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Cyprinid herpesvirus II DNA vaccine based on baculovirus vector as well as building method and application thereof

The technology of a baculovirus vector and carp herpes virus is applied in the field of herpes virus type II DNA vaccine, which can solve the problems of poor immune protection effect, fish injury and stress response, poor immune effect, etc.

Active Publication Date: 2018-11-02
苏州培恩特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Injection immunization is the most common immunization method. This method has good immunization effect, but the workload of immunization is large, and it is easy to cause fish injury and stress response, and the requirement for vaccine purity is also high; oral immunization has little damage to fish and the method is simple. , easy to operate, but the immune protection effect is poor because the vaccine is easy to degrade in the digestive tract; soaking immunization is mainly suitable for immunizing seedlings, and the amount of vaccine required is large, and the immune effect is relatively poor

Method used

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  • Cyprinid herpesvirus II DNA vaccine based on baculovirus vector as well as building method and application thereof
  • Cyprinid herpesvirus II DNA vaccine based on baculovirus vector as well as building method and application thereof
  • Cyprinid herpesvirus II DNA vaccine based on baculovirus vector as well as building method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction method of cyprin herpesvirus type II DNA vaccine based on baculovirus vector and its injection immune effect

[0049] (1) Synthesize the promoter sequence (0.56 kb) as shown in SEQ ID NO:1, clone it into a T-vector, perform sequencing verification, and name the verified correct plasmid as pMD-β-actin;

[0050] (2) use Sma I and xho I digest pMD-β-actin plasmid, recover the promoter fragment, and clone it into pFSATBac digested with the same restriction enzyme TM In Dual, get pFSATBac TM Dual-FA carrier;

[0051] (3) Synthesize ORF72-ORF66-ORF81-ORF82 fusion DNA sequence as shown in SEQ ID NO:2, clone the synthesized sequence into T vector, perform sequencing verification, and name the verified correct plasmid as pMD-D4ORF;

[0052] (4) use xho I and Kpn I digest the pMD-D4ORF plasmid, recover the ORF72-ORF66-ORF81-ORF82 fusion DNA fragment, and clone it into pFSATBac TM In the Dual-FA vector, pFSATBac was obtained TM Dual-FA-D4ORF vect...

Embodiment 2

[0058] Example 2 Construction method and vaccine characteristics of cyprin herpesvirus type II DNA vaccine based on baculovirus vector

[0059] (1) Same as embodiment one step (1);

[0060] (2) Same as embodiment one step (2);

[0061] (3) Same as embodiment one step (3);

[0062] (4) Same as step (4) of the first embodiment;

[0063] (5) Same as Step (5) of Embodiment 1;

[0064] (6) Same as Step (6) of Embodiment 1;

[0065] (7) Use insect needles to pick up the P2-generation recombinant virus, puncture and inoculate the 5th instar silkworm from the intersegmental membrane, and raise it normally at about 25°C. After 5 days of silkworm disease, collect the hemolymph of the silkworm, and freeze and thaw alternately for more than 3 times. Centrifuge at 1000 rpm for 10 minutes, remove cell debris, and then centrifuge at 20,000 rpm at 4 degrees for 60 minutes. Discard the supernatant, dissolve the precipitate with 0.01mol / L phosphate buffer, centrifuge at 1000 rpm for 10 min...

Embodiment 3

[0067] Example 3 Construction method and oral immunization of cyprin herpesvirus type II DNA vaccine based on baculovirus vector

[0068] (1) Same as embodiment one step (1);

[0069] (2) Same as embodiment one step (2);

[0070] (3) Same as embodiment one step (3);

[0071] (4) Same as step (4) of the first embodiment;

[0072] (5) Same as Step (5) of Embodiment 1;

[0073] (6) Same as Step (6) of Embodiment 1;

[0074] (7) Use insect needles to pick up the P2 generation recombinant virus, puncture and inoculate the initial pupation from the intersegmental membrane, protect at 25°C for 5 days, collect the recombinant virus to infect the silkworm chrysalis, add 0.01mol / L at a ratio of 1:10 (W / V) Phosphate buffered saline, homogenate. After the homogenate was filtered through 8 layers of gauze, the filtrate was freeze-dried to prepare silkworm chrysalis freeze-dried powder, and the moisture content of the freeze-dried powder was less than 7%. Store the lyophilized powder ...

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Abstract

The invention relates to a cyprinid herpesvirus II DNA vaccine based on a baculovirus vector as well as a building method and application thereof. Codons of regional sequences 1-186, 993-1197, 603-783and 85-186 nt of cyprinid herpesvirus II ORF72, ORF66, ORF81 and ORF82 are optimized; then, ORF72-ORF66-ORF81-ORF82 fusion sequences are chemically synthesized; next, the promoter control is performed; the clone into the baculovirus transfer vector pFSATBacTMDual is performed to build recombinant plasmids pFSATBacTMDual-FA-D4ORF; after the plasmids are used for converting DH10 / Bac competent cells, Bacmid-FA-D4ORF is obtained; after the Bacmid-FA-D4ORF DNA is used for transfecting silkworm culture cells, BmNPV-FA-D4ORF is obtained and is the cyprinid herpesvirus II DNA vaccine based on the baculovirus vector. The DNA vaccine can realize the immune on carassius auratus gibelio by using an injection or oral administration or soaking method; the gill bleeding diseases of the carassius auratusgibelio can be reduced.

Description

technical field [0001] The invention relates to the technical field of viral genetic engineering, in particular to a method and application for preparing a carp herpesvirus type II DNA vaccine based on a baculovirus vector. Background technique [0002] Cyprinid herpesvirus II (CyHV-2) infection can cause gill hemorrhage outbreaks in heterogeneous gibel crucian carp, resulting in serious economic losses. At present, the prevention and control of the disease is mainly carried out from the aspects of seed detection, enhancement of immunity, reduction of stress, reduction of breeding density, removal of diseased fish, etc., but the prevention and control effect is limited. Immunoprophylaxis is the most direct and effective method for the prevention and treatment of viral diseases. At present, common vaccines mainly include: inactivated vaccines (killed vaccines), attenuated vaccines, subunit vaccines, DNA vaccines, and protein subunit / DNA combined vaccines. Different types of...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N15/38C12N5/10C12N15/62A61K39/245A61P31/22
CPCA61K39/12A61K2039/53A61P31/22C07K14/005C12N15/86C12N2710/14043C12N2710/16022C12N2710/16034C12N2800/105
Inventor 贡成良曹广力李坤刘波胡小龙薛仁宇
Owner 苏州培恩特生物科技有限公司
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