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A method for constructing a lentiviral vector carrying rhesus monkey resistin splice body gene

A technology of lentiviral vector and construction method, which is applied in the field of construction of lentiviral vector carrying rhesus monkey resistin spliceosome gene, can solve the problems of heavy screening workload, high cost, low efficiency of plasmid transfection, etc., and achieve high-efficiency transfection Infect and eliminate the effect of probability restriction

Active Publication Date: 2018-06-01
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for constructing a lentiviral vector carrying a rhesus monkey resistin splice body gene, aiming to solve the problem of heavy workload and high cost of screening by the PCR+1 method, the traditional method of lentiviral packaging, and the presence of plasmids Low transfection efficiency and low titer of lentivirus

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  • A method for constructing a lentiviral vector carrying rhesus monkey resistin splice body gene
  • A method for constructing a lentiviral vector carrying rhesus monkey resistin splice body gene
  • A method for constructing a lentiviral vector carrying rhesus monkey resistin splice body gene

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[0023] In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

[0024] The application principle of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0025] 1. Test materials and reagents

[0026] Plasmids, strains and cells:

[0027] PLV-EF1a-EGFP[2A] Puro / PH1 / PH2 lentiviral system was purchased from Yingmaoshengye Company, competent DH5α (preserved by Institute of Medical Biology, Chinese Academy of Medical Sciences), competent Stbl3 was purchased from GeneCopoeia Company, 293T cells and KMB17 Cells (preserved by the Institute of Medical Biology, Chinese Academy of Medical Sciences).

[0028] Main reagent...

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Abstract

The invention discloses an establishing method of a lentiviral vector carrying rhesus monkey resistin spliceosome gene. Common PCR is used for directly amplifying resistin full length and the spliceosome gene at the same time, a glue recovery method is used for directly recycling a spliceosome strip, and then carrier quick clone sequencing is carried out. A lentiviral vector efficient transfection agent is used for transfecting all lentiviral vector plasmids, an inactivation serum is used for packaging lentiviral particles, and high-titer recombination lentiviral original liquid is obtained. A large amount of work for screening with a PCR + 1 method and high cost can be avoided, the probability limiting for obtaining the spliceosome gene is removed, and the spliceosome gene is directly obtained efficiently, quickly and cheaply. Efficient lentiviral vector plasmid transfection can be achieved, and high recombination lentivirus titer is obtained.

Description

technical field [0001] The invention belongs to the technical field of cloning, and in particular relates to a construction method of a lentiviral vector carrying rhesus monkey resistin splice body gene. Background technique [0002] Resistin is a newly discovered adipose tissue-specific secretion of adipocytokines, which has the effect of resisting insulin. It is considered to be an undiscovered link between obesity and type 2 diabetes. It is even believed that obesity can cause insulin resistance because of fat Cells secrete resistin. The discovery of resistin is a great progress in diabetes research and has become a research hotspot connecting obesity and type Ⅱ diabetes. However, the research results in recent years have diverged in the elucidation of the biological function of resistin in the body's insulin resistance. In the statistical analysis of the human type 2 diabetes population, there are even no correlation results. Most scientific researchers speculate that ...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/66C12N15/65
Inventor 鲁帅尧马开利乞素冬李鸿钧和占龙马进陈瑜吴正存
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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