application in African swine fever virus vaccine production. Background technique [0002] African swine fever virus (ASFV) is the only African virus genus in the African virus family, and it is also the only DNA arbovirus genus. The virus is mainly infected by direct contact with the mouth or nasal cavity, and has multi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0786C12N15/867A61K39/12A61P31/20
CPCC12N5/0645C12N15/86C07K14/005A61K39/12A61P31/20C12N2510/04C12N2740/15022C12N2740/15043C12N2710/12034A61K2039/5252A61K2039/5254A61K2039/552Y02A50/30
Inventor 宋庆庆武瑾贤叶正琴屠颉张健陈坚刘建奇徐丽媛俎红丽赵丽霞张竞张翀宇
Owner JINYUBAOLING BIO PHARMA CO LTD
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