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

Multi-titer live vaccine as well as preparation method and application thereof

A live vaccine, multi-valence technology, applied in the field of preparation of multi-valence live vaccines, can solve problems such as multi-valence vibriosis live bacteria vaccines that have not yet been seen, and achieve obvious multi-valence immune protection and good application foreground effect

Inactive Publication Date: 2010-03-03
EAST CHINA UNIV OF SCI & TECH
View PDF0 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no commercial multi-potency vibriosis live bacteria vaccine that mainly targets Vibrio anguillarum and Aeromonas hydrophila

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
  • Multi-titer live vaccine as well as preparation method and application thereof
  • Multi-titer live vaccine as well as preparation method and application thereof
  • Multi-titer live vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 , Recombinant plasmid construction

[0039] 1.1. Primer design

[0040] Design primers hlyASP-F, hlyASP-R, rtxASP-F, rtxASP-R, vah3SP-F, vah3SP-R, empASP-F, empASP-R, gapA-F1 / 2, gapA-R1, gapA-R2, gapA- F3, gapA-F4 and gapA-R3 / 4, the sequences and restriction sites of the above primers are as follows:

[0041] hlyASP-F: 5′-CACTCAGCAGGACAAAGCACGAAAGATGCAT-3′;

[0042] hlyASP-R: 5′-CGC GGATCC TTATGCTGATGTGGTC-3′;

[0043] BamHI site

[0044] rtxASP-F: 5'-GATTACTTTAATGGTAACCGCGCTCAAGTG-3';

[0045] rtxASP-R:5′-TGC ATGCAT TTACACCATCAATCCTTTTTTGTG-3′;

[0046] NsiI site

[0047] vah3SP-F: 5′-CCG GAATTC GATGACTTCTTCTAAATTTTCG-3';

[0048] EcoRI site

[0049] vah3SP-R: 5′-AGACCACTTTAATTGGGTGTTTATCTCACTAGGG-3′;

[0050] empASP-F: 5′-CCG GAATTC GATGAAAAAAGTACAACGTC-3′;

[0051] EcoR I site

[0052] empASP-R: 5'-AGCTTGTTCTAGTAGACTTGGATCA-3'.

[0053] gapA-F1 / 2: 5′-TAGGA GAATTC GATGACTATCAAAGTAGG-3′;

[0054] EcoR I site

[0055] gapA-R1: 5′-CGTG...

Embodiment 2

[0079] Example 2 , Vibrio anguillaris recombinant strain construction

[0080] Vibrio anguillarum MVAV6203 was inoculated in 50ml of high-salt LB medium, shaken at 30°C overnight, then transferred to 100ml of fresh high-salt LB medium at 1:100 (v / v), at 30°C Shake culture at 200rpm to OD 600 When the value is 0.6, collect the cells by centrifugation, place the cells in an ice bath for 20-30 minutes, wash the cells with 272mM sucrose buffer three times, and then suspend the cells with sucrose buffer to make the final concentration of the cells 1 ×10 9 cfu / ml to obtain electroporation competent cells of Vibrio anguillarum MVAV6203.

[0081]The recombinant strains Top10(pGap-hlyA), Top10(pGap-rtxA), Top10(pGap-vah3), Top10(pGap-empA) and Top10(pGap) were respectively amplified and cultivated, and the bacteria were collected and prepared to obtain the corresponding recombinant strains Plasmids pGap-hlyA, pGap-rtxA, pGap-vah3, pGap-empA and pGap.

[0082] Use the electrotrans...

Embodiment 3

[0085] Example 3 , Multi-potency attenuated live vaccine screening

[0086] The recombinant strains obtained in the steps of Example 2 were respectively inoculated in LB high-salt liquid medium containing 200 μg / ml ampicillin, 200 rpm, and cultivated overnight at 30° C., and inoculated in 100 ml of the next day at 1: 100 (v / v). LB high-salt (Amp) medium was cultured at 30° C. and 200 rpm for 9 hours, and the culture solution was harvested.

[0087] Centrifuge 1 ml of the harvested culture solution at 10,000 g for 10 min, and collect the supernatant as "supernatant fraction".

[0088] At the same time, the bacterial cell pellet was washed three times with PBS (pH 7.0), and then resuspended with 1 ml of PBS. The resuspension was sonicated in an ice bath for 5 minutes until the cells were completely broken, and then the obtained cell lysate was centrifuged at 10,000 g for 5 minutes at 4°C, and the supernatant was collected as "intracellular fraction".

[0089] The obtained "s...

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 provides a recombinant plasmid, a multi-titer live vaccine as well as preparation methods and the application thereof. The provided recombinant plasmid contains a fusion gene sequence ofsignal peptides of vibrio anguillarum metalloprotease and aeromonas hydrophila 3-glycerophosphate dehydrogenase; the preparation method of the recombinant plasmid comprises the following steps: (A) establishing signal peptide-3-glycerophosphate dehydrogenase fusion gene; (B) enzyme-cutting the fusion gene and a carrier; and (C) connecting the enzyme-cutting fusion gene and the enzyme-cutting carrier. The multi-titer live vaccine is prepared by converting the recombinant plasmid into vibrio anguillarum attenuated strains; and the preparation method of the multi-titer live vaccine comprises thefollowing steps: (A) establishing the recombinant plasmid containing signal peptides of vibrio anguillarum metalloprotease and aeromonas hydrophila 3-glycerophosphate dehydrogenase; and (B) converting the recombinant plasmid obtained in the step (A) into vibrio anguillarum attenuated strains. The multi-titer live vaccine is applied to prevent and treat fish diseases caused by vibrio anguillarum and aeromonas hydrophila. The attenuated vaccine provided by the invention has remarkable multi-titer immune protective efficiency, can be used as the live vaccine of vibrio anguillarum and aeromonas hydrophila and has favorable application prospect.

Description

technical field [0001] The invention belongs to the disease prevention technology of aquaculture animals, and in particular relates to a multi-valence live vaccine, its preparation method and application. Background technique [0002] At present, large-scale, intensive, and high-density farming models have gradually become the mainstream of the development of my country's marine fish farming industry. However, with the continuous and steady development of the marine aquaculture industry, various disease problems have become increasingly prominent, which has a serious impact on the production and growth of aquaculture. Sudden and explosive diseases occur frequently, and the average mortality loss rate of mariculture is more than 30%. The disease problem has become an important factor restricting the healthy development of mariculture industry. [0003] Vaccines have the characteristics of strong pertinence, long anti-disease cycle, lifelong immunity, remarkable effect and ac...

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): C12N15/63C12N15/66A61K48/00A61K39/106A61K39/02A61P31/04C12N15/74C12R1/63C12R1/01
Inventor 刘琴张元兴周凌云王启要
Owner EAST CHINA UNIV OF SCI & TECH
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