WO2018170712A1 - Method for constructing human programmed death ligand 2 gene high-expression vector and application thereof - Google Patents

Abstract

Provided are a method for constructing a human programmed death ligand 2 (PD-L2) gene high-expression vector and an application thereof. The construction method comprises the steps of: 1) amplification and purification of a target gene: designing an upper primer and a lower primer, extracting a cell RNA, using a cDNA subjected to reverse transcription as a template for performing PCR amplification, cutting off a target strip, and using a kit for recovery and purification; and 2) construction and identification of a recombinant plasmid pCDNA3.1-PD-L2: performing double enzyme digestion on the purified PCR product and a vector pCDNA3.1 with Xba I and EcoR I, recovering and purifying an enzyme-digested product with the kit separately, ligating fragments, and screening, culturing, cloning, and sequencing, and detecting gene expressions with QPCR.

Description

Title: Method for constructing human programmed death ligand 2 gene gene high expression vector and application thereof

 Technical field

 [0001] The present invention belongs to the technical field of molecular biology, and relates to a method and application for constructing a human PD-L2 gene high expression vector.

 Background technique

 [0002] Non-small cell lung cancer (NSCLC) is a common malignant tumor in the world, and its incidence is increasing year by year. Studying NSCLC tumor recurrence factors and tumor immune escape mechanisms has important theoretical significance and clinical application value. T lymphocytes are important effector cells that mediate tumor immune responses, and T cell activation requires TCR-mediated antigen-specific signaling and costimulatory molecule-mediated costimulatory signals.

 technical problem

 [0003] Programmed death ligand 2 (PD-L2) / B7-H1 is a negative T cell costimulatory molecule in the B7 family, PD-L2 inhibits proliferation of CD4 and CD8 T cells by binding to its receptor PD-1 And activation, negative regulation of the body's immune response process, thereby mediating tumor immune escape, promoting tumor growth, so the study of the role of PD-L2 in tumor escape plays an important role in the prevention and treatment of tumors, but the lack of targets in the prior art Vectors that promote the expression of programmed death ligand 2 genes have hampered the progress of related research. Problem solution

 Technical solution

 [0004] The object of the present invention is to provide a method for constructing a human PD-L2 gene high expression vector, and to investigate the effect of PD-L2 on cell proliferation and apoptosis.

[0005] In order to achieve the above object, the present invention discloses a method for constructing a human PD-L2 gene high expression vector, which is characterized in that: pCDNA3.1 is used as an intermediate vector, a pair of primers are designed, and Xba is introduced into the primer.

I and EcoR

I cleavage site, PCR amplification using cDNA obtained by reverse transcription of RNA containing the gene of interest as a template, After the target band is excised, it is recovered and purified by a kit. After the purified PCR product and the vector pCDNA3.1 are digested with Xba1 and EcoR I, the purified product is recovered by a kit, and then the ligation fragment is The culture and cloning are screened to obtain a human PD-L2 gene high expression vector. The steps of the construction method are as follows:

 [0006] 1) Design primers

 [0007] According to the gene sequence of interest to be amplified, a pair of primers are designed as follows:

 [0008] PD-L2-F: 5'- GTCTAGAATGATCTTCCTCCTGCTAATG -3' (SEQ ID No: l)

[0009] PD-L2-R: 5, - GGAATTCTCAGATAGCACTGTTCACTTC -3' (SEQ ID No: 2

[0010] 2) using reverse transcription of RNA containing the gene of interest as a template, PCR amplification using the above pair of primers and DNA polymerase, and purification by a kit to obtain an amplification product;

[0011] 3) The pCDNA3.1 vector was double digested with Xba I and EcoR I;

 [0012] 4) Gene recombination

 [0013] The amplified product obtained in the step 2) is ligated to the double-digested pCDNA3.1 vector obtained in the step 3), and then cultured and cloned to obtain the high expression of the human PD-L2 gene. Vector pCDNA 3. 1-PD-L2.

 Advantageous effects of the invention

 Beneficial effect

 The human PD-L2 gene high expression vector provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of PD-L2 gene specifically and efficiently, and can be used as a powerful tool for PD-L2. Related drug research and development.

 Brief description of the drawing

 DRAWINGS

 1 is a schematic diagram showing the results of detecting the PD-L2 expression level by transcytometry pCDNA3.1-PD-L2 vector.

