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A kind of perch rhabdovirus recombinant g2 protein and its application

A rhabdovirus and protein technology, applied in the field of perch rhabdovirus recombinant G2 protein, can solve problems such as difficulty in meeting large-scale industrialization requirements, restriction, enzymatic inactivation, etc., and achieve good immunogenicity and immune protection performance. , good protective effect, good protective effect

Active Publication Date: 2021-11-30
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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  • A kind of perch rhabdovirus recombinant g2 protein and its application
  • A kind of perch rhabdovirus recombinant g2 protein and its application
  • A kind of perch rhabdovirus recombinant g2 protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: Recombinant Escherichia coli E. coli Construction and identification of BL21 / pET32a-G2 strain

[0034] G2 target gene amplification

[0035] 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.

[0036] After a large number of designs, the G2 fragment with the amino acid sequence of SEQ ID No: 2 was selected, and ...

Embodiment 2

[0053] Example 2: Evaluation of immune efficacy of recombinant protein

[0054] 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 (the amino acid sequence is SEQ ID No: 5), respectively verifying 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. Subse...

Embodiment 3

[0056] Embodiment 3: the preparation of subunit vaccine

[0057] Functional modification of single-walled carbon nanotubes

[0058] The structure modification of single-walled carbon nanotubes is carried out by the mixed acid oxidation method, and the steps are as follows:

[0059] (1) Put 2g single-walled carbon nanotube sample into 150mL concentrated H 2 SO 4 and 50mL concentrated HNO 3 placed in a magnetic stirrer at 100 rpm for 48 h at room temperature;

[0060] (2) Filter the mixed acid mixture of carbon nanotubes obtained in the previous step with a circulating water vacuum pump, wash with pure water until the pH of the liquid is 7.4, then dry it at 60°C, and grind it into powder in a mortar , through a 300-mesh sieve, and the resulting product is functionalized single-walled carbon nanotubes (o-SWCNTs).

[0061] Preparation of carbon nanotube carrier subunit vaccine

[0062] Take the acidified single-walled carbon nanotube sample (0.5g) and add it to 2-(N-morpholi...

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Abstract

The present invention obtains G2 protein through prokaryotic recombinant expression. The protein fragment has good immunogenicity and can stimulate the body to produce a higher antibody level. Compared with the whole G protein and other fragments, this fragment has better immunogenicity and immune protection Performance, based on the combination of G2 protein and functional carbon nanotubes, the carbon nanotube carrier subunit vaccine prepared by immunizing perch with MSRV FJ985 strain 21 days later, the immune protection rate of perch was 86%, which was higher than the protection rate of existing reports. And based on the selection of specific protein fragments, it has a better protective effect than the original G protein and other G protein fragments based on immune analysis, and can produce better protection after immunization of sea bass, and can make sea bass effectively resist MSRV Poisonous attack.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a perch rhabdovirus recombinant G2 protein and application thereof. Background technique [0002] by perch rhabdovirus ( Micropterus salmoides rhabdovirus, MSRV ) caused the most serious damage to perch rhabdovirus disease, which has a wide range of prevalence, a long onset time, and a mortality rate of more than 85%, which has brought huge economic losses to perch farming. MSRV is a single-stranded RNA virus with a size of (100-430) nm × (45-100) nm and a rod-like or bullet-like shape. The MSRV genome mainly encodes five structural proteins, namely nucleoprotein N, phosphoprotein P, mitrix M, glycoprotein G, and RNA polymerase L. [0003] G protein is glycoprotein, which is mainly responsible for binding to host cell receptors and is the main antigenic protein of viruses. According to the deduced amino acid sequence, it is speculated that the G protein is similar to o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/145C12N15/47C12N15/70A61K38/16A61P31/14
CPCC07K14/005C12N15/70A61P31/14C12N2760/20022A61K38/00
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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