CN105802970A - ShRNA of target-silenced GBeta1 - Google Patents

Abstract

The invention belongs to the technical field of DNA recombination in biological engineering and particularly relates to shRNA of specific-silenced target protein GBeta1. A silencing support of GBeta1 is constructed herein. Firstly, shRNA fragments of 19 bp are designed according to GBeta1 sequence of mouse origin, two ends of the synthesized fragments are provided with suitable restriction enzyme cutting sites, a target fragment is connected to pSUPER support to form a core silencing support, and a male clone is selected for PCR (polymerase chain reaction) verification after transformation. A correct support is verified for DNA sequencing. Then, silencing efficiency is detected through immunoblotting test. Two shRNA fragments capable of targeting at 19 bp of mouse origin GBeta1 are synthesized and designed herein, and the shRNA fragments include GCATCTGGCAAAGATTTAT and GGATGACAATCAGATAGTT.

Description

The shRNA of targeted silent G β 1

Technical field

The invention belongs to the DNA recombinant technique field in biological engineering, be specifically related to the shRNA of specificity silence target proteins G β 1.

Background technology

G-protein β subunit 1(Gprotein β subunit1, G β 1) by Gnb1 gene code, it is the important component part of heterotrimeric G protein, it is possible to and forming dimer with G γ subunit, the common extracellular signal that participates in is at intracellular transductive process.G β 1 is formed seven WDdomain repeated by 340 amino acid residues, and protein sequence forms propeller-type structure after space folding.Due to the wide spectrum expression characterization of G β 1, many researchs about G β γ are all with G β 1 representatively property analysis.Research report proves that G β 1 can participate in the various kinds of cell biological event such as the formation of spindle axis in mitosis process, the migration of neutrophilic granulocyte, cell chemotaxis

The function of the G β γ subunit that G β 1 participates in composition has broad spectrum activity and representativeness, is the one studying the most general G β subunit.Recent studies indicate that, G β 1 can participate in the chemotactic of cell, can regulate formation and the motion transition process of spindle axis in ontogenetic process simultaneously, embryo's ontogeny is had important effect.So the careful accurate functional study in ontogenetic process and mechanism analysis can be played most important by specific reticent G β 1.

Summary of the invention

It is an object of the invention to synthesize specifically can the specific base sequence of targeted silent G β 1, provide effective means for more deep research G β 1 and its effect in cell signalling and cell movement.

What mainly build required in the present invention is the silent carrier of G β 1.Core silent carrier builds and is divided into two big steps, first it is the shRNA fragment that goes out 19bp of G β 1 sequential design according to Mus source, synthesized fragment two ends are with suitable restriction enzyme site, purpose fragment is connected on pSUPER carrier, form the silent carrier of core, after conversion, picking positive colony carries out PCR qualification.Identify correct carrier, carry out DNA sequencing, carried out the detection of silence efficiency subsequently by immunoblotting analysis experiment.

In the present invention, design has synthesized two can shRNA, the shRNA sequence of 19bp of targeted mouse source G β 1 be GCATCTGGCAAAGATTTAT and GGATGACAATCAGATAGTT

Can the structure of expression vector of specificity silence G β 1 comprise the following steps:

The first step, shRNA fragment sequence designs

The sequence of corresponding software design specific recognition G β 1 is applied according to Mus source G β 1 gene order.

Second step, the synthetic of double-strand shRNA fragment

The two of synthetic Sense and Antisense are formed double-stranded DNA by degeneration, annealing.

3rd step, the structure of silent carrier

After silent carrier pSUPER double digestion, utilize and reclaim test kit recovery linearized vector, be attached reclaiming the fragment shRNA fragment with annealing gained, convert, identify that positive colony, order-checking obtain the silent carrier successfully constructed.

4th step, the detection of reticent target protein efficiency

Extract plasmid, extract albumen after transfectional cell, utilize immune-blotting method silence efficiency.

