WO2019033249A1 - Shrna of human btla gene and use thereof - Google Patents

Abstract

The invention relates to a shRNA of the human BTLA gene and its use. The shRNA of the human BTLA gene comprises an RNA-BTLA interference consisting of a sequence coding for SEQ ID NO: 1 and a sequence coding for SEQ ID NO: 2. The shRNA of the human BTLA gene can be used in the preparation of a medicament for the treatment of a disease associated with abnormal expression of the BTLA gene.

Description

 Instruction Name: Human shRNA and application of BTLA gene

 [0001] The present invention relates to a human BTLA gene, and more particularly to a shRNA (short hair clip R NA) of a human BTLA gene and use thereof.

 Background technique

 [0002] B/T lymphocyte attenuating factor (BTLA) is the third inhibitory molecule found in the CD28 family, and is mainly expressed on the cell surface of B cells, T cells, macrophages, and the like. BTLA has a similar structure to CTLA-4 and PD-1, but their expression characteristics are different. CTLA-4 and PD-1 are not expressed on resting T cells, and expression is gradually increased after activation, while BTLA is at rest. Constitutive expression on T cells, continued expression after activation. technical problem

 [0003] BTLA signaling is capable of inhibiting CD3 activated T cell proliferation and secretion of IL-2 and IFN-γ. Research shows that B

TLA plays an important role in maintaining peripheral immune tolerance and transplant immunity, and has a good clinical transformation prospect, which needs further study.

 Problem solution

 Technical solution

 One of the technical problems to be solved by the present invention is to provide a shRNA of a human BTLA gene.

[0005] The second technical problem to be solved by the present invention is to provide a shRNA of human BTLA gene.

 In the preparation of a medicament for treating a disease associated with abnormal expression of the BTLA gene.

[0006] In order to solve the above technical problem, the present invention provides a shRNA of a human BTLA gene, including:

EQ ID NO: The sequence shown by 1 and the BTLA-RNAi consisting of the sequence shown in SEQ ID NO: 2.

 Advantageous effects of the invention

 Beneficial effect

The shRNA provided by the invention has the advantages of high transduction efficiency, high and specific inhibition of BTLA gene expression in Jurkat cells, and can be used as a powerful tool for preparing a medicament for treating diseases related to abnormal expression of BTLA gene. Brief description of the drawing

 DRAWINGS

 Figure 1 is a schematic diagram showing the results of Quantitative PCR detection of Jurkat cells after transduction of pLKO-BTLA vector.

 BEST MODE FOR CARRYING OUT THE INVENTION

 BEST MODE FOR CARRYING OUT THE INVENTION

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

 [0010] Jurkat cells were purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences, Trizol was purchased from Invitrogen, and Endo-free Plasmid Mini Kit was purchased from Omega bio-tek. The complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.

 [0011] Example 1 Design of shRNA Oligonucleotide Sequence Targeting BTLA Gene

 [0012] (1) Designing a silent sequence according to the characteristics of human BTLA gene, the principle of design: 19 bp specific binding B TLA sequence GC content is 45<3⁄4-55<3⁄4, annealing temperature 45 °C-65 °C, the same The first base of the start of the sequence is required to be zero. The selected fragments were BLAST aligned with the human genome to ensure specificity.

 [0013] (2) Design a 59 bp sequence BTLA-RNAi in the form of Age I-GN18-TT-loop-81NC-EcoR I, 81NC is the reverse sequence of NG18, and the 5' end is the Age I restriction site, 3' The end is an EcoR I restriction site, the sense strand sequence is shown in SEQ ID No: 1, and the antisense strand sequence is shown in SEQ ID No: 2. The BTLA-RNAi oligonucleotide chain was synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.

 Example 2 Construction of pLKO-BTLA Vector

 [0015] The synthesized shRNA oligonucleotides are mixed in equal amounts in a single strand and annealed to form a double-stranded BTLA-RNAi.

[0016] pLKO.l-puro vector (Sigma Aldrich) full length 7086 bp, restriction endonuclease site selection Age

I and EcoR I are used as insertion sites for DNA fragments, and the specific steps for constructing each vector using different shRNA corresponding DNA fragments are as follows:

 [0017] 1) Age l and EcoR I double-cleavage pLKO.l-puro vector, Fermentas restriction enzymes Age I and EcoR I double digestion, 50 reaction system is as follows: 5 μL 10x FastDigest Buffer, 2 μL Age I , 2 EcoR I, 2 g pLK0.1-puro plasmid, ddH20 supplemented to 20ul, 37 ° C reaction for 30 min;

[0018] 2) ligation, 2 L annealing of phosphorylated double-stranded DNA oligos, 1 ligase buffer, 1 double-digested pLKO.l-puro plasmid, 1 ligase, 5 L ddH20, reaction at 16 ° C for 5 h, Get connected pLKO-BTLA vector;

[0019] 3) conversion;

 [0020] 4) pick positive clone culture and perform sequencing to verify that the insertion sequence is completely correct, then use Endo-free

Plasmid Mini Kit for endotoxin-free pLKO-BTLA vector extraction.

Example 3 The pLKO-BTLA vector transduced Jurkat cells.

[0022] Jurkat cells cultured, the growth state of the cell to take good 5,000,000, the cells were collected by centrifugation, then resuspended in 500 μL PBS is added and after mixing the cuvette 20 μ β _pLKO-BTLA vector, Invitrogen Neon electroporation application system Electrotransfer, electroporation procedure: 2.1 KV, 25 μΐΌ, pulse shock once; Transfer the cells to a 6 cm dish containing 5 mL DMEM complete medium, gently shake the dish to mix the cells, and detect the expression of TL6 gene after 48 h.

 [0023] Example 4 Fluorescence quantitative PCR was used to detect the expression level of BTLA gene.

 [0024] Jurkat cells inoculated with Jurkat cells and transduced pLKO-BTLA vectors were separately cultured into cell culture flasks, and after 24 hours of culture, total RNA of each group was extracted with Trizol, and mRNA was reverse-transcribed into cDNA using PrimeScrip RT reagent Kit. The cDNA was diluted with 90 RNase-Free dH20 and stored at -20 °C.

 [0025] taking cDNA 1 of each group of cells

 For the template, GAPDH was used as the internal reference, and the relative expression of BTLA 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 SYBR Primescript RT-PCR Kit was used to detect the relative expression of BTLA gene in each group. The results are shown in Figure 1. The results showed that the expression of BTLA gene was significantly inhibited in Jurkat cells transduced with pLKO-BTLA vector, and the inhibitory efficiency of the interference fragment on the target gene was 82.4%±7.6<3⁄4, which proved that the p LKO-BTLA vector used in this experiment can carry shRNA energy. Specific inhibition of BTLA gene expression, and the inhibition effect is very significant industrial applicability

 The shRNA provided by the present invention has the advantages of high transduction efficiency, and can efficiently and specifically inhibit the expression of BTLA gene in Jurkat cells, and can be used as a powerful tool for preparing a medicament for treating diseases related to abnormal expression of BTLA gene.

Claims

Claim  [Claim 1] A shRNA of human BTLA gene, comprising: BTLA-RNAi consisting of a sequence encoding SEQ ID NO: 1 and a sequence encoding SEQ ID NO: 2.  [Claim 2] The use of the shRNA of the human BTLA gene according to claim 1 for the preparation of a medicament for treating a disease associated with abnormal expression of BTLA gene.  [Claim 3] The use according to claim 2, wherein: the shRN of the human BTLA gene  A is BTLA-RNAi.