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BANCR gene overexpression lentivirus vector, BANCR lentivirus, construction methods and application

A lentiviral vector and gene overexpression technology, applied in the field of molecular biology, can solve the problem of no overexpression of BANCR gene in lentiviral vector, and achieve good biological safety, simple process and high positive rate

Inactive Publication Date: 2017-06-09
KUNMING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there are no reports of BANCR gene overexpression lentiviral vectors and related research at home and abroad

Method used

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  • BANCR gene overexpression lentivirus vector, BANCR lentivirus, construction methods and application
  • BANCR gene overexpression lentivirus vector, BANCR lentivirus, construction methods and application
  • BANCR gene overexpression lentivirus vector, BANCR lentivirus, construction methods and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Select the overexpression vector and synthesize the complete sequence of human LncRNA BANCR gene

[0058] LV5 expressing green fluorescent protein was selected as the overexpression vector, and the PCR primers required for the synthesis of the human LncRNA BANCR gene sequence were designed by the oligo online software (http: / / www.oligo.net / ), and the upstream and downstream primers were respectively added to the LV5 vector Homologous sequences flanked by NotI and NsiI for subcloning of vectors. Primers were synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0059]

[0060] After diluting the synthesized primers to 50 μM, carry out PCR reaction, the reaction system is as follows:

[0061] Reagent Volume (μl) LncRNA BANCR template 1 10×Pfu Buffer (+Mg 2+ )

5 Upstream primer BANCR-F 1 Downstream primer BANCR-R 1 dNTP 1 wxya 2 o

41 Pfu DNA polymerase 0.3

[0062] The loop conditions are a...

Embodiment 2

[0066] The target gene human LncRNA BANCR was cloned into the vector LV5

[0067] Double enzyme digestion of LV5 vector: In a sterile 0.2mL EP reaction tube, take 15 μL of LV5 vector and perform double enzyme digestion with NotI and NsiI restriction endonucleases. The enzyme digestion system is prepared as follows according to the instructions:

[0068] Reagent Volume (μl) 10×Buffer 5 dna 15 NotI 1 NsiI 1 wxya 2 o

[0069] After mixing the above system, react at 37°C for 2h. At this point, the double-digested LV5 is a linearized vector, and the linearized LV5 is recovered by Agarose electrophoresis and a DNA gel recovery kit. use Entry OneStep Cloning Kit, the human LncRNA BANCR gene fragment amplified and recovered in Example 1 was recombined and cloned into a linearized LV5 vector, and the reaction system was as follows:

[0070] Reagent Volume (μl) 5×CE Entry Buffer 4 LV5 1 LncRNA BANCR 2 Exnas...

Embodiment 3

[0073] Transformation of DH5α Competent Cells with Recombinant Overexpression Vector and Identification

[0074]Transformation of the recombinant ligation product: Place the centrifuge tube containing the competent cell DH5α on ice for 4 minutes. After the competent cells are thawed, add 10 μl of the recombinant ligation product, mix the contents gently, and place in ice for 30 minutes. Put the centrifuge tube on the test tube rack placed in a water bath preheated to 42°C, and place it for 90 seconds without shaking the centrifuge tube. Quickly transfer the centrifuge tube to an ice bath and let the cells cool for 3 minutes. Add 800 μl of LB medium (without antibiotics) to each centrifuge tube, then transfer the centrifuge tube to a shaker at 37° C., 250 rpm, and incubate for 45 minutes to recover the bacteria. Take 200 μl of cultured cells and spread evenly on LB plates containing 50 μg / ml Ampicillin. After the liquid on the plate is absorbed, place the plate upside down in...

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Abstract

The invention provides a BANCR gene overexpression lentivirus vector, a BANCR lentivirus, construction methods and application, and relates to the technical field of molecular biology. The BANCR gene overexpression lentivirus vector is constructed on the basis of an LV5 lentivirus overexpression vector expressing green fluorescin, is integrated with a BANCR gene, solves the technical problem of an overexpression vector required in BANCR gene study, and allows the BANCR gene study to be more visual. The construction method of the BANCR gene overexpression lentivirus vector is simple in flow and high in positive rate. The BANCR lentivirus has high biological safety. In addition, the invention further provides the construction method of the BANCR lentivirus and the application of the BANCR gene overexpression lentivirus vector and / or BANCR lentivirus in preparation of a BANCR gene overexpression cell.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a BANCR gene overexpression lentiviral vector, BANCR lentivirus, construction method and application. Background technique [0002] Overexpression of target gene is one of the commonly used means to study gene function. The target gene is introduced into the cells by transfection (including electroporation, liposome transfection, etc.), microinjection or virus infection, so as to establish a stable cell line with overexpression of the target gene, which can be widely used in biological research. [0003] Lentiviral vectors can effectively integrate exogenous genes or exogenous shRNAs into the host chromosome, so as to achieve the effect of persistent expression of the target sequence. In terms of infection ability, it can effectively infect various types of cells such as neuron cells, liver cells, cardiomyocytes, tumor cells, endothelial cells, stem cells, etc., so as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N7/01
CPCC12N15/86C12N7/00C12N2740/15021C12N2740/15043
Inventor 朱月春杨丽娟白宏刚蒋露杨雨叶杨慧鑫易子寒张巧
Owner KUNMING MEDICAL UNIVERSITY
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