WO2018170762A1

WO2018170762A1 - Rnai expression vector of tnlg5a gene, construction method therefor, and application thereof

Info

Publication number: WO2018170762A1
Application number: PCT/CN2017/077604
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2017-03-22
Filing date: 2017-03-22
Publication date: 2018-09-27

Abstract

Provided are an RNAi expression vector of TNLG5A gene, a construction method therefor, and an application thereof. Provided are: an isolated polynucleotide having a sequence as shown in SEQ ID NO:1; a vector containing the polynucleotide as represented by SEQ ID NO:1; a recombinant vector containing a sequence as shown in SEQ ID NO:2; and an RNAi expression vector of TNLG5A gene and a preparation method therefor. The preparation method comprises: using pLVX-shRNA1 as a skeleton vector, and inserting in a recombinant site an shRNA oligonucleotide sequence consisting of a Bam HI enzyme cutting site + a target nucleotide sequence + a stem-loop structure sequence + a target nucleotide sequence complementary sequence + a termination site sequence + an EcoR I enzyme cutting site in sequential connection.

Description

Description: The RNAi expression vector of TNLG5A gene, construction method and application thereof

[0001] The present invention belongs to the field of genetic engineering, and relates to an RNAi expression vector, a construction method and application thereof, and particularly relates to an RNAi expression vector of TNLG5 A gene, a construction method and application thereof.

Background technique

[0002] ILA belongs to the TNFR superfamily and is mainly expressed in activated T cells and is an inducible T cell surface receptor; TNLG5A belongs to the TNF superfamily and is mainly expressed in concentrated antigen presenting cells (APC). IL A/TNLG5A is another important costimulatory molecule other than CD28/B7, which may or may not be dependent on CD28/

The B7 pathway mediates the production of costimulatory signals that induce T cell activation, proliferation, and cytokine secretion. technical problem

[0003] ILA and its ligand system have two-way signal transduction, which can transmit cells to T cells through TNLG5A, and can transmit signals to cells expressing ligands, which plays an important role in tumor immunotherapy, and needs to be done. A large number of studies can achieve clinical transformation, but the lack of RN Ai expression vectors that specifically inhibit the expression of TNLG5A gene in the prior art makes the related research not well developed.

Problem solution

Technical solution

[0004] The object of the present invention is an RNAi expression vector, a construction method and application thereof of the TNLG5A gene.

In a first aspect of the invention, an isolated polynucleotide is provided, the nucleotide sequence of which is set forth in SEQ ID NO: 1.

In another aspect of the invention, there is provided a recombinant vector comprising the nucleotide sequence set forth in SEQ ID NO: 2, wherein the recombinant vector is a pLVX-shRNA1 vector as a backbone vector, in a polyclonal enzyme A shRNA oligo consisting of a Bam HI restriction site + a target nucleotide sequence + a stem loop structure sequence + a target nucleotide sequence complementary sequence + a stop site sequence + an EcoR I restriction site at the cleavage site Nucleotide sequence.

In another aspect of the present invention, a method for preparing an RNAi expression vector of the aforementioned TNLG5 A gene is provided, comprising the steps of:

[0008] A) Design of shRNA oligonucleotide sequence: according to the full sequence of TNLG5A mRNA, by BLAST The source alignment confirmed the specificity and then used the RNA structure software to evaluate the secondary structure of the target mRNA sequence to obtain the target nucleotide sequence, and designed and synthesized the shRNA oligonucleotide sequence targeting the TNLG5A gene.

[0009] B) Construction and identification of RNAi expression vector of TNLG5A gene: The single strand of the synthesized shRNA oligonucleotide is mixed in an equimolar ratio, annealed to form a double-stranded shRNA, and the plasmid pLVX-shRNA1 is extracted, and restriction endonuclease is used. Enzyme Bam HI, EcoR I double digestion, electrophoresis, decapitation recovery vector, and then double-stranded shRNA was ligated into pLVX-shRNA1 expression vector by T4 DNA Ligase to obtain ligation product; the ligation product was transformed into competent E. coli Stbl3 Medium, uniformly applied to the ampicillin-containing LB medium plate, picking up the positive monoclonal colony culture and carrying out PCR identification, and preliminary identification results indicate that the shRNA oligonucleotide sequence was inserted into the successful bacterial solution for sequencing and identification;

[0010] C) Extraction of the RNAi expression vector of the TNLG5 A gene: The sequencing result confirmed that the shRNA oligonucleotide sequence was inserted into the successful bacterial cell expansion culture, and a large number of recombinant plasmids were extracted to obtain the RN Ai expression vector of the TNLG5A gene. .

