Tumor targeting recombinant DNA vaccine, preparation method thereof and application thereof

A DNA vaccine and tumor-targeting technology, which is applied in the field of tumor-targeted recombinant DNA vaccines and DNA vaccines, to achieve the effects of fewer vaccinations, easy transportation and use, and low preparation costs

Active Publication Date: 2010-02-17
SHANGHAI PULMONARY HOSPITAL AFFILIATED TO TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But there is no relevant report about COX-2 DNA vaccine

Method used

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  • Tumor targeting recombinant DNA vaccine, preparation method thereof and application thereof
  • Tumor targeting recombinant DNA vaccine, preparation method thereof and application thereof
  • Tumor targeting recombinant DNA vaccine, preparation method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Cloning of COX-2 gene

[0043] Extraction of total mRNA from A549 cells

[0044] A549 cells were cultured in complete 1640 medium containing 10% calf serum at 37°C and 5% CO 2 After trypsinization, the cells were centrifuged to collect the cells in an incubator, and the supernatant was discarded. The total mRNA was extracted according to the Trizol extraction kit (Hua Shun Company), and detected by electrophoresis.

[0045] Primer design and RT-PCR amplification

[0046] Primers were designed according to the COX-2 gene sequence provided by the accession number BC013734 of the GenBank database and the restriction sites on the pVAX1 vector, and BamH I and EcoR I restriction sites were introduced into the upper and lower primers respectively. The primers were provided by Shanghai Saibaisheng Gene Technology Co., Ltd. synthesis:

[0047] Upstream primer (SEQ ID NO 8): 5'-CG GGATCC GGTGGGGGCGGTAACGTTCCTTGCTGTTCCAACCCATGTC-3` (the BamH I site is underlined, pa...

Embodiment 2

[0078] Example 2 Cloning of murine mUbi gene

[0079] Extraction of total mRNA from mouse testis tissue

[0080] The testicular tissues of C57BL / 6 male mice (Shanghai Experimental Animal Center, Chinese Academy of Sciences) were washed under sterile conditions with DEPC-treated double distilled water twice, and 0.5 g of tissues were taken, crushed, and extracted according to the Trizol Kit (Hua Shun Company) The total mRNA was extracted and detected by electrophoresis.

[0081] Primer design and RT-PCR amplification

[0082] Primers were designed according to the mUbi gene sequence provided by the GenBank database X51703 accession number and the restriction site on the pVAX1 vector, and the HindIII and BamH I restriction sites were respectively introduced into the upper and lower primers. The primers were synthesized by Shanghai Saibaisheng Gene Technology Co., Ltd.:

[0083] Upstream primer (SEQ ID NO 10): 5'-CG AAGCTT GCCACCATGCAGATCTTCGTGAAAACC-3` (the underline is the ...

Embodiment 3

[0105] Example 3 Construction of pVAX1-mUbi-ISS-COX-2-ISS Tumor Targeting DNA Vaccine Recombinant Vector

[0106] Construction of pVAX1-mUbi recombinant vector

[0107] Incubate 5 mL of LB containing 20 μg / mL Amp resistance, and culture DH5a-competent Escherichia coli containing pMD19-mUbi plasmid overnight at 37°C with shaking, and extract pMD19-mUbi plasmid DNA according to the third edition of "Molecular Cloning Experiment Guide".

[0108] Also culture 5 mL of LB containing 50 μg / mL Kan resistance, and culture DH5a-competent Escherichia coli containing pVAX1 plasmid overnight at 37°C with shaking, and extract pVAX1 plasmid DNA according to the third edition of "Molecular Cloning Experiment Guide".

[0109] Digest pMD19-mUbi plasmid DNA and pVAX1 plasmid DNA (Invitrogen company) with HindIII and BamH I (TaKaRa company), respectively, and the digestion reaction system is 80 μL:

[0110] Plasmid DNA 20μL

[0111] HindIII 3μL

[0112] BamH I 3 μL

[0113] 10×buffer K 8μL

...

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Abstract

The invention relates to the field of genetic engineering and relates to a tumor targeting recombinant DNA vaccine, a preparation method thereof and application thereof. The preparation method comprises the following steps: recombining murine ubiquitin mUbi, COX-2 and a immunostimulatory DNA sequence ISS by utilizing a genetic engineering method; and inserting a fragment mUbi-ISS-COX-2-ISS at multiple cloning sites MCS of a basic vector pVAX 1 to build encoding murine ubiquitin protein mUbi and COX-2 fusion protein antigens, and linking the fusion protein antigens by a flexible joint so as toprepare the tumor targeting recombinant DNA vaccine. After the vaccine immunizes animals, an organism immune reaction can be activated, and a specific and efficient CTL reaction of the COX-2 is quickly induced in vivo so as to carry out targeting killing of tumor cells abnormally expressed by the COX-2 and fulfill the aim of targeting tumor therapy. The tumor targeting recombinant DNA vaccine hassimple preparation, high security, low production cost, large-scale production and lasting immune response, and can also be used in the fields of inflammation treatment and cancer pain treatment.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a tumor targeting recombinant DNA vaccine. More specifically, the present invention relates to a DNA vaccine of COX-2 related to the occurrence and development of tumors. The vaccine induces a CTL response to tumor-targeted killing in patients to achieve the goal of tumor treatment. Background technique [0002] In the process of tumorigenesis, tumors need promoting factors to accelerate the speed of carcinogenesis. Cyclooxygenase-2 (Cyclooxygenase-2, COX-2) is one of the important promoting factors among many tumor promoting factors. COX-2 and its isoenzyme COX-1 are the key rate-limiting enzymes in the process of decomposing arachidonic acid (Arachidonic acid) to generate various endogenous Prostaglandins (PGs) (Cell, 1995, 83(3): 493 -501; Cell, 1996, 87(5):803-809; Cell, 1998, 93(5):705-716). COX enzyme (EC 1.14.99.1) has two activities of cyclooxygenase and peroxidase, COX-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K48/00C12N15/62C12N15/63A61P35/00A61P29/00
Inventor 王和勇周彩存罗恒唐亮赵应敏
Owner SHANGHAI PULMONARY HOSPITAL AFFILIATED TO TONGJI UNIV
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