CN105907760A - shRNA for targeted silencing of TKS4 - Google Patents

Abstract

Belonging to the technical field of DNA recombination in bioengineering, the invention in particular relates to shRNA for specific targeted silencing of TKS4. Firstly, a 19bp shRNA fragment is designed according to a mouse-derived TKS4 sequence, two ends of the synthesized fragment have appropriate enzyme cutting sites, and a target fragment is connected to a pLL3.7 vector to form a core silencing vector, after conversion, positive clone is selected for PCR identification. The vector that is identified correct is subjected to DNA sequencing, and then immunoblotting experiment is carried out to detect silence efficiency. The 19bp shRNA fragment able to target the mouse-derived TKS4 has a sequence of GCAAAGAGTCCATCATCAA.

Description

The shRNA of targeted silent TKS4

Technical field

The invention belongs to the DNA recombinant technique field in bioengineering, be specifically related to selectively targeted reticent TKS4's shRNA。

Background technology

Frank-Ter Haar syndrome (FTHS), is a kind of rare autosomal recessive hereditary diseases, and patient exists perhaps The most abnormal, such as brachycephalism, forehead is prominent, eyes are prominent and eye distance huge cornea wider, congenital, glaucoma, the congenital heart Dirty defect, scoliokyphosis, skeleton development are bad, hypoevolutism etc., all there is also slight cognitive defect it is believed that ill. By confirmation, this disease is caused by TKS4 gene mutation.TKS4 includes a PX domain and four SH3 domains. It is the substrate of Src EGFR-TK, has the sequence of a series of Pro-rich.Document being reported, TKS4 is positioned at sufficient body, can Affect the F-actin location at foot body

Since self-discovery, TKS4 effect in brain is not by numerous studies, and TKS4 is at the brain of Embryonic Stages and nerve Great expression in unit, experiment finds, TKS4 may have impact on the transition process of neuron in embryo development procedure.So it is specific Reticent TKS4 to study its effect in nervous system most important.

Summary of the invention

It is an object of the invention to synthesize the shRNA of specific targeted silent TKS4, for more in-depth study TKS4 and its Effect in cell signalling and cell movement provides effective means.

Mainly build required in the present invention is the silent carrier of TKS4.Core silent carrier build be divided into two big Step, is first the shRNA fragment that goes out 19bp of the TKS4 sequences Design according to mouse source, and synthesized fragment two ends are with suitable enzyme Cutting site, purpose fragment be connected on pLL3.7 carrier, form the silent carrier of core, after conversion, picking positive colony enters Performing PCR is identified.Identify correct carrier, carry out DNA sequencing, carried out the detection of silence efficiency subsequently by immunoblotting analysis experiment.

In the present invention design synthesized one can the shRNA of 19bp of targeted mouse source TKS4, sequence is GCAAAGAGTCCATCATCAA。

Can the structure of the specific expression vector of reticent TKS4 comprise the following steps:

The first step, shRNA fragment sequence designs

The sequence of corresponding Software for Design specific recognition TKS4 is applied according to mouse source TKS4 gene order.

Second step, the Prof. Du Yucang of double-strand shRNA fragment

The two of Prof. Du Yucang Sense and Antisense are formed double-stranded DNA by sex change, annealing.

3rd step, the structure of silent carrier

After silent carrier pLL3.7 double digestion, utilize and reclaim kit recovery linearized vector, fragment and annealing institute will be reclaimed The shRNA fragment obtained is attached, converts, identifies 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 is pLL3.7 carrier double digestion gel electrophoresis figure；

Fig. 2 is TKS4 shRNA bacterium solution PCR result gel electrophoresis figure, is positive findings in frame；

Fig. 3 is that external source detects TKS4shRNA silence efficiency figure.

The present invention designs the shRNA fragment of the 19bp of synthesis, it is possible to specific reticent TKS4.Show after deliberation, TKS4 master Cell foot body region to be distributed in, plays a significant role during cell movement.Simultaneously in ontogenetic process, as lacked TKS4 albumen, will cause serious disease.Therefore, the research that the application of TKS4 silent carrier can be the finest its in ontogeny And the function during cell movement, for explaining that related mechanism is provided fundamental basis.

Detailed description of the invention

Embodiment one: the expression vector establishment of specific reticent target proteins TKS4

1, the sequences Design of specific reticent target protein TKS4

(1) silencing sequence, the sequence of the specific binding TKS4 of the principle of design: 19bp are designed according to mouse source TKS4 gene expression characteristics G/C content be 45%-55%, annealing temperature 45 DEG C-65 DEG C, first base simultaneously requiring sequence initial is G.By choose Fragment carries out BLAST human genome comparison, selects specific sequence.

(2) synthesizing one section of 59bp sequence with Hpa I-GN18-TT-loop-81NC-Xho I form, 81NC is N G18's Reverse complemental, 5 ' ends are Hpa I restriction enzyme site, and 3 ' ends are Xho I restriction enzyme site.The fragment of design synthesis targeting TKS4's respectively CDNA sequence 1574-1592 (TKS4 shRNA1574)

2, the synthesis of the shRNA sequence of targeted silent TKS4 and storage

(1) utilize annealing buffer to be dissolved to 50 M two sections of Oligo of synthesis, respectively take 10 l mixing.

(2) 95 DEG C of sex change 4min, 72 DEG C of annealing 10min, can be placed on 4 DEG C of preservations afterwards.

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

3, the structure of silent carrier pLL3.7-TKS4

(1) take 2 g pLL3.7 carriers, Hpa I and Xho I double digestion, electrophoresis, reclaim linear pLL3.7 large fragment [Fig. 1]

(2) connect and convert.By linearisation pLL3.7 concentration dilution to 100ng/, the coupled reaction (T4 of Promege company DNA ligase)

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, kit extracts plasmid, is identified by PCR Positive colony [Fig. 2].Identify that primer is pLL3.7 test-F:5 '-GCACAGACTTGTGGGAGAAG-3 ', pLL3.7 test- R:5’CCTGCTGGAATCTCGTGAA-3’.With U6 promoter as sequencing primer, send Beijing Hua Da gene sequencing.

Embodiment two:

The detection of reticent target protein efficiency

(1) control and TKS4 shRNAs is entered in neuron by electrotransfection.

(2) extract total protein of cell, utilize Western blot immunoblotting assay, after proceeding to TKS4 silent carrier, TKS4 expression significantly reduces, and α-Tubulin is internal reference [Fig. 3].

Sequence table

<110>Northeast Normal University

<120>shRNA of targeted silent TKS4

<141> 2015-12-22

<160>19

<210> 2

<211> 19

<212>

<213>artificial sequence

<220>

<221> misc_RNA

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

<223>

<400> 1

GCAAAGAGTCCATCATCAA。

Claims (1)

1. the shRNA of targeted silent TKS4, is characterized in that: sequence is GCAAAGAGTCCATCATCAA.