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Eukaryotic expression recombinant plasmid and construction method thereof and Vero cell line stably expressing the plasmid

A technology for recombinant plasmids and cell lines, applied in the field of bioengineering, can solve the problems of cumbersome construction and identification methods, affect the isolation efficiency of canine distemper virus, and the low sensitivity of virus isolation, so as to maintain biological characteristics and improve combination Efficiency, the effect of enhancing translation efficiency

Inactive Publication Date: 2012-01-18
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the methods used for the establishment and identification of this cell line are relatively cumbersome.
In addition, due to the low expression of canine SLAM in the positive Vero cell line, the sensitivity of virus isolation is not high, which directly affects the isolation efficiency of canine distemper virus.

Method used

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  • Eukaryotic expression recombinant plasmid and construction method thereof and Vero cell line stably expressing the plasmid
  • Eukaryotic expression recombinant plasmid and construction method thereof and Vero cell line stably expressing the plasmid
  • Eukaryotic expression recombinant plasmid and construction method thereof and Vero cell line stably expressing the plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction of eukaryotic expression recombinant plasmid pIRES2-EGFP-rSLAMh

[0057] PCR amplification and gene cloning: the plasmid pMD18-T-rSLAM was used as a template, and

[0058] P 1 5'-CCG ATGGATTCCAGGGGCTTCCTC-3' is the upstream primer, with P 2 5'-CCG TCA GTGATGGTGATGGTGATG GCTCTCTGGGAACGTCAC-3' is the downstream primer (the box is the restriction enzyme site, the bold one is the Kozak sequence, the underline is the histidine tag gene), and PCR amplification is performed to obtain the target gene. The reaction system is: DNA template 1 μL (0.2ng / μL), upstream and downstream primers 1μL (20pmol / L), Ex taq enzyme 0.3μL (5U / μL), dNTP (each 2.5mmol / L) 2μL, 10×PCR Buffer 2.5μL, ddH 2 O to make up 25 μL.

[0059] The amplification program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 61.8°C for 1 min, extension at 72°C for 1 min, 35 cycles; total extension at 72°C for 10 min; preservation at 4°C for...

Embodiment 2

[0064] Example 2 Establishment of a Vero cell line stably expressing pIRES2-EGFP-rSLAMh

[0065] Determination of the optimal concentration of G418: After the resuscitated Vero cells were passaged for 2 to 3 passages in DMEM with 10% FBS, the cells to be passaged were trypsinized, and 1×10 4 The number of cells / well was paved on a 96-well plate, and the medium was aspirated after the plate was confluent on the second day, the cells were washed once with PBS, and the screening medium was added. The concentration of G418 was 0, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μg / mL in sequence, and each gradient was set in 3 replicates, and the screening medium was changed every 3 days depending on the color of the medium. Continuously culture for 10 days, observe the cell death every day, and take the lowest G418 concentration at which all the cells die on the 10th day as the optimal screening concentration. Adding different concentrations of G418 to Vero cells, a small amount...

Embodiment 3

[0077] Sensitivity comparison of cell lines to CDV: when Vero and V-p-rSLAMh cells were in good growth state, two kinds of cells were used to pave 3 wells each in a six-well plate (Nunc Company, Denmark), and Vero cells were treated with DMEM cells of 10% FBS Growth medium, V-p-rSLAMh cell growth medium also added 800 μg / mL of G418, at 37 ° C, 5% CO 2 After culturing in the incubator for 48 hours, the virus was adsorbed and inoculated respectively. The maintenance solution of V-p-rSLAMh cells also contained 800 μg / mL of G418, and the pathological changes of the cells were observed every 12 hours. The 3 wild strains inoculated were 2010 Hebei fox organ virus [HeB(10)f1]; 2010 Jilin Jiutai raccoon organ virus [JL(10)r1]; 2010 Lianning Jinzhou fox organ virus [ LN(10)f1)]. The above three canine distemper samples were all positive for canine distemper virus nucleic acid by RT-PCR. Blind passage was performed on the cells without lesions, and the cells without CPE after 6 passag...

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Abstract

The invention relates to the technical field of bioengineering, and particularly relates to an eukaryotic expression recombinant plasmid and a construction method thereof and a Vero cell line stably expressing the plasmid. The eukaryotic expression recombinant plasmid has the nucleotide sequence shown in SEQ ID NO:1. The Vero cell line stably expressing the plasmid can be used for high-efficiency separation of canine distemper virus virulent strains and the research of the pathogenic mechanism of canine distemper virus.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a eukaryotic expression recombinant plasmid, a construction method thereof and a Vero cell line stably expressing the plasmid. Background technique [0002] Canine distemper (Canine distemper, CD) is caused by canine distemper virus (Canine distemper virus, CDV), a kind of animal such as canidae, mustelidae is susceptible to highly contagious infectious disease. The disease is one of the most harmful infectious diseases in the dog industry and fur animal breeding industry. It is characterized by anorexia, biphasic fever, conjunctivitis, gastroenteritis and neurological symptoms. It can cause a large number of dogs, foxes, raccoon dogs and other animals to become ill , The mortality rate is 30-80%. In recent years, the range of hosts infected by canine distemper virus has been expanding. Primate macaques, pinniped seals, giant pandas, cat lions, African wild dogs, etc. hav...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/66C12N5/10C12N7/00
Inventor 赵建军闫如勋闫喜军
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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