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Recombinant adenovirus, preparation method and application thereof

A technology of recombinant adenovirus and toxin, which is applied in the field of recombinant adenovirus, can solve the problem of undiscovered glioma and achieve the effect of efficient infection time

Inactive Publication Date: 2010-12-22
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there have been reports at home and abroad about the connection of specific genes with adenovirus vectors in glioma, malignant tumors, prostate cancer, liver cancer, esophageal cancer, etc., but no recombinant adenovirus-mediated scorpion chloride ion channel toxin gene therapy has been found. Glioma reports

Method used

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  • Recombinant adenovirus, preparation method and application thereof
  • Recombinant adenovirus, preparation method and application thereof
  • Recombinant adenovirus, preparation method and application thereof

Examples

Experimental program
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Embodiment 1

[0030] Example 1 Construction of recombinant adenovirus

[0031] (1) Optimized synthesis and identification of BmK CT gene and construction and identification of intermediate plasmid pShuttle-IRES-hrGFP-2-BmK CT

[0032] The known BmK CT gene sequence was optimized according to the preferred codon table of mammals, handed over to Shanghai Sangon Bioengineering Technology Co., Ltd. for artificial synthesis and cloned into the vector pUC57, and Xho I and HindIII restriction sites were added to the two ends of the sequence respectively , for enzyme digestion identification ( figure 1 ).

[0033] The cloning vector pUC57 carrying the optimized BmK CT gene was transformed into Escherichia coli DH5a for massive amplification, and the plasmid was extracted and identified by PCR and enzyme digestion.

[0034] Primers were designed, and EcoRV and Xho I restriction sites were added to both ends of the BmK CT gene by PCR method ( figure 2 ), the PCR product was recovered after diges...

Embodiment 2

[0039] Example 2 Experiment of recombinant adenovirus inhibiting the proliferation and spread of glioma cells

[0040] (1) Concentration determination and purity identification of recombinant adenovirus

[0041] Densitometry was used to determine the concentration of adenovirus. The blank tube was 960 μl of PBS, 40 μl of 10% SDS; the measuring tube was 860 μl of PBS, 100 μl of recombinant virus solution, and 40 μl of 10% SDS. After incubation at 56°C for 1 hour, the supernatant was collected by centrifugation. UV spectrophotometer measures OD260 value and OD280 value, virus titer=A260×dilution factor×10 12 The ratio of vp / ml; A260 / A280 reflects the purity of the adenovirus, and >1.3 indicates that the purity of the adenovirus is high. The virus assay A260=0.246 of this experiment package, A280=0.170; Virus titer is 2.46×10 12 , A260 / A280=1.45, the purity is higher.

[0042] The identification of the recombinant adenovirus adopts the extracted and purified Ad-BmK CT virus ...

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Abstract

The invention provides recombinant adenovirus, comprising: using a mammal preference codon table to optimize and synthesize the gene of chloride ion channel toxin (BmK CT) of the scorpion; cloning to a intermediate vector of the adenovirus to obtain a recombinant plasmid pShuttle-IRES-hrGFP-2-BmK CT comprising the toxin gene; transforming the recombinant plasmid to the escherichia coli BJ5183 having the adenovirus skeleton genome, obtaining pAdEasy-1-BmK CT after homologous recombination; transfecting the adenovirus genome to the AD293 cell, obtaining the recombinant adenovirus particles AdEasy-1-BmK CT containing the gene of chloride ion channel toxin of the scorpion. The chloride ion channel toxin can inhibit the glioma; with the adenovirus as the carrier, the infection efficiency and the action time of the glioma are improved; the recombinant adenovirus has good application prospect in the gene treatment aspect of the glioma.

Description

technical field [0001] The invention relates to a recombinant adenovirus, in particular to a recombinant adenovirus containing the gene of the East Asian scorpion chloride ion channel toxin (BmK CT for short), a preparation method thereof, and an application of the recombinant adenovirus in the preparation of drugs for treating glioma. Background technique [0002] Brain glioma is the most common malignant tumor in the central nervous system. The conventional clinical treatment is mainly based on surgical resection, radiotherapy or chemotherapy, which cannot be completely treated and is prone to recurrence. The treatment process is also very harmful to the patient. damage. With the development of gene manipulation technology, gene therapy has been widely used. Adenoviral vectors carrying specific tumor suppressor genes have obvious advantages in cancer treatment. Adenovirus was first discovered in the human body. It can infect most mammalian cells and stably express the ta...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N7/01A61K48/00A61P35/00A61P25/00
Inventor 梁爱华杜军付月君张志云王伟柴宝峰申泉杨仁佳
Owner SHANXI UNIV
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