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

A live vaccine and multi-titer technology, applied in the field of its preparation and multi-titer live vaccine, can solve the problems such as no multi-titer vibrosis live bacteria vaccine, and achieve obvious multi-titer immune protection and good application. Foreground effect

Inactive Publication Date: 2011-09-28
EAST CHINA UNIV OF SCI & TECH
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  • 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

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  • 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 sequence of the above primers and restriction sites 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'-CGTGCTTTGTCCTGCTGAGTGCTTAGAGATGTGAGCGAT-3'

[...

Embodiment 2

[0079] Example 2 , Construction of Recombinant Strains of Vibrio anguillarum

[0080] Inoculate Vibrio anguillarum MVAV6203 into 50ml high-salt LB culture medium, shake culture overnight at 30℃, and then transfer it to 100ml fresh high-salt LB culture medium at 1:100 (v / v) at 30℃ Incubate with shaking at 200 rpm to OD 600 When the value is 0.6, collect the bacteria by centrifugation, place the bacteria in an ice bath for 20-30 minutes, wash the bacteria three times with 272mM sucrose buffer, and then suspend the bacteria with sucrose buffer to make the final concentration of the bacteria 1 ×10 9 cfu / ml, to obtain the electrotransformation 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 amplified and cultured respectively, and the bacteria were collected and prepared to obtain the corresponding recombinant Plasmids pGap-hlyA, pGap-rtxA, pGap-vah3, pGap-...

Embodiment 3

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

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

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

[0088] At the same time, the bacterial pellet was washed three times with PBS (pH 7.0), and then resuspended in 1 ml PBS. The resuspension was sonicated in an ice bath for 5 min until the cells were completely broken, and then the obtained cell lysate was centrifuged at 4° C., 10000 g for 5 min, and the supernatant was collected as the "intracellular fraction".

[0089] The obtained "supernatant fraction" and "intracel...

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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 of signal 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 the following 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 aquaculture animal disease prevention and control technology, and specifically relates to a multi-titer live vaccine, its preparation method and application. Background technique [0002] At present, large-scale, intensive, and high-density aquaculture models have gradually become the mainstream development of my country's marine fish aquaculture industry. However, with the continuous and steady development of the marine aquaculture industry, various disease problems have become increasingly prominent, which have a serious impact on the production and growth of aquaculture. For example, in my country, the development of marine cage aquaculture and industrial fish farming in recent years Sudden and fulminant diseases frequently occur, and the average death loss rate of mariculture is over 30%. Disease problems have become an important factor restricting the healthy development of mariculture. [0003] Vaccines have the characteristics ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/66A61K48/00A61K39/106A61K39/02A61P31/04C12N15/74C12R1/01C12R1/63
Inventor 刘琴张元兴周凌云王启要
Owner EAST CHINA UNIV OF SCI & TECH
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