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Construction scheme and use of recombinant adeno related virus containing 16 type HPV antisense E7 gene

A raav-hpv16e7as, virus technology, applied in gene therapy, genetic engineering, plant gene improvement, etc., can solve problems such as difficult tumor treatment

Inactive Publication Date: 2009-05-27
武汉市奥尼克斯基因技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Based on the above technical background and major medical needs, the present invention will disclose a construction scheme of a recombinant human adeno-associated virus type 5 (AAV5) recombinant containing the antisense E7 gene of HPV type 16. The construct obtained by this scheme has obvious Its therapeutic and preventive effects can solve the difficulties and deficiencies of current tumor treatment

Method used

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  • Construction scheme and use of recombinant adeno related virus containing 16 type HPV antisense E7 gene
  • Construction scheme and use of recombinant adeno related virus containing 16 type HPV antisense E7 gene
  • Construction scheme and use of recombinant adeno related virus containing 16 type HPV antisense E7 gene

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example 1

[0023] Example 1. Construction and identification of the backbone plasmid pUF1-HPV16E7AS containing the target gene

[0024] A 315bp exogenous HPV16E7 cDNA fragment was reversely inserted between the two Not I sites, which corresponds to 858-544nt of HPV16E7 mRNA.

[0025] RT-PCR method to amplify the target gene

[0026] ①TRIZOL TM Reagent one-step extraction of total RNA from SiHa cells; 25cm 2 Discard the culture medium in the culture bottle, wash it once with RNase-free PBS, and absorb the PBS; add TRIZOL TM Reagent 1ml, make it evenly cover the bottom of the bottle, place at room temperature for 5-15min, transfer the liquid in the bottle to EP tube; add 0.2ml of chloroform, vibrate vigorously for 15 seconds, place at room temperature for 10-15min; centrifuge at 4°C, 12000rpm×15min; Transfer the upper aqueous phase to another EP tube, add 0.5ml ice-cold isopropanol, mix well, place at room temperature for 10min; Centrifuge at 7500rpm×15min; suck the supernatant with a ...

example 2

[0097] Example 2, packaging and purification of recombinant adeno-associated virus

[0098] Inoculate 293 cells in logarithmic growth phase to 75cm 2 When nearly 70% confluent in the culture flask, the calcium phosphate precipitation method was used for cell transfection, and pUF1-E7AS, pXX2, pXX6 or pdx11-lacZ, pXX2, pXX6 were co-transfected into 293 cells in a molar ratio of 1:1:1 After 12 hours, change the medium, suck out the medium at 72 hours, collect the cells with a cell curette, collect 60 bottles of cells in each group, place the cells in a -80°C refrigerator, freeze and thaw three times, centrifuge at 12,000rpm at 4°C for 20min, and take the supernatant spare. Viruses were purified and concentrated using cesium chloride gradient centrifugation.

example 3

[0099] Example 3. Determination of recombinant adeno-associated virus titer and activity

[0100] Determination of rAAV-E7AS and rAAV-Laz virus titers by dot blot method.

[0101] ① Dilution of samples: Take 40 μl of the supernatant containing recombinant adeno-associated virus, put it in 200 μl of DNaseI, RNase and corresponding buffer solution at 37°C for 1 hour, then add 400ul of 10ng / ml proteinase K system, and incubate at 37°C for 2-3 hours Equal volumes of phenol, chloroform, and isotropic alcohol were extracted, centrifuged at 12,000 rpm at 4°C for 15 min, and 400 μl of the supernatant was mixed with an equal volume of 0.4M NaOH / 10mmEDTA. The control plasmid pdx11-lacZ, pUF 1 -E7AS and the rAAV-E7AS and rAAV-lacZ treated by the above method were heated at 100°C for 5 minutes, cooled on ice for 2 minutes, and pressed with 0.4M NaOH / 10mM EDTA for 10 11 , 5×10 10 , 2.5×10 10 , 1×10 10 , 5×10 9 , 2.5×10 9 , 1×10 9 , 5×10 8 , 2.5×10 8 、10 8 、10 7 、10 6 Molecules...

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Abstract

The invention discloses a proposal for constructing gland related virus containing 16 type antisense E7 gene of human papilomavirus(HPV), and particular application of a recombination gland related virus constructor obtained by the proposal in tumor treatment. By techniques of full length enlarging by the PCR method, enzyme cutting, connection, homologous recombination, transfection, purification of gland related virus and the like, the recombination gland related virus constructor is obtained. The technical characteristic comprises that a 16 type E7 gene cDNA segment of the full length exogenous HPV is oppositely inserted in a NotI enzyme cutting site. The recombination gland related virus constructor has the following obviously technical advantages: chromosomes can be integrated in a fixed site; the inserted reverse exogenous gene is a full length segment, and has good specificity; and the constructor has strong effect on cells infected with the 16 type HPV, and has prevention function. The recombination gland related virus constructor provides an ideal gene target point specificity treatment and prevention application way for treating and preventing tumor gene.

Description

1. Technical field [0001] The present invention relates to a human adeno-associated virus type 5 (Human adeno-associated virus type 5, AAV5) recombinant construction scheme, which is characterized in that: a 315bp exogenous type 16 is reversely inserted into the Not I restriction site The cDNA fragment of HPV E7 genetic engineering is equivalent to 858-544nt of type 16 HPV E7 mRNA, so as to obtain the recombinant adeno-associated virus construct rAAV-HPV16E7AS with therapeutic effect on tumors. The content of the present invention belongs to the technical field of medical genetic engineering. 2. Background technology [0002] Cervical cancer ranks second among the malignant tumors that cause women's death. Due to the lack of suitable screening methods, the incidence of cervical cancer ranks first in developing countries. Epidemiological survey data show that my country is one of the countries with a high incidence of cervical cancer, with an annual incidence rate of 14.6 / 1...

Claims

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Application Information

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IPC IPC(8): C12N15/861A61K48/00A61K47/42A61P35/00
Inventor 马丁王世宣周剑峰卢运萍邬素芳
Owner 武汉市奥尼克斯基因技术有限公司
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