WO2018170764A1

WO2018170764A1 - Vector for rnai and application thereof

Info

Publication number: WO2018170764A1
Application number: PCT/CN2017/077606
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: a GP34-RNAi sequence and a recombinant vector containing the sequence, the GP34-RNAi sequence being as shown in SEQ ID NO:1; a recombinant vector containing a sequence as shown in SEQ ID NO:2; and an RNA interference vector of GP34 gene and 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

Title of Invention: A Vector for RNAi and Its Application

Technical field

[0001] The present invention belongs to the field of genetic engineering, and relates to a vector for RNAi and application thereof, and particularly to an RNA interference vector of GP34 gene and application thereof.

Background technique

[0002] GP34 is a member of the TNF receptor superfamily and is a type I transmembrane glycoprotein. The expression profile of GP34 is restricted to the surface of activated CD4+ and CD8+ T cells, and is predominantly CD4+ T cells. OX40/GP34 is an important co-stimulatory molecule that plays an important role in the body's immune response and various diseases, and its interaction can promote CD+4.

Activation, proliferation, and migration of T cells prolong their life span and promote the formation of germinal centers and the differentiation and maturation of DCs.

technical problem

[0003] GP34 can synergistically stimulate the activation of T cells, promote the production of high-valent antibodies and class switching of B cells, mediate the infiltration of OX40+ T cells into the inflammatory reaction site, and play an important role in the immunotherapy of tumors. The research can achieve clinical transformation, but the lack of vectors specifically inhibiting the expression of GP34 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 RNA interference vector of GP34 gene and a construction method and application thereof.

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 GP34- consisting of Bam HI restriction site + target nucleotide sequence + stem loop structure sequence + target nucleotide sequence complementary sequence + termination site sequence + EcoR I restriction site at the cleavage site RNAi sequence.

In another aspect of the present invention, a method for preparing an RNA interference vector of the aforementioned GP34 gene is provided , including the following steps:

[0008] 1) Design of GP34-RNAi sequence: According to the full sequence of GP34 mRNA, the interference fragment was designed according to the design principle of RNAi fragment, NCBI BLSAT was used for homology comparison, and the target nucleotide sequence was obtained after confirming specificity, and obtained as SEQ ID NO: sequence shown by l, named GP34-RNAi sequence;

[0009] 2) Construction and identification of RNA interference vector of GP34 gene: The synthesized GP34-RNAi sequence and the extracted plasmid pLVX-shRNA1 were digested with restriction endonucleases Bam HI and EcoR I, electrophoresis and gelatinization. The vector was recovered, and the GP34-RNAi sequence was ligated into the SJpLVX-shRNA1 expression vector by T4 DNA Ligase to obtain a ligation product; the ligation product was transformed into competent E. coli Stbl3 and uniformly coated onto an ampicillin-containing LB medium plate. The positive monoclonal colonies were picked and cultured and identified by PCR. The preliminary identification results indicated that the GP34-RNAi sequence was inserted into the successful bacterial solution for sequencing and identification;

[0010] 3) Extraction of RNA interference vector of GP34 gene: The GP34-RNAi sequence was confirmed to be inserted into a successful bacterial cell expansion culture, and a large amount of recombinant plasmid was extracted to obtain an RNA interference vector of GP34 gene.

[0011] The present invention utilizes RNAi technology to construct an RNA interference vector targeting GP34 gene expression, and after successful identification, electrotransformation of DC2.4 cells, using the real-time fluorescent quantitative PCR technique to verify the inhibitory effect of GP34 gene expression from the mRNA level, the experiment As a result, it was confirmed that the GP34-RNAi sequence provided by the present invention was successfully inserted into the pLVX-shRNA1 expression vector, and the inhibitory effect on the expression of the GP34 gene was remarkable.

In another aspect of the invention, a polynucleotide set forth in SEQ ID NO: 1 or comprising SEQ ID is provided

The use of the polynucleotide of the polynucleotide represented by 1 or the RNA interference vector of the aforementioned GP34 gene for the drug of the GP34 gene expression abnormality-related disease.

Advantageous effects of the invention

Beneficial effect

The RNA interference vector of the GP34 gene provided by the invention has the advantages of high transfection efficiency, low dosage, high and specific inhibition of GP34 gene expression in DC2.4 cells, and can be used as a powerful tool for preparing and treating the G P34 gene. A drug that expresses an abnormally related disease.

Brief description of the drawing

DRAWINGS

1 is a schematic diagram showing the results of real-time quantitative PCR detection of RNA interference vector cells transfected with GP34 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] DC2.4 cells were purchased from Wuxi Yingnuui Biomedical Technology Co., Ltd., RNAi vector pLVX-shRNAl was purchased from Clontech, RNeasy MiniKit was purchased from Qiagen, and endotoxin free plasmid extraction kit was purchased from Omega bio-tek. The complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.

