Bombyx mori recombinant baculovirus, and preparing method and applications thereof in preparation of cancer vaccines

A technology of recombinant baculovirus and baculovirus, applied in the field of biomedicine, can solve the problems of inability to provoke an immune response, insignificant immune effect, low immune effect, etc., and achieves favorable promotion, low cost, and small immune response. Effect

Inactive Publication Date: 2015-06-10
特菲(天津)生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this recombinant virus also has the common problems of the above-mentioned cancer vaccines, that is, the produced immunogenicity is weak, and it cannot stimulate a strong and effective immune response, resulting in insignificant immune effect and low immune effect.

Method used

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  • Bombyx mori recombinant baculovirus, and preparing method and applications thereof in preparation of cancer vaccines
  • Bombyx mori recombinant baculovirus, and preparing method and applications thereof in preparation of cancer vaccines
  • Bombyx mori recombinant baculovirus, and preparing method and applications thereof in preparation of cancer vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Construction and acquisition of baculovirus pFastBacDual-CMV-DLL4-VSVG

[0056] 1.1 PCR amplification of gp64 signal peptide (SP) and transmembrane region (TM) from wild baculovirus genome

[0057] Using gp64-SP-F (shown in SEQ ID NO: 1) and gp64-SP-R (shown in SEQ ID NO: 2) as primers, using the wild baculovirus genome as a template, perform PCR amplification to obtain the gp64 signal The gene sequence of the peptide (SP) is shown in SEQ ID NO:11. in:

[0058] gp64-SP-F:5'-AGTAGGATCCATGGTAGGCGCTATTG-3'

[0059] gp64-SP-R:5'-CTAAGTACAAAAGGCACTTCGCCGCAAAGGCAGAA-3'

[0060] PCR reaction system:

[0061]

[0062] The PCR reaction procedure is:

[0063]

[0064] Using gp64-TM-F (shown in SEQ ID NO: 3) and gp64TM-R (shown in SEQ ID NO: 4) as primers, using the wild baculovirus genome as a template, PCR amplifies the transmembrane region, and the PCR reaction system 1. The reaction procedure is the same as that of the signal peptide, and the gene sequen...

Embodiment 2

[0124] Embodiment 2: Obtaining of Bombyx mori recombinant baculovirus Bm-CMV-DLL4-VSVG

[0125] (1) The Pfastbacdual-CMV-DLL4-VSVG recombinant plasmid was transformed into DH10Bac-sensitive peptide cells, and Bacmid-CMV-DLL4-VSVG was obtained by blue-white screening. Identification of recombinant Bacmid-CMV-DLL4-VSVG as Figure 4 As shown, where M: Mark; 1: gp64-TM-F, M13-F are mutual primers; 2: gp64-SP-F, M13-F are mutual primers; 3: VSVG-F, M13-F are mutual primers ; 4: CMV upstream and downstream primers; 5: CMV-F, M13-R are mutual primers; 6: DLL4-F, M13-R are mutual primers.

[0126] (2) Bacmid-CMV-DLL4-VSVG was transfected into BmN cells for 72 hours to cause onset, the virus genome was extracted and identified by PCR with M13 primers, and the silkworm recombinant baculovirus Bm-CMV-DLL4-VSVG was obtained. Identification of recombinant Bm-CMV-DLL4-VSVG as Figure 5 As shown, where M: Mark; 1: CMV upstream and downstream primers; 2: DLL4, M13-R are mutual primers; 3: ...

Embodiment 3

[0127] Example 3: Application of Bombyx mori recombinant baculovirus Bm-CMV-DLL4-gp64-VSVG in the preparation of cancer vaccines

[0128] 3.1 Preparation of virus samples for injection

[0129] 3.1.1 Amplification of baculovirus

[0130] The obtained first-generation virus was first transferred to a vial of BmN cells to increase the virus titer. After 3 days, the cells became ill, and the virus liquid was collected and stored in a refrigerator at 4°C. Gently blow off the BmN cells in the medium bottle with a Pasteur tube, blow and beat repeatedly, transfer the cells to a large culture flask with a pipette, add new low-sugar DMEM medium, and culture in a 27°C incubator. When the cells cover the culture flask Between 70% and 80%, the virus was inoculated with an inoculation volume of 1:1000.

[0131] 3.1.2 Collection of baculovirus

[0132] Three days after the inoculation of baculovirus, the BmN cells of the silkworm generally became ill, became round, floated up and fell of...

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Abstract

The invention provides a bombyx mori recombinant baculovirus Bm-CMV-DLL4-VSVG. The recombinant baculovirus adopts a baculovirus expression vector Pfastbacdual as a vector, and comprises a CMV-IE promoter, a DLL4 gene, a gp64 signal peptide gene, a VSVG gene and a gp64 transmembrane domain gene. Preparation of the recombinant baculovirus includes steps of: subjecting the CMV-IE promoter sequence, the DLL4 protein bases, the gp64 signal peptide gene sequence, the gp64 transmembrane domain gene sequence and the VSVG gene sequence to PCR amplification, constructing a Pfastbacdual-CMV-DLL4-VSVG recombinant plasmid, and extracting viral genome. The recombinant baculovirus is good in stability, high in host infection rate, high in expression capability and capable of efficiently expressing DLL4 protein in mammal animal cells, thus provoking high and effective immunoreactions. The recombinant baculovirus is suitable for application in preparation of cancer vaccines.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a silkworm recombinant baculovirus capable of highly expressing DLL4 protein in mammalian cells, a preparation method of the virus, and an application of the virus in the preparation of cancer vaccines. Background technique [0002] In recent years, research on therapeutic cancer vaccines developed by targeting certain specific surface antigens of tumors has achieved fruitful results. At present, a number of related cancer vaccines have been approved by the FDA for clinical trials in the United States. However, due to the weak immunogenicity of the cancer cell antigens, the inability to stimulate a strong and effective immune response, and the high production cost of the plasmids used to express the antigens have seriously affected the development of this treatment. [0003] DLL4 protein is a ligand of Notch signaling pathway and plays a key role in angiogenesis. ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/866C12N15/66A61K48/00A61P35/00
Inventor张耀洲张皓陈剑清舒特俊
Owner特菲(天津)生物医药科技有限公司