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Packaging method for constructing oncolytic adeno-associated virus oAAV-SP-GSDM-NT for expressing pyroptosis protein and application of oncolytic adeno-associated virus oAAV-SP-GSDM-NT

A packaging method, the technology of oaav-sp-gsdm-nt, is applied in the field of tumor treatment and can solve the problem of oncolytic virus that does not express pyroptosis protein, cannot be packaged to obtain oncolytic virus vector expressing pyroptosis protein, and bacterial pyroptosis, etc. question

Active Publication Date: 2021-04-02
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing oncolytic virus vector packaging methods cannot avoid the expression of the target gene (GSDM-NT) during packaging, and GSDM-NT has strong cytotoxicity. Oncolytic virus vector expressing pyroptosis protein obtained by packaging
[0004] There are currently no reports of oncolytic viruses expressing pyroptosis protein (GSDM-NT)

Method used

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  • Packaging method for constructing oncolytic adeno-associated virus oAAV-SP-GSDM-NT for expressing pyroptosis protein and application of oncolytic adeno-associated virus oAAV-SP-GSDM-NT
  • Packaging method for constructing oncolytic adeno-associated virus oAAV-SP-GSDM-NT for expressing pyroptosis protein and application of oncolytic adeno-associated virus oAAV-SP-GSDM-NT
  • Packaging method for constructing oncolytic adeno-associated virus oAAV-SP-GSDM-NT for expressing pyroptosis protein and application of oncolytic adeno-associated virus oAAV-SP-GSDM-NT

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Screening for mammalian-specific promoter mCBA

[0107] 1. Using molecular cloning technology, amplify the mammalian-specific promoter mCBA fragment from the plasmid pMD18T-mCBA, amplify the promoter CAG fragment from the plasmid pCAGGS, and replace the plasmid pFastBac-ITR-CMV- with the mCBA fragment and the CAG fragment Promoter CMV fragment in eGFP, obtain plasmids pFastBac-ITR-mCBA-eGFP and pFastBac-ITR-CAG-eGFP;

[0108] 2. pFastBac-ITR-CMV-eGFP, pFastBac-ITR-mCBA-eGFP and pFastBac-ITR-CAG-eGFP were transfected into sf9 insect cells respectively, and the expression of eGFP was observed with green fluorescent protein after 48 hours ( figure 1 );

[0109] The results showed that the mammalian-specific promoter mCBA was not activated in sf9 insect cells, and it could be used for subsequent packaging of oncolytic adeno-associated virus expressing pyroptosis protein using the baculovirus / sf9 insect cell packaging system.

Embodiment 2

[0111] A packaging method for constructing an oncolytic adeno-associated virus oAAV-SP-GSDMD-NT expressing pyroptosis protein comprises the following steps:

[0112] 1. Amplify the mammalian-specific promoter mCBA and construct the donor plasmid pFastBac-ITR-CBA-eGFP

[0113] a. Design amplification primers to amplify the mammalian-specific promoter mCBA fragment from the plasmid pMD18T-mCBA (comprising the mammalian-specific promoter mCBA);

[0114] b. Using molecular cloning technology, replace the promoter CMV fragment in the plasmid pFastBac-ITR-CMV-eGFP with the mammalian specific promoter mCBA fragment to obtain the plasmid pFastBac-ITR-mCBA-eGFP ( figure 2 ).

[0115] 2. Amplify the GSDMD-NT gene

[0116] The GSDMD-NT gene was amplified from a human cDNA library, and its nucleotide sequence is shown in SEQ ID NO: 3:

[0117]5’-atggggtcggcctttgagcgggtagtccggagagtggtccaggagctggaccatggtggggagttcatccctgtgaccagcctgcagagctccactggcttccagccctactgcctggtggttaggaagccctcaagctca...

Embodiment 3

[0132] The above oAAV-mCBA-GSDMD-NT can kill liver cancer Hep3B cells, breast cancer 4T1 cells and glioma C6 cells at the cellular level

[0133] Liver cancer Hep3B cells, breast cancer 4T1 cells and glioma C6 cells were respectively infected with oAAV-mCBA-GSDMD-NT. After 72 hours, adding propidium iodide (Propidium Iodide) and annexin V-APC (Annexin V-APC) to detect the pyroptosis of tumor cells caused by oAAV-mCBA-GSDMD-NT infection, the results showed that oAAV-mCBA-GSDMD -NT infection can lead to pyroptosis in liver cancer Hep3B cells, breast cancer 4T1 cells and glioma C6 cells ( Figure 4 ).

[0134] In addition, after oAAV-mCBA-GSDMD-NT infected tumor cells for 72 hours, the cell death rate and survival rate were detected by lactate dehydrogenase detection kit and ATP detection kit respectively. The results showed that compared with the control group, oAAV-mCBA- The tumor cell death rate in the GSDMD-NT infection group was higher ( Figure 5 ), the survival rate is ...

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Abstract

The invention discloses a packaging method for constructing an oncolytic adeno-associated virus oAAV-SP-GSDM-NT for expressing pyroptosis protein and application of the oncolytic adeno-associated virus oAAV-SP-GSDM-NT. According to the invention, oAAV-SP-GSDM-NT is packaged by utilizing SP and sf9 insect cells of mammal specific promoters, so that the expression of the pyroptosis protein with strong cytotoxicity in the packaging process is avoided, safe, efficient and high-titer oAAV-SP-GSDM-NT is successfully obtained, and the oAAV-SP-GSDM-NT enables various tumor cells to be subjected to pyroptosis in vitro, has a good tumor cell killing effect, and can be used for treating animal tumor models in vivo. The oAAV-SP-GSDM-NT and an immunomodulator are combined to form a combined medicamentfor treating tumors, the combined medicament has a more remarkable oncolytic effect on an animal tumor model than the single use of the oAAV-SP-GSDM-NT or the immunomodulator, and therefore, it indicates that the combined medicament for treating tumors has a very good tumor treatment application prospect.

Description

technical field [0001] The invention relates to the field of tumor treatment, in particular to a packaging method for constructing an oncolytic adeno-associated virus oAAV-SP-GSDM-NT expressing pyroptosis protein and its application. Background technique [0002] Pyroptosis is a programmed cell death in which cells expand and rupture and release a large amount of inflammatory substances mediated by the gasdermins N-segment domain of the porin family. Inducing pyroptosis in tumor cells can not only directly kill tumor cells, but also trigger a strong anti-tumor immune response. Combined with the blocking of PD-1 / PD-L1 at the immune screening site, it can play a good role in oncolysis Effect. [0003] The method of inducing tumor pyroptosis is to use a carrier to deliver the gasdermins N segment domain (GSDM-NT) gene of the porin family into tumor cells. The existing oncolytic virus vector packaging methods cannot avoid the expression of the target gene (GSDM-NT) during pack...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/866A61K48/00A61K38/17A61K45/06A61P35/00C12R1/93
CPCC12N7/00C12N15/86C07K14/47A61K48/005A61K38/1709A61K45/06A61P35/00C12N2710/14043C12N2750/14152C12N2800/105A61K2300/00Y02A50/30
Inventor 曹罡鲁愿何文波黄信刘小可何雨陶大刚张茜姜雪梨徐伟泽
Owner HUAZHONG AGRI UNIV
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