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Perch rhabdovirus subunit vaccine and preparation method thereof

A subunit vaccine and rhabdovirus technology, applied in the biological field, can solve the problems of restriction, difficulty in meeting the requirements of large-scale industrialization, enzymatic inactivation, etc., to achieve improved effect, good immunogenicity and immune protection performance, good protective effect

Active Publication Date: 2021-10-22
深圳万可森生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, injection immunization has the best protective effect, but it is difficult to meet the requirements of large-scale industrialization due to operating technology and production costs; although immersion and oral immunization are simple in operation technology and low in production cost, there are also defects in the body surface skin and intestines. A series of insurmountable technical problems such as barrier restriction, enzymatic inactivation, and low immune protection rate

Method used

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  • Perch rhabdovirus subunit vaccine and preparation method thereof
  • Perch rhabdovirus subunit vaccine and preparation method thereof
  • Perch rhabdovirus subunit vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: Recombinant Escherichia coli E. coli Construction and identification of BL21 / pET32a-G2 strain

[0047] G2 target gene amplification

[0048] Referring to the instructions of the viral RNA extraction kit, the MSRV FJ985 strain virus liquid cultured with CO cells (the MSRVFJ985 strain was provided by Northwest A&F University, see Fei Yang et al. Evaluation on the antiviral activity of ribavirin against Micropterus salmoides rhabdovirus (MSRV) in in vitro and in vivo , Aquaculture, Volume 543 , Internet release time: May 27, 2021) as materials to extract MSRV virus RNA. Agarose gel electrophoresis was used to detect the integrity of the RNA, and a micro-nucleic acid analyzer was used to determine the concentration and quality of the sample RNA. The extracted RNA samples were reversed into cDNA using a reverse transcription kit. The G2 gene was amplified with MSRV-G-F / R and VP7-F / R primer pairs, wherein MSRV-G-F: 5'- GAACTGTGGATGGAATAACG-3' (SEQ ID...

Embodiment 2

[0065] Example 2: Evaluation of immune efficacy of recombinant protein

[0066] During the research and development process, different fragments were designed, and different glycoprotein fragments and complete glycoproteins were prepared using the method of Example 1, glycoprotein fragment G1 (amino acid sequence is SEQ ID No: 3), glycoprotein fragment G3 (The amino acid sequence is SEQ ID No: 4) protein and the complete glycoprotein G (the amino acid sequence is SEQ ID No: 5), respectively verified the immune efficacy of the above protein fragments. Specifically, the following immune efficacy evaluation test was adopted: 300 healthy perch were randomly divided into 6 groups, including the control group (PBS), the G1 protein group (40 mg / L), the G2 protein group (40 mg / L), and the G3 protein group. (40 mg / L), G protein group (40 mg / L), G protein group (80 mg / L), all sea bass were vaccinated with the corresponding vaccines through the bath solution and soaked for 6 hours. Subs...

Embodiment 3

[0067] Embodiment 3: the preparation of intermediate product for vaccine manufacture

[0068] Production of seed bacterial liquid preparation

[0069] recombinant Escherichia coli E. coli After diluting the basic strain of BL21 / pET32a-G2 strain with normal saline, pick a small amount of bacterial liquid with an inoculation loop, inoculate it in LB liquid medium, culture it at 37°C and 180r / min for 12-16 hours, and harvest the bacteria solution, stored below -20°C.

[0070] Preparation of semi-finished products

[0071] Preparation of first-class seeds: Pick a small amount of bacterial liquid with an inoculation loop for the production strain, inoculate it on the LB solid medium plate by streaking, and culture it at 37°C for 12-16 hours, then pick a single colony and inoculate it on the LB liquid culture culture medium at 37°C and 180r / min for 12-16 hours.

[0072] Preparation of secondary seeds: Inoculate primary seeds in LB liquid medium at 1% (V / V), culture at 37°C and ...

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Abstract

Part of protein fragment G2 of MSRV glycoprotein is selected, G2 protein is obtained through prokaryotic recombinant expression, and mannosylation modification is carried out on the G2 protein to prepare a perch rhabdovirus nano-carrier targeting vaccine, and research shows that for perch after immunized by the subunit vaccine for 21 days is attacked by an MSRV FJ985 strain, the perch immune protection rate is 94%-96%, and the vaccine is safe and effective. After the perch is immunized, a better protection effect can be achieved, and the perch can effectively resist the attack of MSRV virulent virus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a perch rhabdovirus subunit vaccine. Background technique [0002] largemouth bass ( Micropterus salmoides ) is commonly known as California perch, which belongs to the genus Perciformes sunfish family. Largemouth bass was introduced in Foshan, Guangdong in 1983, and was successfully artificially bred in 1985. The fish species have been widely cultured and have achieved good economic benefits. Farmers and consumers love it. However, perch is susceptible to many diseases, such as gill rot, canker, nocardiosis, and trichotillomaniasis. Among them, perch rhabdovirus ( Micropterus salmoides rhabdovirus, MSRV ) caused the most serious damage to perch rhabdovirus disease. It is endemic in a wide area, has a long onset time, and has a mortality rate of more than 85%, which has brought huge economic losses to perch farming. [0003] Perch rhabdovirus disease has been repo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/205A61K47/04A61P31/14C07K14/145C07K1/107
CPCA61K39/12A61K47/02A61K9/0017A61P31/14C07K14/005C07K1/1077A61K2039/552C12N2760/20034C12N2760/20022
Inventor 王高学朱斌凌飞郭孜饶焦铁军
Owner 深圳万可森生物科技有限公司
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