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Method for preparing virus analogs of nervous necrosis viruses

A nerve necrosis and analogue technology, applied in the biological field, can solve the problems of time-consuming and expensive, unsuitable for large-scale purification and production, and achieve the effect of low cost of equipment and reagents, and simple purification methods

Active Publication Date: 2011-01-12
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally, the purification method of VLP is ultra-fast density gradient centrifugation (that is, a purification method similar to viruses). The purification process is not only time-consuming but also expensive, and is not suitable for large-scale purification and production.

Method used

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  • Method for preparing virus analogs of nervous necrosis viruses
  • Method for preparing virus analogs of nervous necrosis viruses
  • Method for preparing virus analogs of nervous necrosis viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Production and purification of wild-type oblique-banded grouper neuronecrosis virus (OGNNV) VLPs.

[0039] Under prokaryotic conditions, the modified pQE30 vector was used to express wild-type capsid protein (CP). Since CP needs time and space for self-assembly in E. coli after expression, the speed of expression and Quantities are strictly controlled, requiring a good balance between yield and VLP quality. We performed optimization for prokaryotic expression of VLPs. The overnight culture (OD600=2) was simultaneously inoculated into multiple 1 L media at a ratio of 1:100, cultured at 37°C until OD600=0.4, and then added with IPTG to a final concentration of 900 μM to induce expression for 4 hours , and finally checked the expression level of CP by SDS and identified the quantity and quality of VLP formation by electron microscope negative staining observation after purification. Finally, the production and purification methods of VLP were optimized as follows: 4 L of...

Embodiment 2

[0042] Production and purification of OGNNV His-VLP.

[0043] This is the first study on the ability of CP to form VLP after inserting a tag in the development of Nodavirus VLP, and it can be purified by affinity chromatography. After successfully producing VLP, how to obtain VLP quickly and at low cost becomes another important research direction. Therefore, we try to add tags that can be used for purification to VLP for affinity chromatography purification, which will greatly shorten the time of VLP purification and reduce the high-cost purification process caused by ultracentrifugation. 6×His was used as a purification tag, inserted into the N-terminus and C-terminus of VLP, respectively, His-VLP and VLP-His, and the prokaryotic expression process was similar to that of VLP. Anti-His and polyclonal antibody detection, while only His-VLP and VLP-His with 6×His tag can be detected with anti-His monoclonal antibody ( Figure 4 ). After standard sucrose cushion and sucrose g...

Embodiment 3

[0045] Immunogenicity of VLPs.

[0046] In order to study the immunogenicity and immunoprotection of VLP, the VLP expressed, isolated and purified in Example 1 was used as an antigen to immunize juvenile sea bass (average body weight 1.6 g, average body length 3.3 cm) to detect fish The reaction curve of the anti-VLP antibody produced by the body was used to study the ability of fish serum to neutralize the virus and to detect the protection rate and protection period of the virus on sea bass after immunization. Inject 5 μg of protein per gram of fish body weight, including VLP, GST-CP (recombinant protein), inactivated virus (inactive virus) and recombinant mutant virus (RDV), and immunize 25 juvenile sea bass by intraperitoneal injection (per Tail fish injection was less than 30 μl), and the control group was injected with the same injection volume of PBS. On days 7, 14, 21, and 28 after immunization, blood was collected from 5 fish each, and the changes of antiviral antibo...

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Abstract

The invention belongs to the technical field of biology, and relates to a method for preparing virus analogs of nervous necrosis viruses. The method comprises the following steps of: (1) performing expression of the virus analogs of the nervous necrosis viruses on escherichia coli of pQE30 plasmid vectors coded with nervous necrosis virus capsid protein genes under the pronucleus condition, wherein the expression is performed under the following conditions of: inoculating 1 mass percent of escherichia coli solution of which OD600 is equal to 1.5 into a culture medium, culturing the escherichia coli at 37 DEG C under 250rpm till the OD600 is 0.3 to 0.5, adding isopropyl-beta-D-thiogalactoside into the escherichia coli solution till the final concentration of the isopropyl-beta-D-thiogalactoside is 900muM, transferring the solution, and continuously culturing the escherichia coli for 3 to 4 hours at the temperature of between 25 and 30 DEG C at the rotational speed of 200rpm to finish the expression; and (2) after the expression is finished, breaking, separating and purifying the strains to obtain the virus analogs of the nervous necrosis viruses, wherein the plasmid coded genes also can be nervous necrosis virus capsid protein genes containing histidine tags, and the expression product of the genes can be purified by affinity chromatography. The method provided by the invention can obtain high virus analog expression amount; and the chromatography and purification method is simple and convenient and has low costs in required apparatuses and reagents.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a preparation method of a neuronecrosis virus analogue. Background technique [0002] Viral nervous necrosis (VNN) in fish, also known as viral encephalopathy and retinopathy (VER), brings huge economic benefits to local hatchery farms every time the outbreak occurs The loss is a major obstacle to the development of the aquaculture industry. The causative agent of the disease is Betanodavirus, and the larvae and juveniles of various fish are its sensitive hosts. The mortality rate after the onset of the disease is as high as 90%, or even 100%. The host species of the virus spread over 16 families and more than 40 kinds of fish, most of which are marine fish. In recent years, with the promotion and deepening of research work, it has been reported that there is an increasing trend in the cases of β-noda virus infection in freshwater fish. [0003] In recent years, research on the viru...

Claims

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Application Information

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IPC IPC(8): C12P21/00C07K1/22C07K1/14C12R1/19
Inventor 谢俊锋赖宇雄何建国
Owner SUN YAT SEN UNIV
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