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Nonresistant bifunctional DNA vaccine vector, and construction method and application thereof

A technology of DNA vaccine and construction method, which is applied in the field of non-resistant bifunctional DNA vaccine carrier, can solve the problems of no medicine, social panic, etc., and achieve the effect of high copy number, good safety and easy preparation

Inactive Publication Date: 2015-10-28
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A variety of superbugs that have emerged in recent years are caused by the spread of resistance genes. There is no cure for them clinically, and they have brought great panic to the society.

Method used

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  • Nonresistant bifunctional DNA vaccine vector, and construction method and application thereof
  • Nonresistant bifunctional DNA vaccine vector, and construction method and application thereof
  • Nonresistant bifunctional DNA vaccine vector, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] In this example, the non-resistance bifunctional DNA vaccine vector pcD-asd was constructed based on pcDNA3.1-Zeo(+), and the construction diagram is shown in figure 2 , the specific operation is as follows:

[0034] 1) Amplify the non-antibiotic resistance gene aspartate β-galactose dehydrogenase (SD-asd), using the balanced lethal system vector pYA3493 plasmid (gifted by Dr. Roy Curtiss III, University of Washington, USA) as a template, according to the established Published balanced lethal system vector pYA3493 plasmid gene sequence, designed a pair of upstream and downstream primers respectively introducing XmnI and SalI restriction endonuclease sites (shown in SEQ ID NO.2 and SEQ ID NO.3 respectively) :

[0035] Upstream primer: 5′-CG GAATTAATTCgcacatctctttgcagg-3' (XmnI restriction site),

[0036] Downstream primer: 5′-ACGC GTC GAC ctacgccaactggcgcag-3' (SalI restriction site),

[0037] The SD-asd gene was amplified by PCR with rTaq enzyme. Amplification...

Embodiment 2

[0057] In this embodiment, the application of the non-resistance bifunctional DNA vaccine carrier in the preparation of the non-resistance DNA vaccine comprises the following steps:

[0058] 1) Amplify the whole gene of Newcastle disease HN, use the constructed PMD18-T-HN as a template, and design a pair of restriction genes respectively introduced into HindⅢ and BamH I according to the whole gene sequence of NDV in Genbank (accession number: JF950510.1). The upstream and downstream primers of the endonuclease cleavage site (respectively shown in SEQ ID NO.4, SEQ ID NO.5):

[0059] Upstream primer: 5′-CCCAAGCTTACCATGGACCGCGCCGTTAGC-3′ (HindⅢ restriction site),

[0060] Downstream primer: 5′-CGGGATCCCTAGCCAGACCTGGCTTC-3′ (BamH I restriction site),

[0061] PCR amplification of the HN gene. Amplification system: 10×PCR Buffer 2.5μL; dNTPs (200umol / L) 1μL; rTaq enzyme 2U / μL; upstream primer / downstream primer 100pmol; Mg 2+ 1.5mmol / L 0.5μL; template (PMD-18-T-HN) 2ug; add ddH ...

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Abstract

The invention discloses a nonresistant bifunctional DNA vaccine vector, and a construction method and an application thereof, and belongs to the technical field of animal gene engineering. A nutrition selection marker is used as the screening marker of the nonresistant bifunctional DNA vaccine vector to substitute antibiotic resistance gene in order to construct a eukaryotic expression plasmid pcD-asd and fundamentally block the diffusion propagation of the antibiotic resistance gene, the safety is good, and the expression efficiency and the copy number are high. The DNA vaccine vector can complement with asd gene defect Gram-negative bacteria to construct a chromosome-plasmid balanced lethal system, and the system can highly and stably express exogenous gene in the eukaryotic cell, and can be used as a nucleic acid vaccine expression vector, for example, attenuated Salmonella typhimunum is used as a vector to construct bivalent and multivalent live bacterial vaccines.

Description

technical field [0001] The invention relates to a non-resistance bifunctional DNA vaccine carrier, and also relates to a construction method and application of the DNA vaccine carrier, belonging to the technical field of animal genetic engineering. Background technique [0002] DNA vaccine (DNA vaccine) is also called nucleic acid vaccine or genetic vaccine (genetic vaccine), and its discovery is known as the third vaccine revolution. DNA vaccines can induce cellular immunity and humoral immunity of the body. The preparation is simple, the production cost is low, the genetic background is clear, and it is convenient to construct multivalent vaccines and add various immune adjuvant molecules. At present, most of the plasmid vectors use antibiotic resistance gene as their selectable marker, and this environmental safety problem seriously restricts the development and application of DNA vaccines. With the widespread use and even abuse of antibiotics, the problem of the spread ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66C12N15/65A61K48/00
Inventor 郁川程相朝翟崇凯张春杰余祖华何雷廖成水贾艳艳李静汪洋
Owner HENAN UNIV OF SCI & TECH
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