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Double-specificity anti-tumor recombinant adenovirus and construction method and application thereof

A technology of recombinant adenovirus and adenovirus, which is applied in the field of biotechnology and genetics, can solve the problem of high RCA pollution ratio, achieve the effect of improving tumor suppression speed and tumor suppression specificity, improving operability and reducing possibility

Inactive Publication Date: 2010-10-20
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sometimes high levels of RCA contamination necessitate an additional, time-consuming plaque purification process to purify recombinant adenovirus

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of bispecific anti-tumor recombinant adenovirus shuttle plasmid

[0047] 1. Cloning of E1a and Apoptin

[0048]According to the human type 5 adenovirus E1a gene sequence (NC_001406) and Apoptin gene sequence (NC_001427) registered in GenBank, following the basic principles of primer design, the following two pairs of primers were analyzed, designed, and screened to amplify E1a and Apoptin respectively. Primers for Apoptin:

[0049] E1aS: 5'-GCCTGCAGACCACCATGGGACATAT TATCTGCCAC-3'

[0050] E1aA: 5'-GCGGATCCTTATGGCCTGGGGCGTTTACAGC-3'

[0051] ApoptinS: 5'-GCGATATCACCACCATGGACGCTCTCCAA-3'

[0052] ApoptinA: 5'-GCGAATTCTTACAGTCTTATACACCTTCT-3'

[0053] Optimize the reaction conditions of each step and the optimal concentration of the reagents involved in the reaction, and perform DNA amplification on a PCR machine. The total reaction volume is 50 μL (5 μL of 10×PCR buffer, 1 μL of each 20 μmol / L primer pair, 5 μL of template DNA, 5 μL of dNTP (ea...

Embodiment 2

[0060] Embodiment 2 recombinant adenovirus

[0061] 1. Co-transfection

[0062] Plasmid pAd-VT (or pAd-VP) was digested and linearized with NheI as follows: mix an appropriate amount of plasmid DNA with an appropriate amount of water, and add 4U of restriction endonuclease NheI and 10 μl of the corresponding 10× restriction endonuclease Digest the enzyme reaction buffer to make the total volume 100 μl, flick the tube wall to mix and centrifuge, and put in a 37°C water bath overnight.

[0063] Add 100 μl of saturated phenol to the linearized plasmid, shake moderately, and centrifuge at 4°C and 12,000 rpm for 10 min; take the supernatant, add 100 μl saturated phenol / chloroform / isoamyl alcohol (25:24:1), shake moderately, and Centrifuge at 4°C and 12,000 rpm for 10 min; take the supernatant, add 100 μl of chloroform / isoamyl alcohol (24:1), shake moderately, and centrifuge at 4°C and 12,000 rpm for 10 min; take the supernatant, add 200 μl of absolute ethanol and 20 μl Sodium ace...

Embodiment 3

[0074] Example 3 The killing effect of recombinant adenovirus on tumor cells

[0075] Digest tumor cells in logarithmic growth phase (human lung cancer cells (A549), human liver cancer cells (HepG-2), human cervical cancer cells (Hela)). Count and adjust the cell concentration to 5×10 with complete cell culture medium 4 cells / ml, inoculated in the upper 96-well cell culture plate (i.e. 5×10 3 After the cells adhered to the wall (about 24 hours), the culture medium was discarded and washed twice with Hank’s solution. Adjust the Ad-VT titer to 1×10 with serum-free and antibiotic-free RPMI-1640 culture medium 7 PFU / ml, 1×10 6 PFU / ml and 1×10 5 PFU / ml. Take 50 μl of the above-mentioned virus dilution (i.e. 100moi (multiplicity of infection units), 10moi and 1moi), add the corresponding wells of the tumor cells washed with Hank’s solution and cultured in a 96-well cell culture plate, and store at 37°C, 5% CO 2 After 4 hours in the cell culture incubator, add complete RPMI-164...

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Abstract

The invention provides a double-specificity anti-tumor recombinant adenovirus. The recombinant adenovirus comprises an adenovirus vector and expression boxes introduced therein; the replication of the adenovirus vector depends on gene mutation; and the expression boxes comprise an expression box consisting of tumor specific promoters and E1a genes and an expression box consisting of eukaryotic promoters and Apoptin genes. The invention also provides a construction mode of the double-specificity anti-tumor recombinant adenovirus and tumor treatment medicament application thereof. The recombinant adenovirus of the invention has double specificities of tumor specific replication and tumor specific killing, can be used for preparing medicaments for treating various tumors and medicaments for preventing tumor recurrence after operation, and meanwhile can be used with any medicament or used as a medicament combined with surgical therapy, radiotherapy and chemotherapy.

Description

technical field [0001] The invention belongs to the fields of biotechnology and genes, and specifically relates to a bispecific anti-tumor recombinant adenovirus and its construction method and application. Background technique [0002] The development of malignant tumors is a dynamic process involving multiple factors. If it is not discovered in time and effective control measures are not taken, the astonishing rate of spread caused by its strong reproductive ability will become an inevitable challenge for all therapies. At this time, how to effectively remove the malignant tumor is the last line of defense for the patient's life. [0003] Years of experience in tumor gene therapy research shows that how to win the race against malignant tumors is the key to achieving a Jedi counterattack. Therefore, it can be concluded that malignant tumors are all-conquering, but the only thing that can’t be broken quickly is the conclusion that no matter how rampant the malignant tumor ...

Claims

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Application Information

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IPC IPC(8): C12N15/861A61K48/00A61P35/00C12R1/93
Inventor 金宁一李霄李昌鲁会军田明尧金扩世陈漉刘妍高鹏杨恩成
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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