WO2019237376A1

WO2019237376A1 - Method for site-directed integration of txgp1 gene into raji cell and use thereof

Info

Publication number: WO2019237376A1
Application number: PCT/CN2018/091706
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 TXGP1 gene into a Raji 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 TXGP1 gene in a site-directed manner into the AAVS1 locus of chromosome 19 in a Raji cell.

Description

Method and application of TXGP1 gene site-specific integration into Raji cells

Technical field

The invention belongs to the technical field of genetic engineering. More specifically, the present invention relates to a method for site-integrated TXGP1 gene into Raji cells and its application.

Background technique

TXGP1 is a member of the TNF receptor superfamily and is a type I transmembrane glycoprotein. TXGP1 expression profile is limited to the surface of activated CD4 + and CD8 + T cells, and is predominantly CD4 + T cells. Human TXGP1 ligand belongs to the TNF family and is a type II transmembrane glycoprotein. IMD16 / TXGP1 is an important pair of co-stimulatory molecules that play an important role in the body's immune response and various diseases. Their interactions can promote the activation, proliferation, and migration of CD + 4 T cells, extend their life span, and promote germination. The formation of centers and the differentiation of DCs mature.

technical problem

TXGP1 can synergistically stimulate the activation of T cells, promote the production of high titer antibodies and class conversion by B cells, and mediate the infiltration of IMD16 + T cells into the inflammatory response site, which plays an important role in tumor immunotherapy and its potential clinical transformation value It is very large and requires solid research before it can be put into practical use. However, the lack of a means of knocking out TXGP1 gene expression in the prior art has caused certain obstacles to 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 TXGP1 gene into Raji cells, so that the transformed Raji cells stably overexpress TXGP1 protein.

In order to achieve these objects and other advantages according to the present invention, a method for site-directed integration of TXGP1 gene into Raji cells is provided, which includes the following steps:

1) Design AAV-ITR expression cassette containing TXGP1 gene and commission synthesis;

2) The AAV-ITR expression cassette containing the TXGP1 gene is inserted into a pFastBac1 vector to construct a plasmid pFastBac1-ITR-TXGP1;

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-TXGP1 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-TXGP1 And Bacmid-RC;

5) Use Cellfectin II Reagent transfected Bacmid-ITR-TXGP1 and Bacmid-RC into sf9 cells in logarithmic growth phase. Infection 120 The culture supernatant was collected after h, which is P1. High-titer P3 virus Bac-ITR-TXGP1 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-TXGP1 and Bac-RC, and continue to culture 72 After h, the cells were collected, the DNA was extracted separately and the small molecular weight DNA was isolated;

7) Transfection of small molecular weight DNA obtained in step 6) into Raji cells in logarithmic growth phase by electroporation. After 72 hours of incubation, the expression of TXGP1 and its insertion site were identified.

The sequence of the AAV-ITR expression cassette containing the TXGP1 gene is shown in SEQ ID No.1.

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

Preferably, the ratio of the Bacmid-ITR-TXGP1 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 TXGP1 gene in Raji cells at the AAVS1 site of chromosome 19, so that it can obtain the ability to continuously overexpress TXGP1 protein, and use the insect protein expression system to synthesize the elements necessary for AAV, avoiding the E. coli gene The risk of potential endotoxin contamination brought by the cloning system has greatly enhanced the safety and practicality of Raji 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 TXGP1 gene;

FIG. 2 is a result chart of TXGP1 gene fluorescent quantitative PCR;

FIG. 3 is a result of PCR identification of a TXGP1 gene insertion site, in which M-Marker, 1-control group, 2-experimental 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 Collection Center,

实施例一：Embodiment one: pFastBac1-ITR-TXGP1pFastBac1-ITR-TXGP1 载体的构建Construction of vectors

According to the human TXGP1 gene sequence provided in GenBank, an AAV-ITR expression cassette containing the TXGP1 gene was designed, and its sequence is as shown in SEQ. As shown in ID No. 1, SpeI and SphI digestion site sequences were added to the 5 'and 3' ends, respectively, and Shanghai Biotech was commissioned to synthesize the sequence by gene synthesis.

