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Recombinant vaccinia virus containing genes CCL5 and SSTR2 and preparation method of recombinant vaccinia virus

A pox virus and gene technology, applied in the field of genetic engineering and tumor biotherapy, to achieve the effect of fast replication cycle, safety guarantee, and improvement of targeted oncolytic activity

Active Publication Date: 2017-09-15
FIRST PEOPLES HOSPITAL OF YUNNAN PROVINCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recombinant oncolytic poxvirus rVV-CCL5-SSTR2-Luc+ containing human CCL5 and human SSTR2 genes has not been reported yet

Method used

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  • Recombinant vaccinia virus containing genes CCL5 and SSTR2 and preparation method of recombinant vaccinia virus
  • Recombinant vaccinia virus containing genes CCL5 and SSTR2 and preparation method of recombinant vaccinia virus
  • Recombinant vaccinia virus containing genes CCL5 and SSTR2 and preparation method of recombinant vaccinia virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the preparation method of the recombinant poxvirus containing CCL5 and SSTR2 gene, specific content is as follows:

[0033] 1. Main materials

[0034](1) BS-C-1 (3142C0001000000033), African green monkey kidney cell line, purchased from China Center for Type Culture Collection (CCTCC), BSC-1 cells were transfected with viral vectors Ideal host for homologous recombination and virus titer determination.

[0035] (2) 143B (ATCC ® CRL-8303TM), a human osteosarcoma cell line, purchased from the American Type Culture Collection (ATCC), 143B cells are a human thymokinase-deficient (TK - ) cell line for the screening of recombinant poxviruses.

[0036] (3) HeLa: human cervical cancer cell line, DLD1: colon cancer cell line, HCT116: colorectal cancer cell line, purchased from the Kunming Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences - Kunming Institute of Zoology, Chinese Academy of Sciences.

[0037] (4) pSC65, purchase...

Embodiment 2

[0063] Example 2: After rVV-CCL5-SSTR2-Luc+ infects colorectal cancer cell lines, WB detection of SSTR2 and ELISA detection of CCL5 protein expression levels

[0064] Inoculate human colorectal cancer cell lines (HCT116, DLD1) in six-well plates the day before, at 37°C in 5% CO 2 Incubate overnight in the incubator. The supernatant and cells were collected after the recombinant poxvirus infected HeLa cells for 60 hours. The supernatant was detected by ELISA to detect the expression level of CCL5 protein, and the total protein extracted from the cells was detected by WB to detect the level of SSTR2 protein. The expression level in the cells was significantly higher than that of the control group rVV-Luc+ ( P image 3 ). Figure 4 The results showed that the expression level of CCL5 protein of double-gene virus rVV-CCL5-SSTR2-Luc+ was significantly higher than that of control virus rVV-Luc+ (*P<0.05).

Embodiment 3

[0065] Example 3: In vitro detection of the inhibitory effect of CIK cells loaded with recombinant poxvirus rVV-CCL5-SSTR2-Luc+ on tumor cell proliferation

[0066] The recombinant poxvirus rVV-CCL5-SSTR2-Luc+ prepared by the above method was expanded in vitro for titer determination and then stored at -80°C. CIK cells can be loaded with recombinant poxvirus for human colorectal cancer cells and human cervical cancer. In the killing experiment, the viability of the cells was detected at 24h, 48h, 72h, and 96h respectively. The results showed that CIK cells loaded with recombinant poxvirus had a certain inhibitory effect on the proliferation of colorectal cancer cell DLD1 ( Figure 5A), the CIK group has the weakest inhibitory effect on DLD1 cells, the rVV-Luc+ / CIK group has a slightly enhanced inhibitory effect on DLD1 cells due to oncolytic virus loading, and the rVV-CCL5-SSTR2-Luc+ / CIK group is due to anti-oncogene The inhibitory effect on DLD1 cells was significantly enhan...

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Abstract

The invention discloses a recombinant vaccinia virus rVV-CCL5-SSTR2-Luc+, containing genes CCL5 and SSTR2, constructed by virtue of a homologous recombination method. By utilizing the characteristic that shuttle plasmids containing human target genes CCL5 and SSTR2 and Luciferase reporter genes have same TK side wings with a vaccinia virus WR strain, homologous recombination is carried out in a vaccinia virus susceptible cell BS-C-1 to integrate the target genes to a vaccinia virus genome, so as to construct the recombinant vaccinia virus rVV-CCL5-SSTR2-Luc+. The recombinant vaccinia virus rVV-CCL5-SSTR2-Luc+ is capable of efficiently expressing exogenous genes, meanwhile, CCL5 is matched with highly expressed CCR5 on the surface of a cell CIK, and SSTR2 has the unique advantage on directional induction of octreotide targeted tumor cells, so that the recombinant vaccinia virus is beneficial to clinical expansion and application and is used for treating various tumors.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and tumor biotherapy, and specifically relates to a recombinant poxvirus containing CCL5 and SSTR2 genes and a preparation method thereof. Background technique [0002] Oncolytic poxvirus, as a kind of tumor therapy drug and gene therapy carrier, has developed rapidly in recent years. Poxvirus carrying anti-cancer genes can improve its oncolytic effect. Many genetically modified targeted oncolytic poxviruses have entered clinical trials one after another, showing Broad application prospects. Among the oncolytic poxviruses that have entered clinical trials, the Wyeth strain recombinant poxvirus JX-594 inserted into the GM-CSF gene is the earliest recombinant poxvirus to enter clinical trials, and it is also the most studied recombinant poxvirus in clinical trials. [0003] In the completed Phase I and Phase II clinical trials of JX-594, there was no significant objective curative effect report....

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/863C12R1/93
CPCC07K14/521C07K14/72C12N15/86C12N2710/24043
Inventor 唐慧李丹洋张锦锦方敬敬严新民郭强
Owner FIRST PEOPLES HOSPITAL OF YUNNAN PROVINCE
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