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Fused DNA (Deoxyribonucleic Acid) and vaccine prepared from fused DNA

A DNA vaccine and vaccine technology, applied in the field of biomedicine, can solve the problems of inability to prevent latent infection and treatment of tuberculosis, complex preparation technology of subunit protein vaccines, and inability to be used in immunocompromised people, etc., to shorten the treatment period and reduce the risk of vaccination , easy to produce effect

Pending Publication Date: 2018-07-13
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problems and defects in the prior art, the present invention provides a vaccine fused with DNAKtCMFO and the vaccine prepared therefor. Prevent and treat tuberculosis, and screen out more effective vaccines, thereby solving the clinical problems that BCG cannot be used in immunocompromised populations, preventing latent infection and treating tuberculosis, and solving the complex preparation technology of subunit protein vaccines that limit its large-scale application In clinical practice, as well as the weak immunogenicity and unsatisfactory anti-infection effect of the existing tuberculosis DNA disease vaccine

Method used

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  • Fused DNA (Deoxyribonucleic Acid) and vaccine prepared from fused DNA
  • Fused DNA (Deoxyribonucleic Acid) and vaccine prepared from fused DNA
  • Fused DNA (Deoxyribonucleic Acid) and vaccine prepared from fused DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 Design and synthesis of fusion gene CMFO and single antigen gene sequence and primer

[0033] The coding sequences of Rv0577, Rv2875, Rv3044 and Rv2073c of M.tb H37Rv were searched through Genbank. Use Signalp software to predict and search the UniProtKB database to see if there is a signal peptide in the search sequence, and then use DNAMAN software to analyze the restriction sites. A Kozak sequence (GCCACC) was added before the target gene to enhance the translation efficiency of the target gene. In order to secrete the final expression product outside the cell, a tPA signal peptide sequence is added before the spliced ​​gene sequence. The splicing of the four target genes after the tPA signal peptide should follow the following principles: 1) If there is a signal peptide, the signal peptide should be removed, including the original start codon, and the stop codon should be removed at the same time (the last gene sequence after splicing Then add TAA); 2...

Embodiment 2

[0034] Embodiment 2 Construction of recombinant plasmid pVAX1-Koza-tPA-CMFO (pKtCMFO)

[0035] 1. Acquisition of the target gene:

[0036] (1) Primer design: Design primers based on the full sequence of the fusion gene and the multiple cloning site on the eukaryotic expression vector pVAX1. Store at ℃ for later use. See SEQ ID No.2 and SEQ ID No.3 for primer sequences.

[0037] (2) PCR reaction system:

[0038]

[0039]

[0040] (3) PCR reaction conditions:

[0041] 98℃30s; 98℃10s, 68℃20s and 72℃90s, 30cycles; 72℃10min; 4℃forever

[0042] 2. Construction of recombinant plasmids:

[0043] The PCR product recovered by DNA gel electrophoresis and the vector pVAX1 were subjected to double enzyme digestion respectively. The enzyme digestion system is:

[0044]

[0045] The KtCMFO fragment of the target gene recovered by enzyme digestion and the vector pVAX1 were ligated according to the following conditions:

[0046]

[0047] 3. Transformation and identification ...

Embodiment 3

[0049] Example 3 Recombinant plasmid pVAX1-CMFO (pCMFO), pVAX1-Koza-CMFO (pKCMFO), pVAX1-tPA-CMFO (ptCMFO), pVAX1-Koza-tPA-0577 (pKt0577), pVAX1-Koza-tPA-2875 (pKt2875 ), construction of pVAX1-Koza-tPA-3044 (pKt3044) and pVAX1-Koza-tPA-2073c (pKt2073c)

[0050] 1. Acquisition of the target gene:

[0051] (1) Primer design: Design primers based on the full sequence of the fusion gene and the multiple cloning site on the eukaryotic expression vector pVAX1. Store at ℃ for later use. See SEQ ID No.4-10 for the primer sequence.

[0052] (2) PCR reaction system:

[0053]

[0054] The template for amplifying the CMFO fragment is pDC316-KtCMFO, and the sequences of the upstream and downstream primers are P1: SEQ ID No.4; P2: SEQ ID No.3;

[0055] The template for amplifying the KCMFO fragment is pDC316-KtCMFO, and the upstream and downstream primer sequences are P1: SEQ ID No.5; P2: SEQ ID No.3;

[0056] The template for amplifying the tCMFO fragment is pDC316-KtCMFO, and the ...

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Abstract

The invention relates to fused DNA (Deoxyribonucleic Acid) and a vaccine prepared from the fused DNA and belongs to the field of biomedicines. According to the fused DNA provided by the invention, encoding genes are of a Kozak sequence, a tPA sequence, Rv0577, Rv2875, Rv3044 and Rv2073c; the genes are connected in series according to a sequence of Kozak-tPA-Rv0577-Rv2875-Rv3044-Rv2073c. The fusedDNA provided by the invention can be used for preparing a tuberculosis DNA vaccine and a recombinant adenovirus vector vaccine, wherein the concentration of the fused DNA in the DNA vaccine is 0.2 to0.8mg / ml. The DNA vaccine provided by the invention can be used for carrying out adjuvant treatment of tuberculosis and shortening a chemotherapy drug treatment period; a rAdKtCMFO vaccine does not need to utilize an adjuvant and relatively strong immunological reaction can be generated through inducing an organism by only one time of inoculation; the vaccine can be used for preventing latent infection and adult tuberculosis and has high safety.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a vaccine fused with DNAKtCMFO and the vaccine prepared therefrom. Background technique [0002] BCG (Mycobacterium bovis BCG) is currently the only vaccine used clinically to prevent tuberculosis (TB), which can effectively prevent tuberculosis in infants and young children. Although BCG immunization has been included in the Expanded Immunization Program since 1974, tuberculosis remains the most deadly infectious disease worldwide, and adult tuberculosis in particular is the leading cause of tuberculosis prevalence and mortality. The occurrence of adult tuberculosis is mainly caused by endogenous reactivation of latent infection; the secondary route is through exogenous infection. Moreover, the co-infection rate of Mycobacterium tuberculosis and human immunodeficiency virus (HIV) continues to increase, and the infection rate of multidrug-resistant Mycobacterium tuberculosis and extensiv...

Claims

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Application Information

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IPC IPC(8): C12N15/62A61K48/00A61K39/04A61P31/06
CPCA61K39/04A61K48/005A61K2039/5256A61K2039/53A61K2039/55555A61K2039/57C07K14/35C07K2319/00
Inventor 范雄林
Owner HUAZHONG UNIV OF SCI & TECH
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