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Application of FimHEd in preparation of fish targeted oral vaccine based on bacillus subtilis spore carrier

A technology of Bacillus subtilis and spores, applied in the field of genetic engineering, can solve problems such as difficult immune effects, and achieve the effect of improving the immune protection effect

Active Publication Date: 2020-10-09
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because spores are difficult to break through the mucosal epithelial barrier, oral vaccines based on spore vectors are still difficult to produce ideal immune effects

Method used

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  • Application of FimHEd in preparation of fish targeted oral vaccine based on bacillus subtilis spore carrier
  • Application of FimHEd in preparation of fish targeted oral vaccine based on bacillus subtilis spore carrier
  • Application of FimHEd in preparation of fish targeted oral vaccine based on bacillus subtilis spore carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] FimH Ed202 Oral recombinant spore vaccine against CyHV2 in heterogeneous gibel crucian carp with independent expression of ORF25

[0081] 1. Build FimH Ed202 Integrative plasmid expressing independently of ORF25

[0082] With orf25-1 and orf25-2 as primers (see Table 1 of the present invention), pJS1608a (see Table 2 of the present invention) as template, PCR amplifies the orf25 gene fragment, digests with NdeI and BamHI, and the recovered products are respectively cloned in the integrative Among the platform vectors pJS1700 and pJS1834, the integrative recombinant plasmids pJS2120 and pJS2121 were constructed (see Table 2 of the present invention). Recombinant plasmids pJS2120 and pJS2121 contain gerKB::erm-cotB-orf25 and gerAA::npt-cotC-orf25 recombinant fragments, respectively.

[0083] Digest plasmid pJS2119 with NdeI and BamHI, recover fimH Ed202 The gene fragment was cloned into the integrative platform vector pJS1834 to construct the integrative recombinant p...

Embodiment 2

[0099] FimH Ed202 Oral recombinant spore vaccine against CyHV2 expressed by heterogeneous gibel crucian carp fused with ORF25

[0100] 1. Build FimH Ed202 Integrative plasmid expressed in fusion with ORF25

[0101] Take fimH Ed202 -1 and fimH Ed202 -2 is a primer (see Table 1), pJS2119 (see Table 2) is a template, and PCR amplifies fimH Ed202 The gene fragments were digested with BamHI and KpnI to amplify the products, and after recovery, they were respectively cloned into integrative plasmids pJS2120 and pJS2121 to construct integrative recombinant plasmids pJS2123 and pJS2124 (see Table 2 of the present invention). Recombinant plasmids pJS2123 and pJS2124 respectively contain gerKB::erm-cotB-orf25-fimH Ed202 and gerAA::npt-cotC-orf25-fimH Ed202 Recombinant fragments (see Table 2).

[0102] 2. Build FimH Ed202 A recombinant strain of Bacillus subtilis expressed in fusion with ORF25

[0103] According to the method described in 2 of Example 1 of the present invention,...

Embodiment 3

[0115] FimH Ed202 Grass carp anti-GCRV oral recombinant spore vaccine expressed independently of Vp7 and Vp7

[0116] 1. Build FimH Ed202 Integrative plasmid expressing independently of Vp7 and Vp7

[0117] Using vp7-1 and vp7-2 as primers (see Table 1), and pJS1608a (see Table 2) as a template, PCR amplifies the vp7 gene fragment, digests with NdeI and BamHI, and clones it into the integrative platform vector pJS1700 and pJS1834, respectively. The integrative recombinant plasmids pJS2125 and pJS2126 were constructed (see Table 2). Recombinant plasmids pJS2125 and pJS2126 contain gerKB::erm-cotB-vp7 and gerAA::npt-cotC-vp7 recombinant fragments respectively, and express recombinant proteins CotB-Vp7 and CotC-Vp7 in spores under the control of cotB and cotC promoters.

[0118] 2. Build FimH Ed202 Recombinant strains of Bacillus subtilis independently expressing Vp7 and

[0119] The integrative recombinant plasmids pJS2125 and pJS2126 were sequentially transformed into B. s...

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Abstract

The invention relates to an application of an I-type ciliary protein FimH (FimHEd) of Edwardsiella, in particular to an application of the FimHEd in preparation of a fish targeted oral vaccine based on a bacillus subtilis spore transfer carrier. The targeted oral vaccine is prepared by taking FimHEd as a ligand for targeting intestinal mucosa epithelial cells of the fish oral vaccine, taking bacillus subtilis spores as a targeting ligand and a transfer carrier of specific antigen protein and constructing recombinant spores of which the surfaces display the targeting ligand and the specific antigen by utilizing a new-generation spore surface display technology. The targeted oral vaccine is coated with granulated feed, and specific antigens are extracted by transferring the targeting ligandFimHEd on the surface of a carrier to mediate intestinal mucosa epithelial cells. The fish oral vaccine has the advantages that the targeting effect of the FimHEd is utilized to promote the specific antigen transferred by the spore carrier to break through the intestinal mucosa epithelial barrier, and the immune protection effect of the fish oral vaccine is improved.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to the utilization of type I cilium protein FimH (FimH) of Edwardsiella (Edwardsiella) Ed ) as the targeting ligand of the specific antigen, using Bacillus subtilis spores as the targeting ligand and the delivery carrier of the specific antigen in the digestive tract of fish, and preparing a targeted oral vaccine for fish through a new generation of spore surface display technology. Background technique [0002] Oral vaccines deliver specific antigens to intestinal mucosal immune organs through oral routes, and induce mucosal immune responses including cellular immunity and humoral immunity[1,2]. The oral vaccine is easy to operate and is suitable for large-scale vaccination of fish. However, proteases, mucus on the mucosal surface, and dense mucosal epithelium in the digestive tract of fish constitute a physiological barrier of the digestive tract[3], which prevents oral vaccines f...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/75C12N1/21A61K39/02A61P31/04C12R1/125
CPCA61K39/0208A61K2039/523A61K2039/542A61K2039/552A61P31/04C07K14/195C12N15/75
Inventor 宁德刚师忠港张淼
Owner INST OF AQUATIC LIFE ACAD SINICA
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