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Combined vaccine for anthrax and black death

A combined vaccine and plague technology, applied in the field of medical biology, can solve the problems of high residual virulence, large population reaction to vaccination, and failure to vaccinate

Inactive Publication Date: 2007-05-23
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently used live attenuated plague vaccine strain EV76 (which is also the current plague vaccine for humans in my country) has good immunogenicity, but due to its high residual virulence, the population has a large vaccination response, so it cannot be widely used for vaccination.

Method used

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  • Combined vaccine for anthrax and black death
  • Combined vaccine for anthrax and black death
  • Combined vaccine for anthrax and black death

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1 Preparation of Bacillus anthracis PA Antigen

[0014] The cloning of PA gene refers to the Bacillus anthracis protective antigen (PA) gene sequence (sequence number: ) and literature published by GenBank, design primers, PA 5' end primer: 5'-AGGAGAACCGGTTATTAAATGAATC-3, PA 3' end primer: 5 '-CGCrTATCCTATCTCATAGCCTTTTTTAG-3', for PCR amplification, pick a small amount of bacteria from the agar plate where the A16R strain was cultivated, put them in 100uL pure water, boil for 10 minutes, and after centrifugation, take 5uL supernatant as a template for PCR reaction. The PCR product was cloned into the pMD-T vector for sequence determination. The DNA sequence determination confirmed that the synthesized gene was correct. The gene had the nucleotide sequence of sequence 1 in the sequence table, and the nucleotide sequence from the 1st to 2292nd position of the 5' end was the coding Sequence, which encodes the amino acid sequence of Sequence 2 in the Sequence Listin...

Embodiment 2

[0026] The preparation of embodiment 2 plague candidate vaccines F1 and V

[0027] Plague standard strains and Bacillus anthracis strains were preserved by our laboratory, and DNA restriction enzymes, T4 DNA ligase, Pyrobest DNA polymerase and pMD-T vectors were purchased from TaKaRa Company.

[0028] E.coli plasmid was extracted by using Omega kit; for the operation method of plasmid construction, refer to Molecular Cloning.

[0029] Referring to the Yersinia pestis capsular antigen (Fraction 1, F1) gene sequence published by GenBank (sequence number: AF542378.1), design primers, F1up: 5'-GCC CATATG AAAAAAATCAGTTCCG-3' and F1 down: 5'-GGG GAATTC TTATTGG TTAGATACGGTTACG'-3 (CATATG and GAATTC are restriction sites of Nde I and EcoR I, respectively, for cloning the PCR fragment into the expression vector pET-32a(+)); perform PCR amplification, and clone the PCR product into pMD- The T vector was sequenced, and the DNA sequence was determined to confirm that the cloned gene wa...

Embodiment 3

[0034] Example 3 Preparation and Immunological Evaluation of Anthrax and Plague Combined Vaccine

[0035] Preparation of Combined Anthrax and Plague Vaccine

[0036] Immunization program and dose Design V, F1 and PA immunization groups alone, and F+V, F+V+PA immunization groups, a total of 5 groups. Balb / c mice were immunized with the purified antigenic protein, and the titer of the antibody was measured after 2 weeks. At the same time, the immunization was boosted, and the titer of the antibody was measured after 5 weeks. After the third immunization, the monoclonal antibody was prepared by fusion with SP2 / O. The groups and doses of immunization are given below.

[0037] 1. Immune with F1 antigen, the original protein concentration is about 4-5 mg / mL, 13 μL / rat, 3 rats in total. Inhale 40ulAg+460ul normal saline+0.5ml Freund's complete adjuvant, and immunize 3 Balb / c.

[0038] 2. Immunization with V antigen, the original Ag concentration was 4-5 mg / ml, 3 μL / rat, a total of...

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Abstract

The invention relates to a coupled vaccine for protecting human or animal from bacillus anthracis and Yersinia, wherein it comprises bacillus anthracis PA antigen, Yersinia V antigen, and Yersinia F1, or their protective epitope. The antigen is recombined protein separated and / or purified. The DNA of integral or partial PA antigen, integral or partial F1 antigen, and integral or partial V antigen can be directly used as nucleic acid vaccine.

Description

technical field [0001] The invention relates to a combined vaccine for simultaneously resisting Bacillus anthracis and Yersinia pestis infection, and belongs to the field of medical biotechnology. Background technique [0002] Anthrax is an infectious disease that seriously threatens human health. It has the characteristics of rapid transmission, high fatality rate, and easy to cause public panic and social chaos. Its pathogen is Bacillus anthracis. At present, human anthrax is still sporadically or locally endemic in the old epidemic areas on all continents of the world, mainly in developing countries, especially in West Africa. According to estimates by the World Health Organization, there are approximately 20,000 to 100,000 cases worldwide each year. [0003] Plague is a severe infectious disease caused by Yersinia pestis. There have been three worldwide pandemics of plague recorded in history, which caused great disasters to mankind. Until now, small-scale epidemics h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/116A61P31/04C12N15/31C07K14/195C07K14/24C12N1/21C12N1/19
CPCY02A50/30
Inventor 陈薇李建民徐俊杰侯利华付玲董大勇李冠霖
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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