WO2019037057A1 - SHRNA TARGETING SILENT DD1α - Google Patents

Abstract

A shRNA that specifically silences the target protein DD1α is provided. Constructed is a silencing vector of DD1α. Firstly, a 19 bp shRNA core fragment was designed based on the sequence of human DD1α gene. The synthesized fragment was ligated with a suitable restriction site at both ends, and the target fragment was ligated to the pSUPER vector to form a core silencing vector. After transformation, pick and extract Positive clones were identified by PCR. The correct vector was identified, DNA sequencing was performed, and then the detection of silencing efficiency was performed by immunoblotting experiments. A 19 bp shRNA core fragment capable of targeting human DD1α was designed and synthesized, and its sequence is 5'-GGTGCAGACAGGCAAAGAT-3'.

Description

 Manual Title: Targeted Silencing DDla shRNA

 Technical field

 [0001] The present invention belongs to the field of DNA recombination technology in bioengineering, and specifically relates to shRNA which specifically silences the target protein D Dla.

 Background technique

 [0002] DDla protein is a new member of the PD-L1 protein family and is a novel and structurally different Ig superfamily inhibitory ligand whose extracellular domain has homology to the B7 family ligand PD-L1.

 technical problem

 [0003] Existing studies have found that DDla protein plays an important role in various cancers and autoimmune diseases, allergies, infections and inflammatory diseases such as multiple sclerosis and cellular immunity of joint disorders, and requires extensive transformation studies for clinical application. However, the lack of lentiviral vectors that specifically inhibit the expression of DDla gene in the prior art has made the related studies not well developed.

 Problem solution

 Technical solution

 [0004] The object of the present invention is to synthesize a specific base sequence capable of targeting silencing DDla, and provides an effective means for further research on the action of DDla.

 [0005] The main construct required in the present invention is a silencing vector of DDla. The construction of the core silencing vector is divided into two major steps. The first step is to design a 19 bp shRNA core fragment based on the human DDla sequence. The synthesized fragment has appropriate restriction sites at both ends, and the target fragment is ligated to the pSUPER vector to form The core silencing vector, after transformation, pick positive clones for PCR identification. The correct vector was identified, DNA sequencing was performed, and silencing efficiency was subsequently detected by immunoblotting.

 In the present invention, a 19 bp shRNA core fragment capable of targeting human DDla was designed and synthesized, and its sequence was 5,-GGTGCAGACAGGCAAAGAT-3, (SEQ ID No: 1).

 [0007] Construction of an expression vector that specifically silences DDla includes the following steps:

 [0008] First step, shRNA fragment sequence design

[0009] The sequence of DDla is specifically identified based on the human DDla gene sequence using the corresponding software design. [0010] The second step, the artificial synthesis of double-stranded shRNA fragments

 [0011] The artificially synthesized Sense (SEQ ID No: 2) and Antisense (SEQ ID No: 3) were denatured and annealed to form double-stranded DNA.

[0012] The third step, the construction of the silent carrier

 After double-clearing the silencing vector pSUPER, the linearized vector is recovered by using a recovery kit, and the recovered fragment is ligated with the shRNA fragment obtained by annealing, transformed, and the positive clone is identified, and the successfully constructed silencing vector is obtained by sequencing.

 [0014] The fourth step, detection of silencing target protein efficiency

 [0015] The plasmid was extracted, and the total RNA was extracted after transfecting the cells. After reverse transcription into cDNA, the silencing efficiency was detected by quantitative PCR.

 Advantageous effects of the invention

 Beneficial effect

 [0016] The shRNA fragment designed in the present invention can specifically silence DDloc. The experimental results show that the successful construction of the vector can specifically silence DDlot and reduce the expression of DDlot in cells. Therefore, the application of DDloc silencing vector can study the function of DDloc more elaborately, providing a theoretical basis for explaining related mechanisms.

 [0017].

 Brief description of the drawing

 DRAWINGS

 1 is a schematic diagram showing the results of quantitative PCR detection of DDla gene expression of Jurkat cells after transfection of pSUPER-DDla vector.

 BEST MODE FOR CARRYING OUT THE INVENTION

 BEST MODE FOR CARRYING OUT THE INVENTION

[0019] The present invention will be further described below in conjunction with the drawings and specific embodiments.

