WO2019237374A1

WO2019237374A1 - Method for site-directed integration of mtabc3 gene into a375 cell and use thereof

Info

Publication number: WO2019237374A1
Application number: PCT/CN2018/091704
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2018-06-16
Filing date: 2018-06-16
Publication date: 2019-12-19

Abstract

Disclosed is a method for the site-directed integration of the MTABC3 gene into an A375 cell, which method uses an insect protein expression system to synthesize the necessary components required for a recombinant adeno-associated virus (rAAV), and achieves the aim of integrating the MTABC3 gene in a site-directed manner into the AAVS1 locus of chromosome 19 in an A375 cell.

Description

Method and application of site-specific integration of MTABC3 gene into A375 cells and its application

Technical field

The invention belongs to the technical field of genetic engineering. More specifically, the present invention relates to a method for site-directed integration of the MTABC3 gene into A375 cells and its application.

Background technique

MTABC is a large class of transmembrane proteins. It uses the energy of hydrolyzing ATP to transmembrane a variety of endogenous and exogenous biomolecules. The substrates for translocation include: sugar, amino acid, metal ion, peptide, protein, cell Metabolites and drugs. MTABC transporters are widely found in eukaryotes and prokaryotes, and 49 members of the MTABC transporter superfamily have been identified in the human genome to date.

technical problem

Because multiple members of the MTABC family are associated with tumor multidrug resistance (MDR), MTABC is also known as the MTABC transporter MDR family. As one of them, MTABC3 is not only related to MDR, but also to the pathogenesis of many tumors. However, the function and role of MTABC3 in it have not been clarified so far, and further research is needed, but the existing technology lacks a means to knock out the expression of MTABC3 gene. This has hindered the progress of related research.

Adeno-associated virus (AAV) is a non-enveloped single-stranded DNA virus. It has the advantages of good safety, wide tropism, infection of dividing or non-dividing cells, stable physical and chemical properties, and easy storage. Recombinant adeno-associated virus (rAAV) carrying a foreign gene can integrate the foreign gene into the host genome in a targeted manner to achieve long-term stable expression of the foreign gene in the host cell.

Technical solutions

The purpose of the present invention is to provide a method for site-specific integration of the MTABC3 gene into A375 cells, so that the modified A375 cells stably overexpress the MTABC3 protein.

In order to achieve these objects and other advantages according to the present invention, a method for site-directed integration of the MTABC3 gene into A375 cells is provided, which includes the following steps:

1) Design the AAV-ITR expression cassette containing the MTABC3 gene and commission the synthesis;

2) Inserting the AAV-ITR expression cassette containing the MTABC3 gene into a pFastBac1 vector to construct a plasmid pFastBac1-ITR-MTABC3;

3) Using the pAAV-RC vector as a template, pRC-F and pRC-R as upstream and downstream primers, respectively, to amplify the Rep module and Cap module fusion sequences, and then insert them into the pFastBac1 vector to obtain the pFastBac1-RC vector. The sequence of pRC-F primer is 5’- GACTAGTGCCACCATGCCGGGGTTTTACGAG-3 ', the sequence of the pRC-R primer is 5'-TAGCATGCGCATTAAGCGCGGCGGGTGT-3';

4) The pFastBac1-ITR-MTABC3 vector obtained in step 2) and the pFastBac1-RC vector obtained in step 3) were respectively transformed into competent E. coli DH10Bac. Blue and white spots were selected for positive clones, and recombinant Bacmid was extracted to obtain Bacmid-ITR-MTABC3. And Bacmid-RC;

5) Bacmid-ITR-MTABC3 and Bacmid-RC were transfected into sf9 cells in logarithmic growth phase with Cellfectin II Reagent. The culture supernatant was collected 120 hours after infection, which is P1. High-titer P3 virus Bac-ITR-MTABC3 and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.

6) Co-infection of sf9 cells in logarithmic growth phase with Bac-ITR-MTABC3 and Bac-RC. After continuing to culture for 72 hours, collect the cells, extract DNA and isolate small molecular weight DNA;

7) The small molecular weight DNA obtained in step 6) was transfected into A375 cells in logarithmic growth phase by electroporation. After 72 hours of incubation, the expression of MTABC3 and its insertion site were identified.

The sequence of the AAV-ITR expression cassette containing the MTABC3 gene is shown in SEQ ID No.1.

Wherein, the site-specific integration site is AAVS1 site of chromosome 19 of A375 cells.