 BEST MODE FOR CARRYING OUT THE INVENTION

BEST MODE FOR CARRYING OUT THE INVENTION [0016] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0017] Example 1 Design of PD-L2 Gene Primers

 [0018] According to the PD-L2 gene coding sequence, it was analyzed using 01igo7 to find upstream primers and downstream primers (requires as little primer-free dimer as possible and the annealing temperature difference is small), and then in the upstream primer and downstream primer 5 Adding protective bases and restriction sites Xba I and EcoR

 I. The designed primer sequences are shown in SEQ ID No: 1 and SEQ ID No: 2.

 [0019] Example 2 Construction of PD-L2 gene high expression vector

 [0020] After diluting the synthetic antibody, the coding sequence of the PD-L2 gene was amplified by Premix PrimeSTAR HS enzyme, and Xba I and EcoR were used after electrophoresis recovery.

 The enzyme I is digested, and the double-cut product is recovered by electrophoresis. The pcDNA3.1 vector was electrophoresed and recovered by Xba l and EcoR I enzyme.

 [0021] The double-digested PCR product and the pcDNA3.1 vector were ligated with T4 DNA ligase, transformed into competent E. coli DH50C, uniformly coated onto an ampicillin-containing LB medium plate, and cultured at 37 ° C. After h, pick a single colony culture and send it to Shanghai Yingjun for sequencing. The recombinant E. coli was sequenced and the recombinant plasmid pCDNA3.1-PD-L2 was extracted with Endo-Free Plasmid Mini Kit II.

 Example 3 pCDNA3.1-PD-L2 plasmid transduction A375 cells

 [0023] A375 cells were seeded into six-well plates, cultured 18

 h to a degree of fusion of 80%, using Lipofectamine

 In 2000, pCDNA3.1-PD-L2 was transduced into A375 cells and cultured for 48 h.

 Example 4 Quantitative PCR detection of PD-L2 gene expression

[0025] According to the GAPDH and PD-L2 gene mRNA sequences, the Primer 5 was designed using the primer design software Premier Primer 5.

C product base large primer (^- 33⁄4

 : fcl, 3⁄4 ) Editing Ϊ ■ 19

::幽 L 194

[375] A375 cells and PD-L2 high expressing cells were inoculated separately 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 end of the reverse transcription, 9 (L of RNase Free dH ₂₀ diluted cDNA was added and stored at -20 ° C for later detection.

 [0027] taking cDNA 1 of each group of cells

 Μΐ was used as a template, and GAPDH was used as an internal reference. The relative expression of PD-L2 was detected by real-time fluorescent quantitative PCR, and the reaction conditions were set: 95. C 30s, 1 cycle, 54°C 30s 40 cycles, 95. C 5s, 60 ° C lmin, 95. C 15s, the result is shown in Figure 1. It can be seen that the PD-L2 gene expression level of PD-L2 high expression cells is 190-fold higher than that of normal A375 cells, indicating that the PD-L2 gene cDNA sequence provided by the present invention is successfully inserted into the pCD NA3.1 expression vector. It can promote the high expression of PD-L2 gene specifically, continuously and efficiently.

 Industrial applicability

 The human PD-L2 gene high expression vector provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of PD-L2 gene specifically and efficiently, and can be applied as a powerful tool to PD-L2. Related drug research and development.

Claims

 Claim  [Claim 1] A method for constructing a human PD-L2 gene high expression vector, which comprises: using pCDNA  3.1 is an intermediate vector, design a pair of primers, introduce Xba I and EcoR I cleavage sites in the primers, perform reverse transcription of the RNA containing the gene of interest, and then perform PCR amplification, and then cut the target band and use the kit. After purification and purification, the purified PCR product and the vector pCDNA3.1 are digested with Xba I and EcoR I, and the purified product is recovered by a kit, and then ligated, screened, cloned, and obtained to obtain human PD- The L2 gene high expression vector, the specific operation is as follows:  1) Design primers  According to the sequence of the gene of interest to be amplified, a pair of primers are designed as follows: PD-L2-F: 5'- GTCTAGAATGATCTTCCTCCTGCTAATG -3' (SEQ ID No: 1) ;  PD-L2-R: 5, - GGAATTCTCAGATAGCACTGTTCACTTC -3' (SEQ 2) using reverse transcription of RNA containing the gene of interest as a template, PCR amplification using the above pair of primers and DNA polymerase, and purification by a kit to obtain an amplification product;  3) The pCDNA3.1 vector was double digested with Xba I and EcoR I; 4) Gene recombination  The amplified product obtained in the step 2) is ligated with the double-digested pCDNA3.1 vector obtained in the step 3), and then cultured and cloned to obtain the human PD-L2 gene high expression vector pCDNA3. 1-PD-L2.