[0011] The present invention utilizes RNAi technology to construct an RNAi expression vector targeting TNLG5A gene expression. After successful identification, the Jurkat cells are electroporated, and the inhibitory effect of TNLG 5A gene expression is verified from the mRNA level by using real-time fluorescent quantitative PCR technology. The shRNA oligonucleotide provided by the present invention was successfully inserted into the pLVX-shRNA1 expression vector, and the inhibitory effect on the TNLG5A gene expression was remarkable.

In another aspect of the present invention, a polynucleotide represented by SEQ ID NO: 1 or a vector comprising the polynucleotide represented by SEQ ID NO: 1 or an RNAi expression vector of the aforementioned TNLG5A gene is provided in TNLG5A Use in drugs for diseases with abnormal gene expression.

Advantageous effects of the invention

Beneficial effect

[0013] The RNAi expression vector of the TNLG5 A gene provided by the invention has the advantages of high transfection efficiency, low dosage, high and specific inhibition of TNLG5A gene expression in Jurkat cells, and can be used as a powerful tool for preparing and treating TNLG5A gene expression abnormality. Drugs related to the disease.

Brief description of the drawing

DRAWINGS

1 is a schematic diagram showing the results of real-time quantitative PCR detection of RNAi expression vector cells transfected with TNLG5A gene. BEST MODE FOR CARRYING OUT THE INVENTION

BEST MODE FOR CARRYING OUT THE INVENTION

[0015] The present invention will be further described below in conjunction with the drawings and specific embodiments.

[0016] Jurkat cells were purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences. The RNAi vector pLVX-shRNA1 was purchased from C1 ontech, RNeasy MiniKit was purchased from Qiagen, and the endotoxin-free plasmid extraction kit was purchased from Ome ga bio-tek. The complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.

[0017] Example 1 Design of shRNA Oligonucleotide Sequence Targeting TNLG5A Gene

[0018] The full sequence of SJTNLG5A mRNA was searched 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 was obtained.

[0019] Synthetic shRNAs are designed based on the target nucleotide sequence. The design is as follows: B _a m HI cleavage site +19 _n t target nucleotide sequence + stem loop structure (TTCAAGAGA) + target sequence complementary sequence + RNAPolym polymerase transcriptional stop site (TTTTTT) + EcoR I cleavage site Six regions, the sequence of which is shown in SEQ ID NO: 1. The designed shRNA oligonucleotide chain was synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.

Example 2 Construction of an RNAi expression vector for the TNLG5A gene.

[0021] The synthetic shRNA oligonucleotides are mixed in equal amounts in a single strand, annealed to form a double-stranded shRNA, and the plasmid pL VX-shRNA1 is extracted, and digested with restriction endonucleases Bam HI, EcoR I, electrophoresis, gelatinization The vector was recovered, and the double-stranded shRNA was ligated into the vector pLVX-shRNA1 with T4 DNA Ligase to form an RNAi expression vector of the TNLG5A gene. The ligation product was transformed into competent E. coli Stbl3, plated on a plate containing ampicillin LB medium, and cultured at 37 ° C for 14 h. Pick up the grown colonies and send them to Shanghai Biotech for sequencing. The sequencing results are in complete agreement with the designed sequence and can be used in subsequent experiments.