Preferably, the ratio of the Bacmid-ITR-MTABC3 and Bacmid-RC vectors in step 5) is 3-10.

Preferably, the electrical conversion conditions described in step 7) are: the voltage is 600-900V, and the pulse time is 20-30 ms.

Beneficial effect

The invention can realize the site-specific integration of the MTABC3 gene in A375 cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress the MTABC3 protein, and use the insect protein expression system to synthesize the elements necessary for AAV to avoid the E. coli gene The risk of potential endotoxin contamination brought by the cloning system greatly improves the safety and practicability of A375 cells for preclinical research.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the structure of the AAV-ITR expression cassette containing the MTABC3 gene;

FIG. 2 is a result chart of MTABC3 gene quantitative PCR;

FIG. 3 is a result of PCR for identifying the insertion site of the MTABC3 gene, in which M-Marker, 1-control group, 2-experiment group.

Embodiments of the invention

The invention is further described below with reference to the drawings and specific embodiments.

SpeI and SphI restriction enzymes were purchased from Fermentas, PCR Cleanup kits were purchased from Omega bio-tek, T4 DNA ligase was purchased from NEB, competent E. coli DH5α and DH10Bac were purchased from Invitrogen, pFastBac1 and pAAV-RC vectors were purchased from BioVector NTCC Deposit center,

实施例一：Embodiment one: pFastBac1-ITR-MTABC3pFastBac1-ITR-MTABC3 载体的构建Construction of vectors

According to the sequence of the human MTABC3 gene provided in GenBank, an AAV-ITR expression cassette containing the MTABC3 gene was designed. Its sequence is shown in SEQ ID No. 1. SpeI and SphI were added to the 5 'and 3' ends of the cassette, respectively. Site sequence, commissioned Shanghai Biotech to synthesize the sequence by gene synthesis.

The synthetic AAV-ITR expression cassette containing the MTABC3 gene was integrated on the pUC19-ITR-MTABC3 vector. The pUC19-ITR-MTABC3 vector was digested with SpeI and SphI enzymes, and the ~ 1500 bp target fragment AAV-ITR-MTABC3 was recovered after agarose gel electrophoresis.

The pFastBac1 vector was digested with SpeI and SphI enzymes, and the digested pFastBac1 vector was recovered by PCR Cleanup kit.

Take 50 μg of pFastBac1 vector, and measure AAV-ITR-MTABC3 according to the molar ratio of pFastBac1 vector and AAV-ITR-MTABC3 to 1: 5. After mixing, use T4 DNA ligase and ligate at 16 ° C overnight. The competent E. coli DH5α was transformed, and ampicillin was screened and cultured. Monoclonal strains were selected and identified by sequencing. A large number of cultured and sequenced E. coli were cultured, and the recombinant vector pFastBac1-ITR-MTABC3 was extracted.

实施例二：Embodiment two: pFastBac1-RCpFastBac1-RC 载体的构建Construction of vectors

The pAAV-RC vector was used as a template, and pRC-F and pRC-R were used as the upstream and downstream primers, respectively. The Rep module and Cap module fusion sequences were amplified, purified and recovered, and then digested with SpeI and SphI enzymes. In one step, it was inserted into the pFastBac1 vector to obtain the pFastBac1-RC vector. The sequence of pRC-F primer is 5’- GACTAGTGCCACCATGCCGGGGTTTTACGAG-3 ', and the sequence of the pRC-R primer is 5'-TAGCATGCGCATTAAGCGCGGCGGGTGT-3'.

The competent E. coli DH5α was transformed, and ampicillin was screened and cultured. Monoclonal strains were selected and identified by sequencing. A large number of cultured and sequenced E. coli were cultured, and the recombinant vector pFastBac1-RC was extracted.

实施例三：高滴度杆状病毒Example 3: High titer baculovirus Bac-ITR-MTABC3Bac-ITR-MTABC3 和with Bac-RCBac-RC 的制备Preparation

The pFastBac1-ITR-MTABC3 vector and pFastBac1-RC vector were transformed into competent E. coli DH10Bac, respectively. Positive clones were selected by blue and white spots, and recombinant Bacmid was extracted to obtain Bacmid-ITR-MTABC3 and Bacmid-RC.

