Immortalization chicken embryo hepatocyte line as well as preparation method and application thereof

A technology of chicken embryos and liver cells, which is applied in the fields of biology, bioengineering and veterinary biological products, can solve the problems of backward preparation technology, failure to meet the actual immune needs of poultry farms, and virus-contaminated production waste, so as to improve the preparation Efficiency, maintaining the effect of division and proliferation

Pending Publication Date: 2017-08-04
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, both of these methods have certain disadvantages
For example, the use of SPF chicken embryos will produce a large amount of virus-contaminated production waste, and the acquisition of primary chicken embryo liver cells also requires the use of SPF chicken embryos, and the preparation of primary cells is extremely time-consuming, laborious, and easily polluted
Therefore, the existing poultry adenovirus vaccine preparation technology is backward, the production efficiency is low, and the actual immunization needs of poultry farms cannot be met.

Method used

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  • Immortalization chicken embryo hepatocyte line as well as preparation method and application thereof
  • Immortalization chicken embryo hepatocyte line as well as preparation method and application thereof
  • Immortalization chicken embryo hepatocyte line as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of pCAG-hMRP18S-2 expression vector

[0042] 1.1 Reagents

[0043] The codon-optimized hMRP18S-2 protein-encoding gene fragment (SEQ ID NO.1) was chemically synthesized by GenScript Biotechnology Company and cloned into the pUC57 vector. Molecular biology materials such as pMD19-T vector, various restriction endonucleases, ExTaq enzyme, T4DNA ligase were purchased from TAKARA Company. The eukaryotic expression vector containing the CAG promoter sequence was constructed and preserved by the National Veterinary Biological Products Engineering Technology Research Center. Gel recovery kit and plasmid small and large extraction kit were purchased from Qiagen.

[0044] 1.2 Primer synthesis: The primers used to amplify the ORF sequence of hMRP18S-2 protein were synthesized by GenScript Biotechnology Company.

[0045] p1: GCC GGTACC GCCACCATGGCCGCG (the underline is the KpnI restriction site)

[0046] p2: GCC GCGGCCGC CTAGAGAGCACTCTG (the underlin...

Embodiment 2

[0051] Example 2 Construction of recombinant CEL-hMRP18S-2

[0052] 2.1 Materials:

[0053] Reagents: MEM, G418, Opti-MEM, and transfection reagents were purchased from Invitrogen. The primary rabbit anti-hMRP18S-2 protein antibody was purchased from Santa Cruz Biotechnology Company, FITC-labeled goat anti-rabbit IgG, and HRP-labeled goat anti-rabbit IgG were purchased from Hangzhou Lianke Biotechnology Company. Molecular biology reagents such as RNA extraction kit and reverse transcription kit were purchased from Qiagen.

[0054] Biological material: Primary chicken embryo liver cells were isolated from SPF chicken embryos by the National Veterinary Biological Products Engineering Technology Research Center. The primary cells were prepared and inoculated in MEM medium containing 10% newborn bovine serum for later use.

[0055] 2.2 Screening of the action concentration of G418

[0056] Adjust the initial cell density of primary chicken embryonic liver cells to 5 × 10 5 cel...

Embodiment 3

[0071] Example 3 Determination of continuous growth performance of CEL-hMRP18S-2 cells

[0072] 3.1 Materials and methods

[0073] Primary chicken embryonic liver cells were prepared by the National Veterinary Biological Products Engineering Technology Research Center.

[0074] Recombinant CEL-hMRP18S-2 cells were screened and obtained as described above, and established and preserved.

[0075] Medium: MEM medium containing 10% newborn bovine serum.

[0076] Primary chicken embryonic liver cells and recombinant CEL-hMRP18S-2 cells of different passages were used 3×10 5 The initial density of cells / ml was inoculated into a T150 culture square flask, placed at 37°C, 5% CO 2 cultured in an incubator. Samples were taken every 24 hours to measure cell density and viability, and cell growth curves of different growth generations were drawn.

[0077] 3.2 Experimental results

[0078] The growth curve of recombinant CEL-hMRP18S-2 cells is as follows Figure 4 shown. Compared w...

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Abstract

The invention discloses an immortalization chicken embryo hepatocyte line as well as a preparation method and application thereof. The immortalization chicken embryo hepatocyte line CEL-hMRP18S-2 is preserved in China General Microbiological Culture Collection Center (CGMCC) on December 6, 2016; the preservation number is CGMCC NO.12333; the preservation address is Microbiology Research Institute of China Science Academy, #3, Yard 1, West Beichen Road, Chaoyang District, Beijing. The obtained immortalization chicken embryo hepatocyte retains main characteristics and main functions of the primary generation of chicken embryo hepatocyte; the immortalization chicken embryo hepatocyte can be amplified and cultured without limit in vitro; the hepatocyte line still has high activity after subculturing for 100 generations in vitro, can retain differentiation and proliferation and is free of senescence and apoptosis; when the immortalization chicken embryo hepatocyte line is applied to proliferation of type I aviadenoviruses, the preparation efficiency of virus antigens can be obviously improved.

Description

technical field [0001] The invention relates to an immortalized chicken embryo liver cell line and its preparation method and application, belonging to the field of biology, in particular to the technical field of bioengineering and veterinary biological products. Background technique [0002] Since 2013, avian adenovirus type I has spread in large numbers in poultry farms in my country, causing serious outbreaks and causing significant economic losses to the farms. [0003] For this virus, the main prevention and control method currently adopted in my country is the use of avian adenovirus-related vaccines. In the prior art, we usually use SPF chicken embryos or primary chicken embryo liver cells to proliferate avian adenoviruses, so as to obtain related vaccines. [0004] However, both of these methods have certain drawbacks. For example, the use of SPF chicken embryos will generate a large amount of virus-contaminated production waste, and the acquisition of primary c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N7/00A61K39/235A61P31/20C12R1/91
CPCA61K39/12C12N5/0603C12N5/067C12N7/00C12N15/85C12N2510/00C12N2710/10234C12N2710/10251
Inventor 冯磊恽君雯吴培培陈丽侯继波
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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