Construction method and application of tumor specific adenovirus vector carrying p21ras single-chain antibody gene

A tumor-specific, single-chain antibody technology, applied in the direction of anti-tumor drugs, applications, antibodies, etc., can solve the problems of low virus delivery efficiency, inability to exert anti-tumor effect, damage, etc., and achieve the effect of reducing damage and clearing effect

Active Publication Date: 2014-06-25
成都军区昆明总医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main method of virus administration is intratumoral injection or intravenous injection. Due to various immune, biochemical or physical barriers, the efficiency of virus delive

Method used

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  • Construction method and application of tumor specific adenovirus vector carrying p21ras single-chain antibody gene
  • Construction method and application of tumor specific adenovirus vector carrying p21ras single-chain antibody gene
  • Construction method and application of tumor specific adenovirus vector carrying p21ras single-chain antibody gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Preparation of single-chain antibody gene fragments

[0061] 1. Immunization of Balb / c mice with p21ras protein: Take 5 Balb / c mice aged 6-8 weeks (purchased from the Experimental Animal Center of Chongqing Third Military Medical University), and inject 100 μg of the prokaryotic Express the purified p21ras-K protein (for the preparation method of p21ras-K protein, refer to the paper "Expression, Identification and Purification of Recombinant p21ras Protein and Preparation of Polyclonal Antibody"), add an equal amount of complete Freund's adjuvant for the initial injection, Inject subcutaneously at 5 points. Two weeks later, the second injection was given, with the same dose as the first injection, plus an equal amount of incomplete Freund's adjuvant, and injected subcutaneously at 5 points. Two weeks later, the third injection was given, the dose was the same as the first one, without adjuvant, intraperitoneal injection. The fourth injection was given two w...

Embodiment 2

[0069] Example 2: Establishment and screening and identification of single-chain antibody library

[0070] 1. Construction of recombinant phagemid

[0071] 1.1 Double enzyme digestion of the recombinant pMD-ScFv vector and expression vector: extract the plasmid from the positive pMD-ScFv clone identified by PCR, and follow the instructions of Tiangen Plasmid Extraction Kit for the extraction steps. Recombined pMD-ScFv plasmid and expression vector plasmid pCANTAB-5E (purchased from Pharmacia Company) were digested with Sfi I respectively, and 30 μl of expression vector plasmid / pMD-ScFv vector were added in 200 μl PCR reaction tube respectively; Sfi I enzyme ( 10U / μl) 4μl; 10×Buffer M5μl, ddH 2 O11μl, after the above system is configured, react at 50°C for 4 hours. After gel recovery and purification of the digested product, add 30 μl of the purified product, 2 μl of Not I enzyme (10U / μl), 5 μl of 10×Buffer H, 2 μl of BSA, 2 μl of Trion X-100, ddH into a new 200 μl PCR reacti...

Embodiment 3

[0083] Example 3: Preparation of tumor-specific adenovirus KGHV500

[0084] 1. Construction of tumor-specific adenovirus shuttle plasmid pXC2P

[0085] 1.1 hTERT promoter replaces viral E1A promoter

[0086] 1.1.1 Construction of the E1A gene promoter deletion mutant fragment: using the human adenovirus type 5 shuttle plasmid pXC1 (purchased from Microbix Biosystems Ins) as a template, the E1A promoter deletion fragment was constructed by overlap extension PCR. The specific steps are as follows: Use the E1Af1 forward primer (5'-CGTCTTCAA with the EcoR I restriction site at the end of the primer) GAATTC TCATGTTT-3') and E1Ar1 reverse primer (5'-CCGCTCGAGCGGCGAC GTC GAC GCGTCACTACACGTCAGCTGACTATAATAATA-3') amplifies the partial fragment 894bp upstream of the E1A promoter. The E1Af2 forward primer (5'-ACGCGTCGACGTCGGCCG with the Xho I restriction site at the end of the primer CTCGAG CGG GACTGAAAATGAGACATATTATCTGC-3') and E1Ar2 reverse primer (5'-TGCATTC TCTAGA CACAGGTGAT-3...

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Abstract

The invention discloses a construction method of a tumor specific adenovirus vector carrying a p21ras single-chain antibody gene and application of the tumor specific adenovirus vector to CIK (cytokine induced killer) cells by loading. The prepared conditionally replicating tumor specific adenovirus has double targeting and can infect the CIK cells. The tumor specific adenovirus KGHV500 carrying the p21ras single-chain antibody gene is prepared by cloning a p21ras single-chain antibody and a GFP (green fluorescent protein) gene into a recombinant adenovirus shuttle plasmid pXC2P simultaneously. The prepared recombinant adenovirus can specifically recognize tumor cells and has low toxicity towards normal cells. The p21ras single-chain antibody gene carried by the virus can be expressed for a long term along with virus replication, and the virus shows good anti-tumor effects through in vitro and vivo experiments. The tumor specific adenovirus vector can be used for researching and developing drug preparations of related tumors caused by p21ras protein over-expression, such as breast tumors and the like.

Description

technical field [0001] The invention belongs to the field of medical biology and relates to the fields of genetic engineering and tumor biological treatment. Specifically, it involves the use of molecular cloning technology, using tumor-specific promoters to regulate the expression of viral E1A and E1B proteins, and transforming the ciliary protein of tumor-specific adenoviruses so that the adenovirus shell has type 5 / type 35 adenovirus chimeric ciliary proteins, and at the same time The p21ras single-chain antibody gene is inserted into the tumor-specific adenovirus gene, and the dual-targeted tumor-specific adenovirus carrying the gene is carried to the tumor tissue site through CIK cells, and synergistically exerts the effect of killing tumor cells. Background technique [0002] At present, tumor has become a common and frequently-occurring disease, among which malignant tumor has high mortality rate and short survival period, which has seriously threatened people's healt...

Claims

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Application Information

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IPC IPC(8): C12N15/861A61K48/00A61K39/395A61P35/00
Inventor 杨举伦李静姜英翰刘秀娟冯强
Owner 成都军区昆明总医院
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