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Recombinant adeno-associated virus as well as construction method and application thereof

A virus and application method technology, applied in the field of genetic engineering, can solve the problems of poor specificity and unsatisfactory treatment effect of tumor immunotherapy, and achieve high DC antigen presentation efficiency, high antigen presentation ability, and long antigen sensitization time. Effect

Inactive Publication Date: 2016-05-18
厚朴生物科技(苏州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The first technical problem to be solved by the present invention is the problem of poor specificity and unsatisfactory treatment effect of tumor immunotherapy in the prior art, and further provides a recombinant adeno-associated virus that can achieve highly targeted immunotherapy application method

Method used

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  • Recombinant adeno-associated virus as well as construction method and application thereof
  • Recombinant adeno-associated virus as well as construction method and application thereof
  • Recombinant adeno-associated virus as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction of recombinant adeno-associated virus

[0043] The recombinant adeno-associated virus constructed in this example is a recombinant adeno-associated virus formed by inserting tumor antigen genes into the shuttle expression vector pAAV-MCS.

[0044] The construction steps of the recombinant adeno-associated virus are as follows:

[0045] (1) Insert the tumor antigen gene into the shuttle expression vector pAAV-MCS, so that the tumor antigen gene enters the vector containing ITR to form a recombinant plasmid;

[0046] (2) Mass extraction of the recombinant plasmid described in step 1), co-transfect AAV-293 cells with pHelper and pAAV-RC plasmids by calcium phosphate precipitation method, culture for 2-3 days, collect the culture medium and cells, use The crude virus solution was collected by repeated freezing and thawing methods.

[0047] As an alternative implementation of this embodiment, the construction steps of the recombinant adeno-associated virus can ...

Embodiment 2

[0052] The application method of the recombinant adeno-associated virus described in Example 2:

[0053] In this example, the recombinant adeno-associated virus prepared in Example 1 was used to infect DC cells with the recombinant adeno-associated virus, and the tumor-associated antigen protein was expressed in the DC cells.

[0054] As a preferred embodiment of the present invention, the application method of the recombinant adeno-associated virus further includes the following step (2): Silencing the PD-1 gene by siRNA interference, and blocking the PD-1 / PD-L1 signal.

Embodiment 3

[0055] Example 3 PD-1 gene siRNA sequence design and virus preparation:

[0056] The specific methods for silencing PD-1 gene and blocking PD-1 / PD-L1 signal by siRNA interference are as follows:

[0057] a) According to the design principles of siRNA, according to the PD-1mRNA nucleotide sequence reported in GeneBank, two online design softwares of WMG and Qigen are used to design three pairs of siRNA against PD-1mRNA. The target sequence of siRNA is as follows:

[0058]

[0059]

[0060] The sequence was screened by Gene Blast, which confirmed that there is no homology with human gene exons. SiRNA-1 having the sequence structure shown in SEQIDNo.1, siRNA-2 having the sequence structure shown in SEQIDNo.2, and siRNA-3 having the sequence structure shown in SEQIDNo.3, the above three pairs are directed to PD-1 mRNA SiRNA targets, synthesize hairpin-like template DNA with paired siRNA at both ends, connected by a loop of 6 deoxynucleotides in the middle, add stop signal polyT at the 3...

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Abstract

The invention relates to a recombinant adeno-associated virus. The recombinant adeno-associated virus is formed by inserting a tumor antigen gene into a shuttle expression vector pAAV-MCS. The invention also provides a construction method and an application of the recombinant adeno-associated virus. According to the invention, the recombinant adeno-associated virus carrying a tumor-associated antigen gene is utilized for infecting DC cells and expresses tumor-associated antigen protein in the DC cells, and a PD-1 gene is also combined for silencing CTL cells in the application method of the recombinant adeno-associated virus.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a recombinant adeno-associated virus and its construction method and application. Background technique [0002] Immune cell tumor treatment technology is a new type of treatment method with significant curative effect that stimulates the autoimmune system to fight tumors by modern biotechnology. It is the fourth largest tumor treatment technology after the traditional treatment methods such as surgery, radiotherapy and chemotherapy. . [0003] At present, a large number of studies have shown that for patients with advanced malignant tumors, adoptive cellular immunotherapy has superiority that other treatment methods cannot compare. It is one of the effective methods for the treatment of advanced malignant tumors and has a very broad clinical application prospect. Although many medical institutions in China have conducted related research and clinical practice of tumor immu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/864C07K14/47
CPCC07K14/4748C12N7/00C12N15/86C12N2750/14121C12N2750/14143
Inventor 孙振华曹晖
Owner 厚朴生物科技(苏州)有限公司
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