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Plasmid type adenovirus vector pAd-NRIP1 and construction method thereof

A pad-nrip1, adenovirus technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, introduction of foreign genetic material using vectors, etc.

Inactive Publication Date: 2012-04-04
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, the functions of RD3 and RD4 have not been reported

Method used

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  • Plasmid type adenovirus vector pAd-NRIP1 and construction method thereof
  • Plasmid type adenovirus vector pAd-NRIP1 and construction method thereof
  • Plasmid type adenovirus vector pAd-NRIP1 and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The construction of embodiment 1 pAd-NRIP1 recombinant adenovirus plasmid

[0034] 1. Materials and methods

[0035] 1.1 Instrument

[0036] Ultra-clean bench, biochemical incubator, gene amplification instrument, PTC-200 single-slot gradient gene amplification instrument, Heraeus refrigerated high-speed centrifuge (Germany), Bio-Rad gel imaging analyzer (USA), CO2 incubator, HZS-H water bath oscillator (Harbin), Eppendorf pipette, DYY-III type steady voltage and current electrophoresis instrument (Beijing Liuyi), DYY-III31A and DYY-III28D electrophoresis tank (Beijing Liuyi), ice maker, MDF-382E ultra-low temperature refrigerator (Sanyo, Japan), Eppendorf desktop high-speed centrifuge, Sartorious electronic balance (Germany), conventional refrigerator, etc.

[0037] 1.2 Main biochemical reagents and kits

[0038] Long Taq polymerase, PrimeSTAR DNA polymerase, DNA restriction endonuclease (NotI, PacI, PmeI, BglII, etc.), collagen, trypsin, DNA Marker, DNA ligase, Tri...

Embodiment 2

[0111] Example 2 Carrying Qinchuan cattle NRIP1 gene recombinant adenovirus transfection primary cultured Qinchuan cattle muscle cells

[0112] 1 Primary culture of Qinchuan cattle muscle cells

[0113] (1) Place the fresh tissue in a petri dish, wash it three times with Hank's solution in a sterile operating table, and remove fat, blood and other sundries;

[0114] (2), use surgical scissors to cut the muscle into small pieces (1mm 2 ), washed three times with Hank’s solution, and transferred to a small penicillin bottle;

[0115] (3) Add 5-6 times of 0.25% trypsin solution depending on the amount of tissue block, digest at 37°C for 20-40 minutes, shake once every 5 minutes, or blow once with a straw to separate the cells;

[0116] (4) Add 3-5ml of culture medium to stop trypsin digestion (or add trypsin inhibitor);

[0117] (5) Let stand for 5-10 minutes to make the undispersed tissue pieces sink, and take the suspension and add it to the centrifuge tube;

[0118] (6), 1...

Embodiment 3

[0123] The preparation of embodiment 3 competent cells

[0124] 1 step

[0125] (1) In a 15ml centrifuge tube, shake the bacteria overnight with 4ml of LB culture medium;

[0126] (2) The next day, 1:100 inoculated and expanded culture in 200ml Rich LB (see Table 5) culture medium (37°C) to OD600=0.6-0.7;

[0127] (3) Place on ice for 10 minutes, shake to cool down, and dispense into 50ml centrifuge tubes, 42ml per tube;

[0128] (4), 4°C, 2000rpm, 15min, collect the cells (remove the supernatant);

[0129] (5), 1 / 6 volume of CCMB 80buffer (see Table 6) resuspended, placed on ice for 20min;

[0130] (6), 4°C, 2000rpm, 15min, collect the cells (remove the supernatant);

[0131] (7) Resuspend with 1 / 24 volume of CCMB80 buffer, put each 100ul in a centrifuge tube, and freeze at -80°C.

[0132] Table 5 Components of Rich LB

[0133]

[0134] Table 6 Formula of CCMB80 buffer

[0135]

[0136] The various reagents in CCMB80 should be weighed accurately, first use a cert...

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Abstract

Construction of a recombinant adenovirus vector of an NRIP1 (nuclear receptor interacting protein 1) gene of Qinchuan cattle can be applied to gene function research and dentification on Qinchuan cattle NRIP1, reconstruction of seed cell, regulation on gene metabolism of muscle, fat and reproduction, etc. The Qinchuan cattle NRIP1 gene is inserted under a CMV promoter of a pAdTrack-CMV adenovirusshuttle plasmid to construct a recombinant shuttle plasmid pAdTrack-CMV-NRIP1. After linearization, a PmeI transforms an E.coli BJ5183 competent cell containing a pAdEasy-1 plasmid; an efficient homologous recombinant system of a Cre recombinase in the E.coli BJ5183 is utilized to complete homologous recombination of pAdTrack-CMV-NRIP1 and pAdEasy-1 in bacteria. A correct recombinant adenovirus plasmid is named as pAd-NRIP1. After digestion linearization of pAd-NRIP1 by a PacI, a liposome is transfected to a 293A cell, so as to obtain a recombinant virosome.

Description

technical field [0001] The invention relates to a plasmid type adenovirus vector containing NRIP1 gene, the construction of the plasmid type adenovirus vector adenovirus vector and its application in transforming seed cells and bovine NRIP1 function identification. Background technique [0002] The AdEasyTM system is a shortcut system constructed by T.C.He et al. (1998) to replace the traditional adenovirus recombination system. In this system, recombinant adenovirus can be produced in only two steps: first, the expression cassette is loaded into the transfer vector, and then inserted into the adenovirus genome by homologous recombination. The most efficient way to insert foreign genes into adenovirus is through homologous recombination, because: 1) adenovirus DNA is a large linear molecule containing almost all endonuclease sites; 2) the genome is too large (36kb) , difficult to operate. [0003] In the AdEasyTM vector system, the vector containing most of the adenovirus ...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/66
Inventor 陈宏刘栋马伟李密杰蓝贤勇潘传英
Owner NORTHWEST A & F UNIV
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