[0022] Example 3 Electrostatic transfer of Jurkat cells

[0023] Jurkat cells were cultured, and 5,000,000 cells in good growth state were taken, and the cells were collected by centrifugation, and then resuspended in 500 μL of PBS, mixed with the RNAi expression vector of 20 μ _β TNLG5 Α gene, and then added to an electric shock cup, and BTX ECM830 was applied. Electrorotation instrument for electrical rotation, electroporation procedure: 2.5 KV, 25 FD, pulse shock once; transfer cells to 6 mL of DMEM complete medium

In the cm dish, the cells were gently shaken to mix the cells, and the expression of TNLG5A gene was detected 48 h later. Example 4 Detection of TNLG5A Gene Expression by Fluorescence Quantitative PCR

[0025] Jurkat cells of normal Jurkat cells and RNAi expression vector of TNLG5A gene were cultured for 48 h, and total RNA of each group was extracted with RNeasy Mini Kit, and mRNA was reverse-transcribed into cDNA using PrimeScript RT reagent Kit. 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 for later detection. Take cDNA 1 from each group of cells

For the template, GAPDH was used as the internal reference, and the relative expression of TNLG5A 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, 54 ° C for 30 s, for 40 cycles. The relative expression of TNLG5 A gene in each group was detected by SYBR PrimeScript RT-PCR Kit. The results are shown in Figure 1. The results showed that the expression of TNLG5 A gene was significantly inhibited in Jurkat cells transfected with the RNAi expression vector of TNLG5 A gene. The inhibition efficiency of shRNA fragment on the target gene was 77.1%±6.8<3⁄4, which proved that the RNA of TNLG5A gene used in this experiment was RNAi. The expression vector can specifically inhibit the expression of the TNLG5A gene, and the inhibitory effect is very remarkable.

The above is a further detailed description of the present invention in conjunction with the specific preferred embodiments. 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

The RNAi expression vector of the TNLG5 A gene provided by the invention has the advantages of high transfection efficiency, low dosage, high and specific inhibition of TNLG5A gene expression in Jurkat cells, and can be used as a powerful tool for preparing and treating TNLG5A gene expression abnormality. Drugs related to the disease.

Claims

Claim

An isolated polynucleotide characterized by having a nucleotide sequence as shown in SEQ ID NO: 1.

A vector comprising the polynucleotide of claim 1.

A recombinant vector containing SEQ ID

The sequence shown by NO: 2, wherein the recombinant vector is pLVX-shRNA1 as a backbone vector, and the Bam HI cleavage site + target nucleotide sequence 歹 ij + stem loop structure sequence is inserted at the recombination site. ShRNA oligonucleotide sequence consisting of a target nucleotide sequence complementary sequence + a stop site sequence + an EcoR I restriction site.

A method for preparing an RNAi expression vector of the TNLG5A gene according to claim 3, comprising the steps of:

A) Design of shRNA oligonucleotide sequence: According to the full sequence of TNLG5A mRNA, the specific post-application RNA was confirmed by BLAST homology alignment.

_S tmct _ure 4.4 software evaluates the secondary structure of the target mRNA sequence to obtain a target nucleotide sequence, and designs and synthesizes a shRNA oligonucleotide sequence targeting the TNLG5 A gene;

B) Construction and identification of the RNAi expression vector of the TNLG5A gene: The single strand of the synthesized shRNA oligonucleotide is mixed in an equimolar ratio, annealed to form a double-stranded shRNA, and the plasmid pLV X-shRNA1 is extracted, and the restriction endonuclease Bam is used. HI, EcoR I double digestion, electrophoresis, gel extraction vector, and then double-stranded shRNA was ligated into SJpLVX-shRNA 1 expression vector with T4 DNA Ligase to obtain ligation product; the ligation product was transformed into competent E. coli Stbl 3 Uniformly applied to the ampicillin-containing LB medium plate, picking up the positive monoclonal colony culture and carrying out PCR identification, and preliminary identification results indicate that the shRNA oligonucleotide sequence was inserted into the successful bacterial solution for sequencing and identification;

C) Extraction of RNAi expression vector of TNLG5A gene: The sequencing result confirmed that the shRNA oligonucleotide sequence was inserted into a successful bacterial cell expansion culture, and a large amount of recombinant plasmid was extracted to obtain an RNAi expression vector of TNLG5A gene.

The polynucleotide of claim 1 or the recombinant vector of claim 2 or the RNAi expression vector of the TNLG5 A gene of claim 3 for treating TNLG5 A gene expression The application of often related diseases.