Replicon DNA (deoxyribonucleic acid) vaccine vector, and construction method and application thereof

A DNA vaccine and replicon technology, applied in the direction of recombinant DNA technology, the use of vectors to introduce foreign genetic material, pharmaceutical formulations, etc., can solve the problems that are not suitable for the development of clinical biotherapeutic drugs, etc., and achieve rapid measurement, broad application prospects, and high sensitivity Effect

Inactive Publication Date: 2013-08-28
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the modified pSCA1, like the original vector pSFV1, is an ampicillin-resistant carrier, and ampicillin-like drugs are still commonly used clinically. Therefore, the ampicillin-resistant carrier will have certain risks in clinical application and is not suitable for use. Research and development of clinical biotherapeutic drugs

Method used

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  • Replicon DNA (deoxyribonucleic acid) vaccine vector, and construction method and application thereof
  • Replicon DNA (deoxyribonucleic acid) vaccine vector, and construction method and application thereof
  • Replicon DNA (deoxyribonucleic acid) vaccine vector, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1, Construction of Replicon DNA Vaccine Vector pSVK

[0061] The construction method of the replicon DNA vaccine vector pSVK of the present invention comprises the following steps:

[0062] 1. Construction of recombinant vector pUC19-PS-KANA carrying PS and KANA fusion gene (PS-KANA)

[0063] Such as figure 1 As shown, the construction method of the recombinant vector pUC19-PS-KANA is as follows:

[0064] 1) Amplify the PS gene: use the replicon DNA vaccine vector pSCA1 containing the ampicillin resistance gene (for the construction method, refer to the literature Yu Y Z, Sun Z W, Yu W Y. Chinese Journal of Biotechnology, 2005, 21(5): 33- 38) as a template, under the guidance of primers PSCA-F (5'-CCGTCTAGAGATCATAATCAGCCAT-3') and PSCA-R (5'-CCGGCATGCCTCGAGACTAGTCTGTCAGACCAAG-3'), PCR amplifies the flanking sequence of the ampicillin resistance gene, the The side sequence at the 5' end contains a restriction endonuclease Xba I recognition site, and the side s...

Embodiment 2

[0069] Example 2, Construction and in vitro expression of replicable luciferase expression plasmid pSVK-luc

[0070] 1. Construction of replicable luciferase expression plasmid pSVK-luc

[0071] The firefly luciferase (luciferase) gene (GenBank No.: JN244035.1) was cloned into the replicon DNA vaccine vector pSVK modified for resistance in Example 1 to obtain the replicable luciferase expression plasmid pSVK-luc, specifically constructed Methods as below:

[0072] Using firefly luciferase reporter vector pGL-3-CMV (Zhang Liang, Li Xiaoxiao, Han Gang, Dong Jinkai, Yan Jinqi, Xiao Yi, Yu Jiyun. Experimental study on the effect of delivering plasmid DNA in different ways to induce expression in vivo. Journal of Military Medical Training College. 20101;31 (1):48-49) was used as a template, and the firefly luciferase gene luc was amplified by PCR under the guidance of primers Luc-F (5'-ATAGGATCCGCCACCATGGAAGACGC-3) and Luc-R (5'-ATTCCCGGGTTACACGGCGATCTTTC-3'); The PCR reaction sy...

Embodiment 3

[0076] Example 3. In vivo expression function verification of pSVK-luc and study on electroporation delivery conditions

[0077] Replicon DNA vaccine is a new type of vaccine with great development potential, which is extremely beneficial to the research of new therapeutic vaccines for major chronic diseases, but the research on how to efficiently deliver new plasmid DNA vaccines in vivo is still very limited. The recently developed high-efficiency technology uses high-intensity electric fields to instantly increase the permeability of cell membranes and introduce nucleic acids, proteins, and other foreign molecules into cells. In order to further study the specific conditions of in vivo electroporation delivery of the replicon DNA vaccine and the dynamic expression of the vaccine in vivo, the present invention simulates the immunization process of the replicon DNA vaccine through bioluminescent technology in vivo, and studies the possible delivery of the replicon DNA vaccine. ...

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Abstract

The invention discloses a replicon DNA (deoxyribonucleic acid) vaccine vector, and a construction method and application thereof in preparation of a replicon DNA vaccine. The replicon DNA vaccine vector having kanamycin resistance is obtained by replacing an ampicillin resistance gene in the replicon DNA vaccine vector pSCA1 with a kanamycin resistance gene, so that the replicon DNA vaccine prepared by using the vector can meet the physical clinical demands for ampicillin medicaments and improve the safety of clinical application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a replicon DNA vaccine carrier and its construction method and its application in preparing the replicon DNA vaccine. Background technique [0002] Because DNA vaccines can induce cellular immunity through the MHC class I antigen presentation pathway, especially the ability to induce antigen-specific cytotoxic T lymphocyte (cytotoxicity T lymphocyte, CTL) activity, it is recognized as an immunotherapy method with good application prospects , which is extremely beneficial to the study of new therapeutic vaccine drugs for the treatment of major chronic diseases. However, recent studies have shown that DNA vaccines are not effective in large animal models and human trials, and it is necessary to upgrade or enhance the efficacy of traditional DNA vaccines. [0003] In recent years, a new type of gene vaccine carrier system has been gradually developed in the world, called re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66C12N15/85A61K48/00A61P31/20A61P35/00A61P31/14A61P31/18A61P31/06A61P31/04
Inventor 于继云阎瑾琦贾锐张亮徐元基张巍王宇朱晓明
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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