Stable cell line MDCK for amplifying recombinant canine adenovirus (CAV2) and construction method of stable cell line

A canine adenovirus and a construction method, which are applied in the field of stable cell line construction, and can solve the problems of low proliferation rate of canine adenovirus and low stability of cell lines.

Inactive Publication Date: 2018-07-31
BRAINVTA (WUHAN) CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the direct construction of the E1 gene into MDCK has the problems of low cell line stability and low proliferation rate of canine adenovirus

Method used

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  • Stable cell line MDCK for amplifying recombinant canine adenovirus (CAV2) and construction method of stable cell line
  • Stable cell line MDCK for amplifying recombinant canine adenovirus (CAV2) and construction method of stable cell line
  • Stable cell line MDCK for amplifying recombinant canine adenovirus (CAV2) and construction method of stable cell line

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Experimental program
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Effect test

Embodiment 1

[0059] Construction of MDCK-E1A and MDCK-E1A-E1B stable cell lines

[0060] (1) Reagents and instruments

[0061] pfu polymerase, dNTPs and 5×Fast pfu buffer were purchased from Quanshijin Biotechnology Co., Ltd.; pFUGW vector was purchased from Addgene; pCDH-puro plasmid was a gift from the Laboratory of Neurovirology, Wuhan Institute of Virology, Chinese Academy of Sciences; primers were synthesized by Shanghai Sangong ; DMEM medium and serum Opti-MEM were purchased from Invitrogen; fetal bovine serum was purchased from Hyclone; plasmid miniprep kit Plasmid Miniprep Kit was purchased from Qiagen; RNA extraction kit was purchased from TaKaRa; FuGene6 transfection reagent was purchased from From Promega; Puromycin (10 mg / mL) solution was purchased from Shanghai Sangon Bioengineering Co., Ltd.

[0062] MDCK cells were a gift from the Influenza Laboratory of Wuhan Institute of Virology, Chinese Academy of Sciences; the inverted microscope was purchased from OLYMPUS; the PCR ins...

Embodiment 2

[0137] Amplification of canine adenovirus CAV2 on MDCK, MDCK-E1, MDCK-E1A, MDCK-E1A-E1B cell lines

[0138] (1) Reagents and instruments

[0139] Primers were synthesized by Shanghai Sangon; DMEM medium was purchased from Invitrogen; fetal bovine serum was purchased from Hyclone; plasmid miniprep kit Plasmid Miniprep Kit was purchased from Qiagen; RNA extraction kit was purchased from TaKaRa; OLYMPUS; PCR instrument was purchased from Bio-rad; 2×T5 Fast qPCR Mix (SYBRGreenI) was purchased from Qingke Bio.

[0140] (2) Cell culture

[0141] The cells used for CAV2 amplification were MDCK, MDCK-E1, MDCK-E1A and MDCK-E1A-E1B, respectively. Among them, MDCK is a dog kidney cell, and MDCK-E1 is a cell line introduced into the E1 region, which is the most commonly used cell line for amplifying CAV2. The construction of this cell line is not described in detail here, and the cell line does not have fluorescence. MDCK-E1A is a stable cell line constructed by introducing E1Aregions ...

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Abstract

The invention provides a stable cell line for expressing canine adenovirus genes E1A and E1B and a construction method and application of the stable cell line. The two genes E1A and E1B in an E1 region are constructed in a lentivirus carrier, then an MDCK cell stably expresses the genes E1A and E1B respectively, and the MDCK-E1A-E1B stable cell line is constructed. The E1 gene which is long in segment and not easy to construct is split into the genes E1A and E1B which are short in segment and easy to construct, so that the success rate of constructing stable cells is increased; the genes E1A and E1B carry fluorescent proteins with different colors so that the stability of the stable cells can be monitored in real time. Experiments prove that the MDCK-E1A-E1B stable cell line is higher in titer of the amplified canine adenovirus than that of an MDCK-E1 stable cell line, and a good foundation is laid for application of the canine adenovirus in the fields of virus live carrier vaccines, gene therapy, cancer treatment and the like.

Description

technical field [0001] The invention belongs to the field of construction of stable cell lines, and more specifically relates to a stable cell line MDCK for amplifying recombinant canine adenovirus CAV2 and a construction method thereof. Background technique [0002] Since the 1980s, the adenovirus vector has become a research hotspot because of its good development prospect. At present, adenoviral vectors are widely used in the production of viral live vector vaccines, gene therapy, cancer treatment and other fields. However, the safety and immunogenicity of adenovirus itself also limit the clinical application of adenovirus vectors. Through continuous research and learning, people have found that canine adenovirus CAV2 can only replicate and grow in canine cells, but not in humans and monkeys. Growth in other cells, even in 293, Hela cells have a low level of replication but cannot be packaged into virus particles or spread, high safety, and there is no immune response to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N7/00C12N15/65C12R1/91
CPCC07K14/005C12N5/0686C12N7/00C12N15/65C12N15/86C12N2510/00C12N2710/10322C12N2710/10351C12N2740/15043
Inventor 潘杏李良凤高娟杨甜黄晓苹
Owner BRAINVTA (WUHAN) CO LTD
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