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Preparation method of recombinant adeno-associated viruses containing EB virus latent membrane protein 1 and 2 genes and application thereof

An EB virus, virus technology, applied in the field of bioengineering

Active Publication Date: 2013-04-10
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recombinant adenovirus vaccine containing EB virus latent membrane protein 2 gene, publication number: CN1548532; combined immunization preparation containing recombinant plasmid and replication-defective recombinant adenovirus, publication number: CN1927405; Ad5F35- containing EB virus latent membrane protein 2 gene LMP2 recombinant adenovirus vaccine, publication number: CN1907493, but this research is also based on the adenovirus vector system, but adenovirus is pathogenic, and application to immunocompromised tumor patients will bring infection risks

Method used

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  • Preparation method of recombinant adeno-associated viruses containing EB virus latent membrane protein 1 and 2 genes and application thereof
  • Preparation method of recombinant adeno-associated viruses containing EB virus latent membrane protein 1 and 2 genes and application thereof
  • Preparation method of recombinant adeno-associated viruses containing EB virus latent membrane protein 1 and 2 genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of rAAV-LMP2 / 1-HSP recombinant adeno-associated virus

[0036] 1. Construction of pAAV-LMP2 / 1-HSP recombinant adeno-associated virus vector plasmid

[0037] (1) Preparation of Escherichia coli Competent Cells

[0038] Take out the E.coli DH5α strains stored at -80°C, pick a small amount of bacterial solution with an inoculation loop and streak it on the LB agar plate, and culture it upside down at 37°C overnight; pick a single DH5α colony the next day, and inoculate it in 3ml LB medium ( No antibiotics,) in a shaker at 37°C at 280rpm overnight; the next day, take 0.5ml of the above bacterial solution and inoculate it into 50ml LB medium, shake at 280-300rpm at 37°C until the OD value of the bacterial solution is A 600 Take it out when it reaches 0.4~0.6; centrifuge at 5,000rpm at 4°C for 10min, discard the supernatant, and pre-cool with 20ml CaCl 2 (0.1mol / L) resuspended bacteria, ice-bathed for 30min, centrifuged at 5,000rpm at 4°C for 10min, ...

Embodiment 2

[0072] Embodiment 2: the determination of rAAV-LMP2 / 1-HSP and rAAV-GFP virus titer:

[0073] (1) Probe labeling

[0074] The CMV promoter fragment in the vector was labeled [α- 32 p]dCTP as a probe:

[0075] Dissolve CMV in sterilized TE at a concentration of 25 μg / mL, boil at 100°C for 2 minutes and place on ice immediately;

[0076] Add the following reactants sequentially on ice:

[0077] Component Amount Final Concentration

[0078] 5×labeling buffer 10μl 1×

[0079]dNTPs mixture 2μl 20μM / each

[0080] CMV template 1μl 500ng / ml

[0081] BSA 2μl 400μg / ml

[0082] [α- 32 P]-dCTP (3000Ci / mmol) 5μl 333nM

[0083] Klenow enzyme 1μl 100u / ml

[0084] Sterilized water 29μl

[0085] Total volume 50μl

[0086] Gently mix the above reactants and incubate at room temperature for 60 min;

[0087] After boiling at 100°C for 2 minutes, place it on ice immediately;

[0088] Add EDTA to a final concentration of 20mM to terminate the reaction, and use it directly in the hybrid...

Embodiment 3

[0105] Example 3: Analysis of immune activity of rAAV-LMP2 / 1-HSP

[0106] BALB / c myeloma cells (SP2 / 0 cells) were routinely cultured. When the cells grew to the mid-log phase, they were transfected with the recombinant plasmid pCDNA3.1-LMP2 carrying the LMP2 gene sequence by liposome method (Lipofectamine 2000 Reagent, Invitrogen). After transfection, they were cultured with RPMI 1640 medium containing 10% bovine serum at 37°C for 24 hours, and then replaced with selective medium containing 600 μg / ml G418 to continue culturing. Three weeks later, positive cells with stable resistance were obtained. After continuing to expand the culture, they were used as target cells. 4 weeks old BALB / c(H-2 d ) male mice were randomly divided into 2 groups, 6 in each group, and each animal was 5×10 10 The number of virus particles was injected intramuscularly with the recombinant adeno-associated virus of this name and the control virus, respectively, to immunize animals. Booster immuniz...

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Abstract

The invention provides a preparation method of recombinant adeno-associated viruses containing EB virus latent membrane protein 1 and 2 genes and application thereof. The recombinant adeno-associated viruses of the invention carry DNA sequences of encoding EB virus latent membrane protein 1 and 2 as shown in CCTCC NO: V200907, wherein the virus titer of the recombinant adeno-associated viruses is not smaller than 1*10<11> / ml viral particles. The recombinant adeno-associated viruses can induce the EB virus specific cell cytotoxic lymphocyte reaction in vivo. The preparation method of the invention comprises the following steps: 1) cloning DNA sequences for encoding the proteins LMP1, LMP2 and HSP70 to proper adeno-associated virus carrier plasmids to obtain recombinant adeno-associated virus carrier plasmids containing the DNA encoding sequences of LMP1, LMP2 and HSP70; and 2) using the recombinant adeno-associated virus carrier plasmids in the first step and auxiliary plasmids for cotransfecting proper incasing cells to obtain required recombinant adeno-associated viruses.

Description

technical field [0001] The invention relates to the technical field of bioengineering. Specifically, it relates to a preparation method of recombinant adeno-associated virus containing Epstein-Barr virus latent membrane protein 1, 2 and heat shock protein 70 genes, and the application of the recombinant adeno-associated virus in vaccines for preventing and treating Epstein-Barr virus-related tumors. Background technique [0002] Nasopharyngeal carcinoma is a malignant tumor of epithelial origin. The traditional treatment of nasopharyngeal carcinoma is based on radiation therapy, supplemented by induction chemotherapy. This traditional treatment model has played a great role in the past few decades, saving the lives of many patients. However, advanced nasopharyngeal carcinoma accounts for 70% of the total cases of NPC, and its 5-year survival rate has not been improved. The main reasons for treatment failure are local uncontrolled, recurrence and distant metastasis. How to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/85A61K39/245A61K48/00A61P35/00C12R1/93
Inventor 汪道文潘建青肖啸
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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