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Cattle plasmid adenovirus vector pAd-ZBED6 as well as construction method and use of cattle plasmid adenovirus vector pAd-ZBED6

A pad-zbed6, adenovirus technology, applied in the field of plasmid-type adenovirus vectors, can solve the problem of slow virus plaque formation and other problems

Inactive Publication Date: 2013-04-10
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the slow formation of viral plaques, the whole process takes a long time

Method used

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  • Cattle plasmid adenovirus vector pAd-ZBED6 as well as construction method and use of cattle plasmid adenovirus vector pAd-ZBED6
  • Cattle plasmid adenovirus vector pAd-ZBED6 as well as construction method and use of cattle plasmid adenovirus vector pAd-ZBED6
  • Cattle plasmid adenovirus vector pAd-ZBED6 as well as construction method and use of cattle plasmid adenovirus vector pAd-ZBED6

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Construction of pAd-ZBED6 recombinant adenovirus plasmid

[0048] 1. Materials and methods:

[0049] 1.1. Instrument:

[0050] 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), CO 2 Incubator, HZS-H water bath shaker (Harbin), Eppendorf pipette, DYY-Ⅲ steady voltage and steady current electrophoresis instrument (Beijing Liuyi), DYY-Ⅲ31A and DYY-Ⅲ28D electrophoresis tank (Beijing Liuyi), manufacturing Ice instrument, MDF-382E ultra-low temperature refrigerator (Sanyo, Japan), Eppendorf desktop high-speed centrifuge, Sartorious electronic balance (Germany), conventional refrigerator, etc.

[0051] 1.2. Main biochemical reagents and kits:

[0052] Long Taq polymerase, PrimeSTAR DNA polymerase, DNA restriction enzymes (XbaI, PacI, PmeI, KpnI and XbaI, etc.), collagen, trypsin, DNA Marker,...

Embodiment 2

[0130] Example 2 : Recombinant adenovirus carrying Qinchuan cattle ZBED6 gene transfected primary cultured Qinchuan cattle muscle cells:

[0131] The purpose of this example is to infect bovine primary muscle cells with the recombinant adenovirus plasmid pAd-ZBED6, and detect the effects of overexpression of the ZBED6 gene on muscle cell proliferation and differentiation and muscle development marker genes (MyoD, MyoG, MEF) through real-time quantitative PCR and Western blotting techniques. -2D, Pax7, MHC) differential expression in bovine muscle cells.

[0132] 1. Primary culture of Qinchuan cattle muscle cells:

[0133] (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;

[0134] (2), use surgical scissors to cut the muscle into small pieces (1mm 3 ), washed three times with Hank’s solution, and transferred to a small penicillin bottle;

[0135] (3) Add 5-6 times of ...

Embodiment 3

[0144] Example 3: Preparation of E.coli DH5α competent cells:

[0145] The preparation steps of the competent cell E.coli DH5α in the embodiment of the present invention:

[0146] 1. Steps:

[0147] (1) In a 15mL centrifuge tube, shake the bacteria overnight with 4mL of LB culture medium;

[0148] (2) The next day, 1:100 inoculated and expanded culture in 200mL RichLB (see Table 6) culture medium (37°C) to OD 600 =0.6~0.7;

[0149] (3) Place on ice for 10 minutes, shake to cool down, and dispense into 50mL centrifuge tubes, 42mL per tube;

[0150] (4), 4°C, 2000r / min, 15min, collect the cells (remove the supernatant);

[0151] (5), 1 / 6 volume of CCMB80Buffer (see Table 7) was resuspended, and placed on ice for 20 minutes;

[0152] (6), 4°C, 2000r / min, 15min, collect the cells (remove the supernatant);

[0153] (7) Resuspend with 1 / 24 volume of CCMB80Buffer, place 100 μL in a centrifuge tube, and freeze at -80°C.

[0154] Table 6 Components of Rich LB

[0155]

[0...

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Abstract

The invention relates to a cattle plasmid adenovirus vector pAd-ZBED6 as well as a construction method and use of the cattle plasmid adenovirus vector pAd-ZBED6, which belongs to the technical field of gene engineering. The construction method of the cattle plasmid adenovirus vector pAd-ZBED6 comprises the follow steps: (1) inserting Qinchuan cattle ZBED6 gene into a CMV (cytomegalovirus) promoter of a pAdTrack-CMV adenovirus shuttle plasmid to construct a recombinant shuttle plasmid pAdTrack-CMV-ZBED6; (2) after linearization of Pme I, converting an E.coil BJ5183 competent cell containing a pAdEasy-1 plasmid, completing homologous recombination of pAdTrack-CMV-ZBED6 and pAdEasy-1 in bacteria by means of an efficient homologous recombination system of Cre recombinase in the E.coil BJ5183, and naming correct recombinant adenovirus plasmid as pAd-ZBED6; and (3) after Pac I digestion linearization of pAd-ZBED6, transfecting a 293A cell and an original muscle cell of Qinchuan cattle by lipidosomes to obtain recombinant virus particles. The cattle plasmid adenovirus vector pAd-ZBED6 can be applied to functional study and authentication of Qinchuan cattle ZBED6 gene as well as modification of seed cells so as to regulate metabolism of genes related to growth and development of muscle.

Description

technical field [0001] The invention relates to a plasmid type adenovirus vector containing ZBED6 gene, the construction of the plasmid type adenovirus vector adenovirus vector and its application in transforming seed cells and bovine ZBED6 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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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/66C12N5/10C12Q1/68G01N33/68
Inventor 陈宏黄永震王璟李明勋赖新生蓝贤勇雷初朝
Owner NORTHWEST A & F UNIV
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