Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Whooping cough genetic engineering blending second unit vaccine and preparing method thereof

A technology of genetic engineering vaccine and pertussis, which is applied in the field of pertussis genetic engineering fusion subunit vaccine and its preparation, can solve problems such as difficult heterologous expression, and achieve the effects of overcoming difficult expression, reducing biological toxicity, and good immune reactivity

Inactive Publication Date: 2008-06-18
ARMY MEDICAL UNIV
View PDF0 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies at home and abroad have confirmed that S1' is extremely difficult to express heterologously

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Whooping cough genetic engineering blending second unit vaccine and preparing method thereof
  • Whooping cough genetic engineering blending second unit vaccine and preparing method thereof
  • Whooping cough genetic engineering blending second unit vaccine and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Construction of embodiment 1.S1 mutant 9K / 129G

[0107] 1. Primer Design

[0108] According to the AY879289 gene sequence registered in GenBank, two pairs of primers I and II were designed by using the mutation kit MutanBEST Kit (TaKaRa) based on the principle of overlap extension to obtain a recombinant containing a double-site (9K / 129G) mutation. In addition, primer III was designed to introduce restriction sites for BamH I and Pst I, respectively. Primers were synthesized by Shanghai Sangon Bioengineering Company.

[0109] I 5'-ATAGCCGCTCTGGTAGGTGGCCCAT-3'

[0110] 5'-CTGGCACACCGGCGCATTCCG-3'

[0111] II 5'-CGCCCGCCGGAGGACGTTTTCCAG-3'

[0112] 5'-GGAGTCATACTTGTATACGGTGGCCAT-3'

[0113] III 5'-GTAGGATCCGACGATCCTCC-3'BamH I

[0114] 5'-CTGCAGCTAGAACGAATACGCGATG-3'Pst I

[0115] 2. Amplification of target gene fragments

[0116] Using the plasmid extracted from PT-PGEM transformed into DH5α bacterial liquid as a template, the mutated PT fragment was obtained by ...

Embodiment 2

[0119] Example 2. Prediction and Identification of Fs Fragments

[0120] Using DNAstar software to predict, the inventors of the present invention made a comprehensive prediction on the structure and antigenicity of the FHA' protein, and the results are shown in Figure 5: it can be found from the figure that the amino acids after the 320th position of FHA' are rich in a helix, The hydrophilicity, flexibility, and surface accessibility are all high, and the antigenicity analysis is significantly higher than other segments. Based on this, it can be judged that this segment (containing 138 amino acids) has good antigenicity, so it is determined to clone and express this segment.

[0121] 1. Primer Design

[0122] The strain used in the present invention is the pertussis CS strain, which has not yet completed the whole genome sequencing, but it is reported that the homology of the FHA gene in the pertussis strain is relatively high. Therefore, the inventors of the present invent...

Embodiment 3

[0135] Embodiment 3. Construction of recombinant engineering bacteria pET-22b(+)-FsS1' / BL21

[0136] 1. Primer design and PCR amplification

[0137] The recombinant plasmids pMD18-T-S1' and pMD18-T-Fs constructed in the above Examples 1 and 2 were used as templates, and the software PrimerPremier5.0 was used to design and analyze primers. linker( CCTCAAAGATCCGCCA ) sequence was designed at the 3' end of the Fs-encoding gene and the 5'-end of the S1'-encoding gene, and the Fs-encoding gene and the S1'-encoding gene were connected through a linker by using an overlap extension PCR method. The primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the primer sequences are as follows:

[0138] Fs upstream primer P1: 5-CATATGCTGAAGAACCTCGACCTGGGC-3 Nde1

[0139] Downstream primer P2: 5-TCTGGCGGATCTTGAGGCTGCAGTCGCACCG-3 linker

[0140] S1' upstream primer P3: 5-CAGCCTCAAGATCCGCCAGACGATCCTCCCGC-3 linker

[0141]Downstream primer P4: 5-CTCGAGTGTGTAGGGGTTGG-3 X...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a pertussis genetic-engineering fused subunit vaccine, which is fused protein produced by connecting pertussis toxin S1 subunit mutant with a peptide Fs. The invention also relates to a preparation method of bordetella pertussis double-subunit genetic engineering vaccine. The invention, which overcomes a variety of deficiencies of the traditional vaccine and the traditional preparation method, has the advantages of mass production potential, easy operation, high security, small side effect and no reverse mutation.

Description

technical field [0001] The invention relates to a vaccine and a preparation method thereof, in particular to a pertussis genetic engineering fusion subunit vaccine and a preparation method thereof. Background technique [0002] Pertussis is a highly contagious respiratory disease caused by the Gram-negative bacillus pertussis bacilli. Its epidemic cycle is a regional disease of 2 to 5 years. Droplets spread through the air, and the sick are mainly infants and young children under the age of 5. Pertussis is highly contagious, and 90% to 100% of unimmunized members who have close contact with pertussis patients will develop pertussis; More than 50% of people may develop subclinical infection. Although the application of vaccines has played a significant role in controlling the prevalence of whooping cough, the prevalence of whooping cough has shown a clear upward trend in recent years, and the so-called "resurgence" phenomenon has appeared. Moreover, the proportion of adult...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): A61K39/10A61P31/04A61P11/00C07K19/00
Inventor 邹全明章金勇张晓丽张卫军毛旭虎郭刚葛迪石云郭鹰罗平
Owner ARMY MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com