 [0020] Jurkat cells were purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences, and the RNeasy Mini Kit was purchased from Qiagen.

. The complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.

[0021] Example 1 Design of shRNA Oligonucleotide Sequences [0022] The full sequence of DDla mRNA was found in GenBank, and the specificity was confirmed by BLAST homology alignment. The secondary structure of the target mRNA sequence was evaluated by RNAstmcture 4.4 software, and finally the target nucleotide sequence, such as SEQ ID, was obtained. NO: 1 is shown.

[0023] The 59 bp sequence was designed in the form of Bgl II-GN18-TT-loop-81NC-Hind III, 81NC is the reverse complement of NG18, the 5' end is the Bgl II restriction site, and the 3' end is the Hind III cleavage site. Point, the obtained sequences are respectively SEQ ID

NO: 2 and SEQ ID NO: 3.

Example 2 Construction of shRNA Lentiviral Expression Vector

 [0025] The synthesized shRNA oligonucleotides are mixed in equal amounts in a single strand and annealed to form a double-stranded shRNA. Take 2 g

 The pSUPER vector, Bgl II and Hind III were digested, electrophoresed, and the linear pSUPER large fragment was recovered. Dilute the linearized pSUPER concentration to 100

 Ng/μί, and the double-stranded shRNA obtained by annealing were ligated overnight at 4 °C with T4 DNA ligase (NEB). The ligation product was transformed into ToplO competent cells and plated on LB plates with Kan resistance. Monoclonal shakes were picked on the transformation plates and cultured overnight before being sent to Shanghai Biotech for sequencing. The correct bacteria were sequenced for the extraction of endotoxin-free plasmids, and the pSUPER-DDla vector was obtained.

Example 3 The pSUPER-DDla vector transduced Jurkat cells.

[0027] Jurkat cells were cultured, and cells grown in good condition were inoculated into a six-well plate at 10 ⁶ cells per well, and the cell density was about 60% after 18 h. The extracted pSUPER-DDla vector was transferred to 3 g using Lipofectamin 2000. Guide Jurkat cells. After 6 h, the medium was changed to fresh complete medium and cultured for 48 h.

 [0028] Example 4 DDlot gene expression level detection

 Jurkat cells and Jurkat cells transduced with pSUPER-DDla vector were separately inoculated into 6-well plates. Cell density reached 80<3⁄4-90<3⁄4吋, total RNA was extracted from each group using RNeasy Mini Kit, using PrimeScrip RT reagent

 Kit reverse transcribes mRNA into cDNA, reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C, ∞. After the reverse transcription was completed, the cDNA was diluted with 90 μl of RNase-Free dH20 and stored at -20 °C.

[0030] taking cDNA 1 of each group of cells

For the template, GAPDH was used as the internal reference, and the relative expression of DDlot was detected by real-time quantitative PCR (QPCR). The reaction conditions were set: 95 ° C for 10 s, 1 cycle; 95 ° C for 5 s, 60 ° C for 30 s, for 40 cycles. WoU used SYBR Primescript RT-PCR Kit to detect DDlot in each group The relative expression of the gene, the results are shown in Figure 2. The results show that Jurkat cells transduced with pSUPER- DDlot vector, DDla

 Gene expression was significantly inhibited, and the inhibitory efficiency of the interference fragment was 81.9%±6.3<3⁄4, which proved that the pSUPER-DDla vector used in this experiment can specifically inhibit the expression of DDla gene, and the inhibitory effect is very significant.

 The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and it is not intended that the specific embodiments of the invention are limited to the description. It will be apparent to those skilled in the art that the present invention may be practiced without departing from the spirit and scope of the invention.

 Industrial applicability

 [0032] The shRNA fragment synthesized in the present invention is designed to specifically silence DDla. The experimental results show that the successful construction of the vector can specifically silence DDlot and reduce the expression of DDla in cells. Therefore, the application of DDla silencing vector can more closely study the function of DDla, providing a theoretical basis for explaining related mechanisms.

 [0033].

Claims

Claim  [Claim 1] A shRNA targeting a human DDlot gene, characterized in that the shRNA core sequence is 5'- GGTGCAGACAGGCAAAGAT -3,.