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Canine-SLAM/Vero cell line and constructing method

A construction method and cell line technology, applied in the field of Canine-SLAM/Vero cell line, can solve the problems of high cost, long time, and low probability of successful separation, and achieve the effect of safe communication and transportation

Inactive Publication Date: 2010-10-06
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the fact that traditional methods are still used in China to isolate and cultivate canine distemper virus, the probability of successful isolation is small, and the time is long and the cost is high

Method used

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  • Canine-SLAM/Vero cell line and constructing method
  • Canine-SLAM/Vero cell line and constructing method
  • Canine-SLAM/Vero cell line and constructing method

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Establishment of Canine-SLAM / Vero cell line

[0036] 1. Isolation of Canine Peripheral Blood Lymphocytes

[0037] Collect canine distemper-positive dog anticoagulant blood 7ml from sterile jugular vein, use canine lymphocyte separation medium to separate peripheral blood lymphocytes according to the instructions, and then centrifuge at 2500rpm / min for 10min. Resuspend in Hank's solution and store at -20°C for later use.

[0038] 2. RT-PCR amplification of canine SLAM gene cDNA encoding

[0039] According to the instructions of the classic total RNA extraction kit, the total mRNA of canine peripheral lymphocytes was extracted, and cDNA was synthesized by using Random Primer(6 mer)pd(N)6 and Oligo d(T)15 reverse transcription primers, and the reference GenBank: AF390108 .1 Designed primer pairs:

[0040] Upstream primer SLAM-F: 5'-GCCTCGAGACAGGTGAGAGCTTGATGAAT-3'(Xho I)

[0041] Downstream primer SLAM-R: 5'-GCAGATCTCAGCTCTCTGGGAACGTCAC-3'(Bgl II)

[0042] Amplified can...

Embodiment 1

[0049] Isolation of canine distemper virus by Canine-SLAM / Vero cells

[0050] The Canine-SLAM / Vero cell monolayer was used to adsorb and separate the canine source (see Figure 5 A) and monkey-derived wild-type CDV (see Figure 5 B) with the vaccine strain CDV (see Figure 5 C), Vero cells as a control (see Figure 5 D, E, F), found that these two strains of wild-type CDV can grow on Canine-SLAM / Vero cells, and produce obvious cytopathic changes at 24-36h after inoculation, but cannot produce pathological changes on Vero cells; vaccine strains can Lesions were generated in both Canine-SLAM / Vero cells and Vero cells, but lesions were found to appear faster in the former.

[0051] The experiment found that the virus titer cultured with Canine-SLAM / Vero cells was at least 10 times higher than that of ordinary Vero cells. All in all, using Canine-SLAM / Vero cells to isolate canine distemper wild strains has the advantages of short time and high toxin production.

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Abstract

The invention provides a Canine-SLAM / Vero cell line and a constructing method, wherein the constructing method comprises the following steps of: collecting canine distemper positive canine anticoagulated blood, separating peripheral blood lymphocytes by applying a canine lymphocyte separation liquid, extracting total RNA (Ribonucleic Acid), and obtaining a canine SLAM gene by uisng a PCR (Polymerase Chain Reaction) method; constructing pCAGGS-Neo / SLAM plasmids; transfecting a constructed pCAGGS-Neo / SLAM expression cassette into a Vero cell, and screening the cell line for stably expressing an SLAM by adopting G418; and identifying and detecting the expression condition of the SLAM on the surface of a cell membrane by utilizing an indirect immunofluorescent method to obtain the cell line. The invention not only can be used for the separation of canine distemper viruses and the titration of the viruses, but also markedly shortens the time for separating the viruses, improves the yield of the viruses, and forcefully promotes researches on the molecular characteristics, such as phenotypes, genotypes and the like of the canine distemper viruses.

Description

Technical field: [0001] The invention provides a Canine-SLAM / Vero cell line. The invention also provides a construction method of the above cell line, which is used for the isolation of canine distemper virus wild strain and the study of its etiology, and belongs to the technical field of bioengineering. Background technique: [0002] Canine distemper virus (CDV) is an enveloped single-stranded negative-sense RNA virus belonging to the Morbillivirus genus of the Paramyxoviridae family. In recent years, with the change of ecological environment and the evolution of animals and viruses, the natural infection hosts of CDV have expanded from the traditional Canidae, Weaselidae and Procyonidae to all 8 families of Carnivora, Artiodactyla Suidae, Primate Rhesus and pinniped seals and other animals, and the host range of CDV natural infection is still expanding. [0003] Although the host range of canine distemper virus is constantly expanding, it has always been difficult to succ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/45C12N15/63C12R1/91
Inventor 高玉伟刘玉秀夏咸柱杨松涛黄耕王承宇侯小强王铁成冯娜
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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