Cellfectin II Reagent was used to transfect Bacmid-ITR-MTABC3 and Bacmid-RC into sf9 cells in logarithmic growth phase. The culture supernatant was collected 120 hours after infection, which is P1. High-titer P3 viruses Bac-ITR-MTABC3 and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.

实施例四：定点插入Embodiment 4: Fixed-point insertion MTABC3MTABC3 基因的genetic A375A375 细胞的构建及鉴定Construction and identification of cells

The sf9 cells in the logarithmic growth phase were co-infected with the P3 virus Bac-ITR-MTABC3 and Bac-RC obtained in Example 3. After the culture continued for 72 h, the cells were collected, DNA was extracted and small molecular weight DNA was isolated, and It was transfected into A375 cells in logarithmic growth phase by electrotransfection, and culture was continued for 72 h.

The quantitative expression of MTABC3 gene in A375 cells (experimental group) and normal A375 cells (control group) was detected by real-time quantitative PCR. The results are shown in Figure 2. It can be seen that the expression level of MTABC3 gene in the cells of the experimental group was significantly higher. In control cells, the MTABC3 gene sequence was successfully integrated into A375 cells.

Next, the insertion site of the MTABC3 gene was identified. Using the partial sequence at the AAVS1 site of A375 cells (accession number: S51329.1) as the upstream primer (sequence: 5’- GAATTCCTAACTGCCCCGGGGC -3 '), using the 5' end partial sequence of the MTABC3 gene as a downstream primer (sequence: 5'- CTGGCTCCGCTCCACGACAAGC -3 ') PCR was performed, and the results are shown in FIG. 3. It can be seen that a band of ~ 1000 bp appeared in the cells of the experimental group, but no band appeared in the cells of the control group, indicating that the MTABC3 gene has been successfully integrated into the AAVS1 site.

Industrial applicability

Claims

A method for site-specific integration of the MTABC3 gene into A375 cells, which comprises the following steps:

1) Design the AAV-ITR expression cassette containing the MTABC3 gene and commission the synthesis;

2) Inserting the AAV-ITR expression cassette containing the MTABC3 gene into a pFastBac1 vector to construct a plasmid pFastBac1-ITR-MTABC3;

3) Using the pAAV-RC vector as a template, pRC-F and pRC-R as upstream and downstream primers, respectively, to amplify the Rep module and Cap module fusion sequences, and then insert them into the pFastBac1 vector to obtain the pFastBac1-RC vector. The sequence of the pRC-F primer is 5'-GACTAGTGCCACCATGCCGGGGTTTTACGAG-3 ', and the sequence of the pRC-R primer is 5'-TAGCATGCGCATTAAGCGCGGCGGGTGT-3';

4) The pFastBac1-ITR-MTABC3 vector obtained in step 2) and the pFastBac1-RC vector obtained in step 3) were respectively transformed into competent E. coli DH10Bac. Blue and white spots were screened for positive clones, and recombinant Bacmid was extracted to obtain Bacmid-ITR-MTABC3. And Bacmid-RC;

5) Use Cellfectin II Reagent transfected Bacmid-ITR-MTABC3 and Bacmid-RC into sf9 cells in logarithmic growth phase. The culture supernatant was collected 120 hours after infection, which is P1. High-titer P3 virus Bac-ITR-MTABC3 and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.

6) Co-infection of sf9 cells in logarithmic growth phase with Bac-ITR-MTABC3 and Bac-RC. After continuing to culture for 72 hours, collect the cells, extract DNA and isolate small molecular weight DNA;

7) The small molecular weight DNA obtained in step 6) was transfected into A375 cells in logarithmic growth phase by electroporation. After 72 hours of incubation, the expression of MTABC3 and its insertion site were identified.
The method for site-directed integration of the MTABC3 gene into A375 cells according to claim 1, wherein, in step 1), the sequence of the AAV-ITR expression cassette containing the MTABC3 gene is shown in SEQ ID No.1.
The method for site-specific integration of the MTABC3 gene into A375 cells according to claim 1, wherein the site-specific integration site is the AAVS1 site of chromosome 19 of A375 cells.
The method for site-specific integration of the MTABC3 gene into A375 cells according to claim 1, characterized in that, in step 5), the ratio of the Bacmid-ITR-MTABC3 and Bacmid-RC vectors is 3-10.
The method for site-integrating the MTABC3 gene into A375 cells according to claim 1, characterized in that, in step 7), the electrotransformation conditions are: a voltage of 600 to 900V and a pulse time of 20 to 30 ms.