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PcrDNA vaccine, and its preparing method and use

A vaccine and universal primer technology, applied in the biological field, can solve the problems of reducing the immune effect, harming the body, reducing the immune efficiency of DNA vaccines, etc., and achieve the effects of good biological safety, safe use and high biological safety.

Inactive Publication Date: 2006-10-25
广东省农业科学院兽医研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the excessive number of genes or DNA sequences carried, there will be many disadvantages: one is the expression of other antigens, which may cause harm or uncertainty to the body; The third and most important thing is that the existence of too many other genes or non-essential DNA sequences constitutes a great biosafety problem, which increases the probability of exogenous DNA integrated into the host genome. Increases the chance of inducing tumors and other problems, greatly increasing the uncertainty of the use of DNA vaccines; in addition, in the case of the presence of too many other genes or non-essential DNA sequences, the copy of DNA within a unit mass This will reduce the immune efficiency of the DNA vaccine and increase the production cost of the vaccine. At the same time, other foreign genes carried on the carrier may affect the processing and modification of the target antigen protein to form a natural conformation

Method used

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  • PcrDNA vaccine, and its preparing method and use
  • PcrDNA vaccine, and its preparing method and use
  • PcrDNA vaccine, and its preparing method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of pcrDNA vaccine universal vector pcrDNAV (such as figure 1 Shown)

[0037] Based on the pMD 18-T vector, the human cytomegalovirus promoter (CMV) and the PolyA tail of simian virus 40 (SV40) were added at the multiple cloning site to construct a universal vector pcrDNAV ( Such as image 3 Shown).

[0038] 1. The CMV promoter and SV40 PolyA were cloned into the pMD 18-T vector to construct the PCRDNA vaccine universal vector pcrDNAV.

[0039] (1) Design of primers for amplification of CMV and SV40 PolyA fragments

[0040] According to the pAdTrack-CMV gene sequence in GenBank, a pair of primers were designed for each of the CMV promoter and SV40 PolyA gene. The primers were synthesized by Shanghai Boya Biotechnology Co., Ltd.

[0041] CMV upstream primer P1: 5’-CGGAATTCATTCTTTCCCACCCTTA-3’ (contains EcoR I restriction site)

[0042] CMV downstream primer P2: 5’-TTGGTACCAGATCTCTAGCGGATC-3’ (contains KpnI, BgI II restriction sites)

[0043] SV40 PolyA...

Embodiment 2

[0050] Example 2 Design of universal primers for amplifying PCRDNA vaccine from PCRDNAV vector

[0051] Design the universal primers for the amplified vaccine at the upstream of the CMV promoter and downstream of the SV40 PolyA of the constructed pcrDNAV vaccine universal vector:

[0052] Upstream primer: 5’-ACAGCTATGACCATGATTACG-3’

[0053] Downstream primer: 5’-AACGACGGCCAGTGCCAAG-3’

Embodiment 3

[0054] Example 3 General process for the construction of pcrDNA vaccine

[0055] Design primers to amplify the desired target gene by PCR or use enzyme digestion method, according to the conventional gene cloning method (enzyme digestion method or PCR amplification method) cloned into the pcrDNA vector (high-efficiency PCR where the PCR product is directly connected to the pcrDNA vector The product ligation site or ligation of the digested product into the multiple cloning site of the PCRDNA vector), the recombinant plasmid pcrDNA-Target is obtained.

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Abstract

The present invention discloses a new type DNA vaccine-pcr DNA vaccine, its preparation method and application. The pcr DNA vaccine directly uses the PCR product in which the complete gene expression element is carried as DNA vaccine. Said DNA vaccine does not carry any indifferent gene or nucleic acid sequence except for target gene, so that it has better biological safety. Said invention adopts PCR technique to prepare DNA vaccine. The pcr DNA vaccine can be used for immunizing human body and animal, and can be used for preventing and curing human and diseases, such as various infectious diseases, parasitic diseases, tumor, nutritional metabolic disease and gene defect disease.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a PCRDNA vaccine (a PCR DNA vaccine, that is, a PCR product containing a complete gene expression element cassette is used as a vaccine), and a preparation method and application thereof. Background technique [0002] In 1990, Wolff et al. (Wolff et al, 1990) accidentally discovered a DNA vaccine when conducting gene therapy experiments on mice. In 1992, four laboratories in the United States developed and researched DNA vaccines at the same time and made a report at the conference on new advances in vaccinology in September of the same year. Since then, DNA vaccines have attracted great attention and are considered to be the third vaccine revolution after attenuated, inactivated vaccines and subunit vaccines. DNA vaccine is to recombine a foreign gene (DNA) encoding a certain antigen protein with a eukaryotic expression vector plasmid containing necessa...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P35/00A61P43/00
Inventor 宋长绪张洪利王建龙黄忠蒋智勇
Owner 广东省农业科学院兽医研究所
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