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HPV16, 18L1 recombinant DNA vaccine for preventing and treating esophagus cancers

A HPV16, gene technology, applied in the field of tumor DNA vaccine preparation, can solve the problem that the safety cannot be guaranteed

Inactive Publication Date: 2014-05-28
BEIJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But it is precisely because E6 and E7 integrate the host genome and can induce cancer, the safety cannot be guaranteed

Method used

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  • HPV16, 18L1 recombinant DNA vaccine for preventing and treating esophagus cancers
  • HPV16, 18L1 recombinant DNA vaccine for preventing and treating esophagus cancers
  • HPV16, 18L1 recombinant DNA vaccine for preventing and treating esophagus cancers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 recombinant VR-HPV16, the construction of 18L1 DNA plasmid

[0025] 1.1 PCR amplification of HPV16, 18L1-pVR gene fragment

[0026] 1.1.1 The HPV16, 18L1-pVR gene fragment was amplified by PCR using pMK-RQ containing the codon-optimized HPV18L1 gene as a template and the synthetic sequence as a primer. After the completion of PCR, 1 μL of the reaction solution was taken for DNA gel (1%) electrophoresis detection. The PCR product was recovered after a good detection result was obtained.

[0027]1.1.2 Construction of HPV16L1 gene sequence: PCR amplification was carried out with the PUC-HPV16L1 plasmid containing HPV16L1 gene as template and the synthetic sequence as primer. dNTP8μl, Pfu2μl, ddH2O77μl, total system 100μl. After 30 cycles (denaturation at 94°C for 30s, annealing at 58°C for 30s, and extension at 72°C for 30s), the HPV16L1 gene sequence was obtained, and the PCR reaction product was purified by gel cutting.

[0028] 1.2 Ligation of HPV18L1-p...

Embodiment 2

[0033] Example 2 Detection of pVR-HPV16, 18L1 expression in vitro

[0034] 2.1 Culture of HEK293 cells The medium used for culturing HEK293 cells is DMEM medium containing 10% FBS and 1% double antibody, and the culture conditions are 37°C, 5% CO 2 Constant temperature incubator.

[0035] Transfection of cells with the plasmid mentioned in 2.2

[0036] (1) The day before transfection, 293 cells were trypsinized and counted, transferred to a six-well plate, and the density was controlled so that the density on the day of transfection was close to 90%. Cells were plated in 2 mL of serum-containing normal growth medium without antibiotics.

[0037] (2) For each well of cells, dilute 4 μg of plasmid (pVR-HPV16, 18L1) with 250 μL of OPTI-MEM I medium. Mix Lipofectamine2000 reagent before use, and dilute 10 μL Lipofectamine2000 with 250 μL OPTI-MEM I medium. Mix gently, let stand at room temperature for 5 minutes, then mix with diluted plasmid, and let stand at room temperature ...

Embodiment 3

[0042] Example 3 Experiment of Recombinant DNA Vaccine pVR-HPV16, 18L1 Immune Mice

[0043] 3.1 Preparation and immunization plan for immunized mice

[0044] Twenty healthy female BALB / cH-2Kd mice of SPF grade 4-6 weeks old obtained from commercial sources were randomly divided into 3 groups, 10 mice in each group, and immunized twice at the 0th and 3rd week by intramuscular injection into the thigh. The immune response was measured one week after the booster immunization. See Table 1 for specific immunization contents and doses.

[0045] Table 1 Mouse immunization groups and doses

[0046]

[0047] 3.2 Detection of HPV16, 18L1 humoral immune response in immunized mice

[0048] One week after the last booster immunization (day 28), the venous blood of the mice was collected, and the serum was separated. HPV16 and 18 pseudovirions were prepared according to the method reported in the literature (Buck, C.B., D.V. Pastrana, D.R. Lowy, et, al. J. Virol. 2004, 78:751-757.). ...

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Abstract

An HPV16, 18L1 recombinant DNA vaccine for preventing and treating esophagus cancer belongs to the technical field of biological medicines. The invention provides an optimal gene sequence coding the major capsid protein L1 of HPV16, 18, a pVR-HPV16, 18L1 DNA vaccine containing the gene sequence, and a preparation method for the pVR-HPV16, 18L1 DNA vaccine. The HPV16, 18L1 recombinant DNA vaccine can also be used for preparing a vaccine for preventing and treating diseases such as cervical cancer and head and neck cancer, caused by HPV.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals. The invention relates to a preparation method of a tumor DNA vaccine containing codon-optimized HPV16, 18L1 gene. Background technique [0002] Cervical cancer is the second largest gynecological malignancy in the world, second only to breast cancer. There are about 500,000 new cases of cervical cancer worldwide, and about 200,000 deaths from cervical cancer, of which more than 90% are from developing countries. The mortality rate is the first among all kinds of gynecological tumors. There are 131,500 newly discovered cases in my country every year, accounting for about 28.8% of the global total. In the 1970s, ZurHausen proposed that HPV is the virological cause of cervical cancer. After that, domestic and foreign scholars conducted a lot of research on the relationship between the two and proposed that more than 90% of cervical cancer is caused by HPV infection. The International Association ...

Claims

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Application Information

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IPC IPC(8): C12N15/37C12N15/85C12N15/66A61K48/00A61K39/12A61P31/20A61P35/00
Inventor 周玉柏曾毅方军刘海庭沈思嗣李劲涛李泽琳
Owner BEIJING UNIV OF TECH
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