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Construction method of protein microcrystal embedded CyHV-2 (cyprinid herpesvirus II)-type-GCRV (grass carp reovirus) subunit vaccine based on yeast expression vector

A yeast expression vector and carp herpes virus technology, which is applied in the field of genetic engineering technology to express vaccines, can solve the problems of many influencing factors, complicated preparation process of sodium alginate antigen microspheres, etc., and achieves high biological safety, good biological activity of the product, The effect of low production cost

Pending Publication Date: 2018-11-06
SUZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the preparation process of sodium alginate antigen microspheres is more complicated, and there are many influencing factors.
The prior art discloses a method of encapsulating the expressed cyprin herpesvirus type II antigen into the silkworm cytoplasmic polyhedron by using a baculovirus expression system through genetic engineering technology; however, there is no simultaneous encapsulation of cyprin herpesvirus Type II-grass carp hemorrhagic virus subunit combination vaccine, and the combination vaccine is not equal to random mixing of any vaccines. The first problem to be solved is how to ensure the stability of the final product of the combination vaccine so as to achieve a shelf life of more than 18 to 24 months

Method used

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  • Construction method of protein microcrystal embedded CyHV-2 (cyprinid herpesvirus II)-type-GCRV (grass carp reovirus) subunit vaccine based on yeast expression vector
  • Construction method of protein microcrystal embedded CyHV-2 (cyprinid herpesvirus II)-type-GCRV (grass carp reovirus) subunit vaccine based on yeast expression vector
  • Construction method of protein microcrystal embedded CyHV-2 (cyprinid herpesvirus II)-type-GCRV (grass carp reovirus) subunit vaccine based on yeast expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 A method for constructing a protein microcrystal-embedded carp herpesvirus type II-grass carp hemorrhagic virus subunit vaccine based on a yeast expression vector

[0046] (1) Chemical synthesis of BmCPV(polh)-TT CYC- P TEF - VP6-VP7-ORF72-ORF66-ORF81-ORF82-BmCPV (VP3) fusion sequence SEQ ID NO: 1, cloned into the pMD-19T vector, and then verify whether the synthesized sequence is consistent with the sequence of SEQ ID NO: 1 by sequencing, Name the correct recombinant plasmid as pMD-Target.

[0047] (2) For pMD-Target plasmid Eco R with not Enzyme digestion, the digested product was electrophoresed on 1% agarose gel, and the exogenous fragment with a molecular weight of 2.86 kb was recovered, and cloned into the vector pPICZA (Invitrogen Company) Eco R with not site, construct the recombinant plasmid pPICZA-BmCPVpolh-GCRV6 / 7-CyHV72 / 66 / 81 / 82-BmCPVVP3. The structure of the plasmid is as figure 1 shown. Among them, 5'AOX1 is the promoter of AOX...

Embodiment 2

[0056] Example 2 A method for encapsulating carp herpesvirus type II-grass carp hemorrhagic virus subunit vaccine based on protein microcrystals of yeast expression vector

[0057] Compared with Example 1, the difference is that when synthesizing SEQ ID NO: 1 in step (1), the "GGTCCTGGA" at positions 2320-2328 is changed to "GGACCTGGT" for artificial synthesis, and the result is SEQ ID NO: 4. Other steps are the same as in Embodiment 1. The purified polyhedron was detected by Western blot to observe the signal band representing the polyhedrin protein ( Figure 7 ). For Western blot detection, mouse-derived BmCPV polyclonal antibody was used as the primary antibody, and goat anti-mouse IgG was used as the secondary antibody. Lane C is the silkworm cytoplasmic polyhedron, and lanes 1-8 are yeast expression vector-based protein microcrystal-encapsulated carp herpesvirus type II-grass carp hemorrhagic virus subunit vaccines purified from different yeast engineering bacteria.

Embodiment 3

[0058] The preparation of embodiment three herpes virus type II-grass carp hemorrhagic virus subunit vaccine

[0059] Example 1 The purified protein microcrystal-embedded carp herpesvirus type II-grass carp hemorrhagic virus subunit vaccine based on yeast expression vector was cleaved with sodium carbonate-sodium bicarbonate solution with pH 10.8 at 37°C, and then adjusted to pH with hydrochloric acid solution At about 7.12, after a large number of flocculent precipitates appeared, centrifuge at 12000 rpm, and the supernatant obtained was the combined subunit vaccine antigen of carp herpesvirus type II-grass carp hemorrhagic virus. Figure 8 It is the western blot detection diagram of the antigen purified from the polyhedron, lanes 1-5 are the antigen purified from the polyhedron in different yeast engineering bacteria, the primary antibody is CyHV-2 ORF72 mouse antibody, and the secondary antibody is goat anti-mouse IgG.

[0060] In the present invention, the sequence is as f...

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Abstract

The invention relates to a construction method of a protein microcrystal embedded CyHV-2 (cyprinid herpesvirus II)-type-GCRV (grass carp reovirus) subunit vaccine based on a yeast expression vector. An AOX1 promoter is used for controlling a silkworm cypovirus protein gene; a TEF1 promoter is used for controlling VP6 and VP7 from GCRV and CyHV-2 ORF72, ORF66, ORF81, ORF82 and an antigenic epitopefusion sequence VP6-VP7-ORF72-ORF66-ORF81-ORF82-VP3 obtained after codon optimization of cypovirus VP3. The vector transforms yeast, inducible expression is performed through methanol, expressed poly-antigen epitope fusion protein is embedded in recombinant cypovirus protein under the guide of cypovirus VP3, a protein microcrystal of embedded CyHV-2-GCRV is formed, the protein microcrystal is split by a sodium carbonate-sodium bicarbonate solution, the pH is regulated to 7.12 with a hydrochloric acid solution, and a supernatant obtained after centrifugation is CyHV-2-GCRV joint subunit vaccineantigen.

Description

technical field [0001] The invention relates to the field of expressing vaccines using genetic engineering technology, in particular to a method for encapsulating carp herpesvirus type II-grass carp hemorrhagic virus subunit vaccines in protein microcrystals based on yeast expression vectors. Background technique [0002] Gill hemorrhagic disease of heterogeneous gibel crucian carp caused by Cyprinid herpesvirus II (CyHV-2) infection has caused serious economic losses and greatly affected the healthy development of heterogeneous gibel carp aquaculture. At present, the prevention and control of the disease is mainly carried out from the aspects of detection of seed origin, enhancement of immunity, reduction of stress response, reduction of breeding density, scientific polyculture, early diagnosis, and timely removal of diseased fish, etc., but the prevention and control effect is not satisfactory . Grass carp is a large-scale farmed freshwater fish in my country. Grass carp ...

Claims

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Application Information

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IPC IPC(8): A61K39/245A61P31/22A61K39/15A61K47/42A61P31/14A61P7/04C12N15/81C12N15/62
CPCA61K39/12A61K47/42A61K2039/70A61P7/04A61P31/14A61P31/22C12N15/815C12N2710/16034C12N2720/12034
Inventor 贡成良曹广力薛仁宇胡小龙张婷婷刘波
Owner SUZHOU UNIV
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