Internal reference plasmid suitable for silkworm cell double luciferase reporter gene system and preparation method and application of internal reference plasmid

A dual-luciferase and reporter gene technology, applied in the field of preparation of the internal reference plasmid, can solve the problems of low relative luciferase activity, unsuitable for low-activity cell level research, unstable internal reference plasmid activity, etc., and achieve moderate expression. Effect

Inactive Publication Date: 2017-05-24
SOUTHWEST UNIVERSITY
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Problems solved by technology

[0004] At present, the dual luciferase reporter gene system used in silkworm cells is commonly used as an internal reference in addition to PRL-CMV, PRL-SV40, PRL-TK, etc., and p-IE1-Rlucp, but PRL-CMV, PRL-SV40, PRL-TK, etc. The RLUC enzyme activity expressed by the vector in silkworm cells is relatively low. Generally, the value of the enzyme activity measured by transfection of 0.1 μg is only between tens and hundreds. In addition, the activity of this series of internal reference plasmids in silkworm cells is unstable; p-IE1-Rlucp internal reference can be stably expressed in silkworm cells, but the value of RLUC enzyme activity measured by transfection of 0.1 μg is too high, up to 10 4 to 10 6 , so the value of relative luciferase activity calculated is very low, which is not suitable for the study of the cell level of the promoter with low activity

Method used

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  • Internal reference plasmid suitable for silkworm cell double luciferase reporter gene system and preparation method and application of internal reference plasmid
  • Internal reference plasmid suitable for silkworm cell double luciferase reporter gene system and preparation method and application of internal reference plasmid
  • Internal reference plasmid suitable for silkworm cell double luciferase reporter gene system and preparation method and application of internal reference plasmid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1, Bombyx mori BmVg P78M gene promoter acquisition

[0024] Previous studies have shown that in the 78bp sequence upstream of the translation start site of the silkworm BmVg gene (including the 40bp 5'untranslated region and the 38bp sequence upstream of the transcription start site, a total of 78bp, named BmVgP78), there is only one with Hormone-related cis-acting element Estrogen receptor α ( figure 1 ), whose element sequence is shown in SEQ ID NO.1.

[0025] First, PCR amplification was performed using the adult genome of the silkworm variety dazao as a template. The upstream primer was: BmVgP78-F: 5'-cctatataaaggggggtgacctg-3' (SEQ ID NO.2); the downstream primer was: BmVgP78-R: 5' -tgtactagctccgctgtc-3' (SEQ ID NO. 3). The amplification conditions were pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 10 seconds, a total of 30 cycles; final extension at 72°C for 10 minu...

Embodiment 2

[0027] Embodiment 2, the construction of PRL-VgP78M internal reference carrier

[0028] Construction of an internal reference vector driven by the BmVgP78M promoter to express Renilla luciferase gene: the pMD19T simple vector containing the BmVgP78M promoter and the cell transfection vector internal reference vector PRL-SV40 were simultaneously treated with BglⅡ and NheI restriction endonucleases. The obtained promoter was connected to the PRL-SV40 backbone vector after restriction treatment, and positive recombinants were screened. The constructed recombinant plasmid is named PRL-VgP78M, and its structure is as follows: figure 2 As indicated, the sequence was verified, and the ultrapure plasmid was extracted for use.

Embodiment 3

[0029] Example 3, Analysis of the Stability of PRL-VgP78M Internal Reference and Its Influence from Hormones and Overexpression

[0030] a. Stability analysis of PRL-VgP78M internal reference

[0031] PRL-VgP78M internal reference plasmid and 0.1 μg each of PRL-SV40 and p-IE1-Rlucp internal references were transfected into BmE, an embryonic cell line commonly used in silkworms, and luciferase was analyzed by dual luciferase reporter system (Promega, USA) at 24h and 48h respectively activity, the result is as image 3 shown. The results showed that the enzyme activity of the PRL-SV40 internal reference carrier was too low, which was similar to that of the control, the enzyme activity of the p-IE1-Rlucp internal reference was too high, and the enzyme activity of the PRL-VgP78M internal reference was moderate, and the standard deviation among the six groups of data was not Large and relatively stable.

[0032] b. Analysis of the response of PRL-VgP78M internal reference to ecd...

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Abstract

The invention relates to an internal reference plasmid suitable for a silkworm cell double luciferase reporter gene system and a preparation method and application of the internal reference plasmid. The internal reference plasmid contains a constitutive promoter BmVgP78M suitable for the silkworm cell double luciferase reporter gene system, wherein the promoter is obtained by a BmVg gene promoter mutant hormone element to constitute the internal reference plasmid which does not respond to a silkworm hormone, can be stably expressed in silkworm cells, has moderate activity, and can be effectively used for the silkworm cell double luciferase reporter gene system to study the promoter activity or gene regulation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an internal reference plasmid suitable for a silkworm cell dual-luciferase reporter gene system, and also relates to a preparation method and application of the internal reference plasmid. Background technique [0002] The dual-luciferase reporter gene system is widely used in genotoxicity detection, signaling pathway research, and transcriptional activity analysis at the cellular level. That is, two luciferase reporter genes are simultaneously expressed in one signaling system (usually a cell), and the activities of the two luciferases expressed can be independently measured. In a dual reporter system, changes in the activity of one reporter gene correlate with specific experimental conditions of gene expression, while the constitutive activity of the other reporter gene provides an internal control (internal control) to normalize experimental values. Compared with the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/65C12N15/66
CPCC07K14/43586C12N15/65C12N15/66C12N15/85C12N2800/105C12N2800/60
Inventor 夏庆友刘红玲林英沈关望
Owner SOUTHWEST UNIVERSITY
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