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Method for establishing canine distemper sensitive cell line based on Nectin4 receptor and application

A sensitive cell and establishment method technology, which is applied in the field of canine distemper sensitive cell line establishment, can solve the problems of CDV growth cycle being too long and not growing, and achieve the effects of improving virus isolation rate, enhancing affinity ability, and increasing localization efficiency

Inactive Publication Date: 2018-01-09
SHANDONG SINDER TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although MDCK cells are virus target animal cells, the growth cycle of CDV in passaged MDCK cells is too long or does not grow, and does not produce obvious cytopathic changes

Method used

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  • Method for establishing canine distemper sensitive cell line based on Nectin4 receptor and application
  • Method for establishing canine distemper sensitive cell line based on Nectin4 receptor and application
  • Method for establishing canine distemper sensitive cell line based on Nectin4 receptor and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Establishment of canine distemper sensitive cell line based on Nectin4 receptor

[0064] 1. Method

[0065] 1 Nectin4 gene preparation and modification

[0066] 1.1 Handling of tissue samples and experimental equipment

[0067] The placental tissue samples of Beagle dogs were collected in a sterile environment, quickly frozen with liquid nitrogen and stored in a -70°C refrigerator. Take 5g of tissue samples and crush them at low temperature in advance and place them in a mortar pre-cooled with liquid nitrogen. Grind thoroughly until no tissue particles are visible to the naked eye. During this period, add an appropriate amount of liquid nitrogen to maintain the frozen state of the tissue. Take every 0.1g of the ground tissue Aliquot the sample into 1.5ml RNase-free EP tubes, add 1ml Trizol, vortex well and store in a -70°C refrigerator or directly use for total RNA extraction.

[0068] 1.2 Sample total RNA extraction and cDNA synthesis

[0069] 1.2.1 Extra...

Embodiment 2

[0135] Embodiment 2 adopts pCI-Neo plasmid transfection method

[0136] The IRES-Puro fragment was amplified by the PCR method, and the IRES-Puro fragment was connected to the downstream of the Nectin4 sequence of the pMD18T-Nectin4 plasmid through the restriction site to obtain the Nectin4-IRES-Puro tandem sequence. The recombinant plasmid was amplified, and the pCI-Neo vector and the recombinant plasmid were double-digested respectively through restriction sites, and the Nectin4-IRES-Puro sequence was inserted into the pCI-Neo vector to obtain the pCI-NIP plasmid.

[0137] The pCI-NIP plasmid liposome method was used to transfect Vero cells, and culture was continued for 24 hours after transfection. After 24 hours, the transfected cells were digested and diluted at a ratio of 1:10 to pass into a 10cm culture dish, and culture was continued for 24 hours. 48 hours after the cells were transfected, the medium was replaced with a complete medium containing 6 μg / mL puromycin, and...

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Abstract

The invention discloses a method for establishing a canine distemper sensitive cell line based on a Nectin4 receptor. The method comprises the following steps: (1) extracting total RNA of a sample andsynthesizing cDNA; (2) amplifying and cloning Nectin4; (3) modifying Nectin4; (4) connecting and converting dNectin4-Hatag and extracting recombinant plasmid; (5) packaging recombinant slow virus; abd (6) transfecting cells and screening positive cell line. The Vero-Nectin4 established according to the method disclosed by the invention can stably express the Nectin4 receptor and is high in universality; the CDV virulent strain is capable of growing in the cell line and acquiring obvious CPE lesion; and the method is suitable for wide application in culturing or separating the canine distempervirus.

Description

technical field [0001] The invention belongs to the technical field of constructing sensitive cell lines for virus cultivation, and in particular relates to a method and application for establishing a canine distemper sensitive cell line based on Nectin4 receptor. Background technique [0002] Canine distemper (Canine Distemper, CD) is an acute, highly contagious infectious disease caused by Canine Distemper Virus (CDV). CDV belongs to the family Paramyxoviridae and the genus Morbillivirus. It mainly infects canines and mustelids. In recent years, it has also been reported to infect wild animals such as monkeys, lions, and pandas. The scope of host infection is expanding. CDV has a high morbidity and mortality rate, and is the main infectious disease of pet dogs and fur economic animals, which can easily cause heavy economic losses. CDV can be divided into Asian type and American type according to genome differences, but there is only one serotype. [0003] CDV is a single...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66C12N15/70C12N5/10C12N7/00
Inventor 李明义于泽坤刘阳单学强孙化露李思菲栾志舫马礼照段笑笑吴海燕朱岩丽
Owner SHANDONG SINDER TECH
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