Accompanying drawing illustrates:

Fig. 1: pSUPER carrier double digestion gel electrophoresis figure；

Fig. 2: bacterium solution PCR result gel electrophoresis figure, 1-5 is that G β 1shRNA159 identifies clone, and 8-12 is that G β 1shRNA456 identifies clone, is positive findings in white box；

Fig. 3: WesternBlot silence efficiency testing result.

The present invention designs the shRNA fragment of the 19bp of synthesis, it is possible to specific reticent G β 1, G β 1 is the important component part of trimer G-protein.Showing after deliberation, G β 1 plays an important role in ontogeny and cell movement process, the carrier successfully constructed, it is possible to specific reticent G β 1, reduces the expression of G β 1 in cell.Therefore, its function in ontogeny cell movement process of research that the application of G β 1 silent carrier can be more fine, for explaining that related mechanism is provided fundamental basis.

Detailed description of the invention:

Embodiment one: the expression vector establishment of specificity silence target proteins G β 1

1, the sequential design of specificity silence target protein G β 1

(1) designing silencing sequence according to Mus source G β 1 gene expression characteristics, the G/C content of the sequence of the specific binding G β 1 of the principle of design: 19bp is 45%-55%, annealing temperature 45 DEG C-65 DEG C, and first base simultaneously requiring sequence initial is G.The fragment chosen is carried out BLAST human genome comparison, selects specific sequence.

(2) synthesizing one section of 59bp sequence with Bgl II-GN18-TT-loop-81NC-Hind III form, 81NC is the reverse complemental of NG18, and 5 ' ends are Bgl II restriction enzyme site, and 3 ' ends are Hind III restriction enzyme site.The cDNA sequence 159-177(G β 1shRNA159 of the fragment of design synthesis targeting G β 1 respectively) and 456-474(G β 1shRNA456)

2, the synthesis of the shRNA sequence of targeted silent G β 1 and storage

(1) two sections of oligo of synthesis are dissolved to 50 μMs, respectively take 10 μ l mixing.

(2) 95 DEG C of degeneration 5min, hatch 10min for 72 DEG C, close PCR instrument.

(3) take out mixing, be placed on standby on ice, or-20 DEG C long-term preservations.

3, the structure of silent carrier pSUPER-G β 1

(1) take 2 μ gpSUPER carriers, Bgl II and Hind III double digestion, electrophoresis, reclaim linear pSUPER large fragment [Fig. 1]

(2) connect and convert.By linearisation pSUPER concentration dilution to 100ng/ μ, coupled reaction (the T4DNA ligase of Promege company)

4 DEG C overnight connect, and will connect product and convert DH5 α competent cell, and be coated on the LB flat board with Kan resistance.

(3) positive clone identification.Shaking bacterium converting picking monoclonal on flat board, test kit extracts plasmid, identifies positive colony [Fig. 2] by PCR.Identify that primer is pSUPERtest-P1:5 '-gcgtgaattcgaacgctgac-3 ', pSUPERtest-R:5 ' gaattttaacaaaatattaacgctt-3 '.With pSUPERtest-P1 for sequencing primer, send Beijing Hua Da gene sequencing.

Embodiment two:

The detection of reticent target protein efficiency

(1) control and G β 1shRNAs is entered NLT cell by electrotransfection

(2) extracting total protein of cell, utilize Westernblot immunoblotting assay, after proceeding to G β 1 silent carrier, G β 1 expression significantly reduces, and α-Tubulin is internal reference [Fig. 3].

Sequence table

<110>Northeast Normal University

<120>shRNA of targeted silent G β 1

<141>2015-01-15

<160>19

<210>2

<211>19

<212>

<213>artificial sequence

<220>

<221>misc_RNA

<222>(1)...(19)

<223>

<400>1

GCATCTGGCAAAGATTTAT and GGATGACAATCAGATAGTT.

Claims (1)

1. the shRNA of targeted silent G β 1, is characterized in that shRNA sequence is GCATCTGGCAAAGATTTAT and GGATGACAATCAGATAGTT.