[0017] Example 1 Design of GP34-RNAi Sequence

[0018] The complete sequence of GP34 mRNA was found in GenBank. According to the full sequence of GP34 mRNA, the interference fragment was designed according to the design principle of RNAi fragment, NCBI BLSAT was used for homology comparison, and the target nucleotide sequence was obtained after confirming specificity.

[0019] The GP34-RNAi sequence was designed as follows: Bam

HI restriction site +19nt target nucleotide sequence + stem loop structure (TTCAAGAGA) + target sequence complementary sequence + R NAPolym polymerase transcription stop site (TTTTTT) + EcoR I restriction site six regions, the sequence of which SEQ ID NO: 1 is shown. The designed GP34-RNAi sequence chain was synthesized by Shanghai Biotech Engineering Services Co., Ltd.

Example 2 Construction of an RNA interference vector for the GP34 gene.

[0021] The synthesized GP34-RNAi sequence and the extracted plasmid pLVX-shRNA1 were digested with restriction endonucleases Bam HI and EcoR I, electrophoresed, and the gel was recovered, and the GP34-RNAi sequence was further amplified by T4 DNA Ligase. An RNA interference vector that forms the GP34 gene is ligated into the vector pLVX-shRNA1. 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 fully consistent with the designed sequence and can be used in subsequent experiments.

[0022] Example 3 Electrical rotation of DC2.4 cells

[0023] DC2.4 cells were cultured, and 35,000 cells in good growth state were taken, and the cells were collected by centrifugation, and then resuspended in 500 L of PBS with 20 g.

The RNA interference vector of GP34 gene was mixed and added to the electric shock cup, and the electric rotation was performed by BTX ECM830 electro-rotation instrument. The electroporation procedure was: 2.1 KV, 25 μ¥Ό, pulse electric shock once; the cells were transferred to 5 mL DMEM. In a 6 cm dish of complete medium, the cells were gently shaken to mix the cells, and the expression of GP34 gene was detected 48 h later.

[0024] Example 4 Fluorescence quantitative PCR detection of GP34 gene expression

[0025] DC2.4 cells of normal DC2.4 cells and RNA interference vector of GP34 gene were cultured for 48 h, respectively, and PrimeScrip RT reagent was used for He ¹ J

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 for later detection. Take the cDNA of each group of cells

Ιμί is the template, GAPDH is used as the internal reference, and the relative expression of GP34 is detected by real-time quantitative PCR (QPCR). The reaction conditions are set: 95 ° C for 10 s, 1 cycle; 95 ° C 5 s, 54 ° C

30s, a total of 40 cycles, using SYBR Prime script RT-PCR Kit to detect the relative expression of GP34 gene in each group, the results are shown in Figure 1. The results showed that the expression of GP34 gene was significantly inhibited in DC2.4 cells transfected with GP34 gene by RNA interference vector. The inhibitory efficiency of GP34-RNAi sequence on the target gene was 71.6%±6.2%, which proved that GP34 was used in this experiment. The RNA interference vector of the gene specifically inhibits the expression of the GP34 gene, and the inhibitory effect is very remarkable.

Industrial applicability

The NP34 gene RNA interference vector provided by the invention has the advantages of high transfection efficiency, low dosage, high and specific inhibition of GP34 gene expression in DC2.4 cells, and can be used as a powerful tool for preparing and treating G P34 gene. A drug that expresses an abnormally related 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 RNA interference vector of the GP34 gene according to claim 3, comprising the steps of:

1) Design of GP34-RNAi sequence: According to the full sequence of GP34 mRNA, the interference fragment was designed according to the design principle of RNAi fragment, NCBI BLSAT was used for homology comparison, and the target nucleotide sequence was confirmed after specificity, and SEQ ID was obtained.

NO: sequence shown by 1, named GP34-RNAi sequence;

2) Construction and identification of RNA interference vector of GP34 gene: The synthesized GP34-RNAi sequence and the extracted plasmid pLVX-shRNA 1 were digested with restriction endonucleases Bam HI and EcoR I, electrophoresis and gel-removed recovery vector. Then, the GP34-RNAi sequence was ligated into the pLVX-shRNA1 expression vector by T4 DNA Ligase to obtain a ligation product; the ligation product was transformed into competent E. coli Stbl3, and uniformly coated onto an ampicillin-containing LB medium plate. The positive monoclonal colonies were picked and cultured and identified by PCR. The preliminary identification results indicated that the GP34-RNAi sequence was inserted into the successful bacterial solution for sequencing and identification;

3) Extraction of RNA interference vector of GP34 gene: The sequencing result confirmed that the GP34-RNAi sequence was inserted into a successful bacterial cell expansion culture, and a large number of recombinant plasmids were extracted to obtain an RNA interference vector of GP34 gene.

Use of the polynucleotide of claim 1 or the recombinant vector of claim 2 or the RNA interference vector of the GP34 gene of claim 3 for the treatment of a disease associated with abnormal expression of GP34 gene.