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
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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

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

Example Embodiment

[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'

[...

Example Embodiment

[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, and the bacteria were collected and prepared to obtain the corresponding recombinant Plasmids pGap-hlyA, pGap-rtxA, pGap-vah3, pGap-empA and pGap...

Example Embodiment

[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 200μg / ml ampicillin, cultured at 200rpm and 30℃ overnight, and inoculated into 100ml 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., 10,000 g for 5 min, and the supernatant was collected as the "intracellular fraction".

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

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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 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

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Application Information

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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
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