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Construction and application of immortalized primary generation sheep lung cell line

A technology of immortalization and cell lines, which is applied in the construction of immortalized primary sheep lung cell lines and its application fields, can solve the problems that hinder the research of virus molecular biology and vaccine development, and the slow progress of research work, and achieve stable transformation. Staining and cytotoxic effect

Inactive Publication Date: 2017-11-24
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Passage cell lines are an important platform for the application and basic research of animal viruses. However, many animal viruses lack host cell lines that can support their stable proliferation, such as Peste des petits ruminants virus and rabbit plague virus, which seriously hinders the molecular biology of these viruses Research and Vaccine Development Efforts
However, the limited number of passages of some primary cells makes the progress of research work slow. At present, the immortalized cell lines successfully constructed by using telomerase include: bovine thyroid cell line, CCTCC preservation number C2014109; skin fibroblast cell line, application publication number It is CN 101921729 A; Immortalized cell line of pig-derived cells, application publication number CN 103923942 A

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  • Construction and application of immortalized primary generation sheep lung cell line
  • Construction and application of immortalized primary generation sheep lung cell line
  • Construction and application of immortalized primary generation sheep lung cell line

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specific Embodiment

[0035] The construction of the immortalized primary sheep lung cell line of the present invention comprises the following steps: using the Dispase-collagenase perfusion method to obtain fresh primary sheep lung fibroblasts with a high activity rate, and after 24 hours of culturing, in the concentration of 5ug / ml In the presence of polybrene (polybrene), the concentrated and purified cell supernatant of recombinant lentivirus containing hTERT gene was used to infect primary sheep lung fibroblasts, and after 1 week, 2ug / ml puromycin (puromycin) was used Carry out drug pressure screening, in the presence of polybrene with a concentration of 5ug / ml, repeat infection once, a week later, culture with 1ug / ml puromycin maintenance solution until the monoclonal appears and grows to about 2.0cm, pick the cells of the monoclonal to expand Cultured to obtain immortalized sheep lung fibroblasts capable of passage. Specific examples are as follows:

Embodiment

[0037] This embodiment relates to the construction and application of an immortalized primary sheep lung cell line, specifically as follows:

[0038] 1. Materials and methods

[0039] 1.1 Materials

[0040] 1.11 Strains, plasmids and sheep lung cells

[0041]The pCI-Neo-hTERT vector, recombinant plasmid pLOV-GFP-3Flag, ps PAX2, pMD2.G and other lentiviral packaging vectors are preserved by our laboratory. DH5a competent, 293T cells, primary sheep lung cells are preserved in our laboratory;

[0042] 1.12 Main reagents and instruments

[0043] rTaq DNA polymerase, LA Taq polymerase, infusion ligase, BamI, NotI and other restriction enzymes, genome extraction kit and Reverse Transcriptase M-MLV kit were purchased from Bioengineering Dalian Co., Ltd.; DMEM medium, MEM Culture medium and 1×PBS were purchased from Hyclone Company; fetal bovine serum (FBS) was purchased from Gibco Biological Company; calcium transfection reagents were self-prepared (CaCl2 and Heps reagents were p...

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Abstract

The invention discloses construction and application of an immortalized primary generation sheep lung cell line. Construction comprises the following steps that fresh primary generation sheep lung fibroblasts with the high viability are obtained through a Dispase-collagenase perfusion method; after being cultured for 24 h, under the condition that polybrene with the concentration being 5 ug / ml exists, the primary generation sheep lung fibroblasts are infected through concentrated and purified cell supernatant of recombinant lentivirus containing hTERT genes; after one week, puromycin with the concentration being 2 ug / ml is used for drug pressure screening, and under the condition that the polybrene with the concentration being 5 ug / ml exists, repeated infection is conducted one time; and after one week, the primary generation sheep lung fibroblasts are cultured through puromycin maintenance media with the concentration being 1 ug / ml till monoclonal cells appear and grow to be about 2.0 cm, the monoclonal cells are selected for culture expansion, and thus the immortalized sheep lung fibroblasts with a passage characteristic can be obtained.

Description

technical field [0001] The present invention relates to the field of construction and application of cell lines, in particular to the construction and application of an immortalized primary sheep lung cell line. Background technique [0002] Passage cell lines are an important platform for the application and basic research of animal viruses. However, many animal viruses lack host cell lines that can support their stable proliferation, such as Peste des petits ruminants virus and rabbit plague virus, which seriously hinders the molecular biology of these viruses Research and vaccine development efforts. However, the limited number of passages of some primary cells makes the progress of research work slow. At present, the immortalized cell lines successfully constructed by using telomerase include: bovine thyroid cell line, CCTCC preservation number C2014109; skin fibroblast cell line, application publication number It is CN 101921729 A; the immortalized cell line of pig-der...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12R1/91
CPCC12N9/1276C12N15/86C12N2510/04C12N2740/15043C12Y207/07049
Inventor 刘光清刘腾朱杰陈宗艳李传峰
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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