The synthetic AAV-ITR expression cassette containing the TXGP1 gene was integrated on the pUC19-ITR-TXGP1 vector. The pUC19-ITR-TXGP1 vector was digested with SpeI and SphI enzymes, and the ~ 1500 bp target fragment AAV-ITR-TXGP1 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-TXGP1 according to the molar ratio of pFastBac1 vector and AAV-ITR-TXGP1 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 correctly sequenced E. coli strains were extracted, and the recombinant vector pFastBac1-ITR-TXGP1 was extracted.

实施例二Example 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 the 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Example three ：高滴度杆状病毒: High titer baculovirus Bac-ITR-TXGP1Bac-ITR-TXGP1 和with BacBac -RC-RC 的制备Preparation

The pFastBac1-ITR-TXGP1 vector and pFastBac1-RC vector were transformed into competent E. coli DH10Bac, respectively. Positive clones were selected from blue and white spots, and recombinant Bacmid was extracted to obtain Bacmid-ITR-TXGP1 and Bacmid-RC.

Cellfectin II Reagent was used to transfect Bacmid-ITR-TXGP1 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-TXGP1 and Bac-RC were obtained after P1 was continuously infected with sf9 cells twice.

实施例四Example 4 ：定点插入: Fixed-point insertion TXGP1TXGP1 基因的genetic RajiRaji 细胞的构建及鉴定Construction and identification of cells

The P3 virus Bac-ITR-TXGP1 and Bac-RC obtained in Example 3 were used to co-infect sf9 cells in the logarithmic growth phase, and continued to culture 72 After h, the cells were collected, DNA was extracted separately and the small molecular weight DNA was isolated, and it was transfected into Raji cells in the logarithmic growth phase by electroporation, and culture was continued for 72 h.

TXGP1 gene expression levels of transfected Raji cells (experimental group) and normal Raji cells (control group) were detected by real-time quantitative PCR. The results are shown in Figure 2. It can be seen that the TXGP1 gene expression level of the experimental group cells was significantly higher. In control cells, the TXGP1 gene sequence was successfully integrated into Raji cells.

Next, the insertion site of TXGP1 gene was identified. The 5′-end sequence of the AAVS1 site of Raji cells (accession number: S51329.1) was used as the upstream primer (sequence: 5’- GAATTCCTAACTGCCCCGGGGC -3 ’), using the 5’ end of the TXGP1 gene as a downstream primer (sequence: 5’- GAAACCTTTCTCCTTCTTATA -3 ') PCR, the results are shown in Figure 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 TXGP1 gene has been successfully integrated into the AAVS1 site.

Industrial applicability

Claims

A method for site-specific integration of TXGP1 gene into Raji cells, which comprises the following steps:

1) Design AAV-ITR expression cassette containing TXGP1 gene and commission synthesis;

2) The AAV-ITR expression cassette containing the TXGP1 gene is inserted into a pFastBac1 vector to construct a plasmid pFastBac1-ITR-TXGP1;

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-TXGP1 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-TXGP1 And Bacmid-RC;

5) Use Cellfectin II Reagent transfected Bacmid-ITR-TXGP1 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-TXGP1 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-TXGP1 and Bac-RC. After incubation for 72 h, collect the cells, extract DNA and isolate small molecular weight DNA;

7) Transfection of small molecular weight DNA obtained in step 6) into Raji cells in logarithmic growth phase by electroporation. After 72 hours of incubation, the expression of TXGP1 and its insertion site were identified.
The method for site-integrating the TXGP1 gene into Raji cells according to claim 1, wherein, in step 1), the sequence of the AAV-ITR expression cassette containing the TXGP1 gene is shown in SEQ ID No.1.
The method for site-integrating the TXGP1 gene into Raji cells according to claim 1, wherein the site-specific integration site is the AAVS1 site of chromosome 19 of Raji cells.
The method for site-integrating the TXGP1 gene into Raji cells according to claim 1, wherein, in step 5), the ratio of the Bacmid-ITR-TXGP1 and Bacmid-RC vectors is 3-10.
The method for site-integrating the TXGP1 gene into Raji cells according to claim 1, wherein, in step 7), the electrotransformation conditions are: a voltage of 600 to 900V and a pulse time of 20 to 30 ms.