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

A genetically engineered vaccine, pertussis technology, applied in the field of pertussis genetically engineered fusion subunit vaccine and preparation thereof

Inactive Publication Date: 2012-11-28
ARMY MEDICAL UNIV
View PDF3 Cites 0 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 5

[0176] 2. Detection of Specific Immune Response

[0177] (1) Preparation of ELISA antigen-coated plates:

[0178] The PT antigen was diluted to 5 μg / ml with coating solution, and 100 μl / well was coated on the ELISA plate, overnight at 4°C. Wash with washing solution 4 times. Add 300 μl of 1% BSA to each well, block at 37°C for 2 hours, wash with washing solution 4 times, and store at 4°C for use.

[0179] (2) Collection of mouse serum samples:

[0180] Serum collection: Before immunization and 7 days after 2 and 3 times of immunization, about 100 μl of blood was collected from each mouse by docking the tail, collected in a 1.5ml sterile EP tube, placed at room temperature for 1 hour, 5000g×10min, collected serum, and packed, Store at -70°C

[0181] (3) Detection of serum specific antibody IgG:

[0182] Detection of serum antigen-specific IgG: starting from 1:5000, dilute the serum to be tested with antibody diluent, dilute the negative serum 1:100, 100μl / well, incubate at...

Embodiment 6

[0190] Downstream primer P2: 5'-TC AGGATCCTGCGGCGCGTA CTGCAGTCGCACCG-3’ linker

[0191] S1' upstream primer P3: 5'-CAG TACGCGCCGCAGGATCCT GACGATCCTCCCGC-3’ linker

[0192] Downstream primer P4: 5'- CTCGAG TGTGTAGGGGTTGG-3' Xho1

[0193] 2. PCR amplification of the target gene

[0194] Using the recombinant plasmids pMD18-T-S1' and pMD18-T-FsS1' constructed in Examples 1 and 3 as templates, S1' was amplified with the upstream and downstream primers of S1' and FsS1' respectively B and FsS1' B Gene fragment. The PCR reaction conditions were: pre-denaturation at 94°C for 5 minutes, 30 cycles of 94°C for 50s→60°C for 40s→72°C for 1min, and finally extension at 72°C for 10min.

[0195] 3. Cloning identification and expression

[0196] (1) S1’ to be obtained B and FsS1' B Gene fragments were cloned into pMD18-T vector to construct recombinant plasmid pMD18-T-S1' B and pMD18-T-FsS1' B , transforming E. coli DH5α, ampicillin resistance screened positive recombinants...

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 adul...

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
Patent Type & AuthorityPatents(China)
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
OwnerARMY MEDICAL UNIV