Preparation and application of baculovirus expression system-based duck tembusu virus subunit vaccine

A technology of baculovirus and recombinant baculovirus, applied in the direction of microorganism-based methods, applications, vaccines, etc., can solve the problems of immune effect being easily affected by various factors, poor protein biological activity, strong virulence, etc. , to achieve good protective effect, high biological safety and good immunogenicity

Inactive Publication Date: 2017-06-13
HUAZHONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The attenuated vaccine has the possibility of strong virulence, which poses a great risk to the immunized duck population. Its immune effect is easily affected by many factors, and there are certain restrictions on transportation and storage.
However, the biological activity of the protein expressed by the prokaryotic protein expression system is not good, and the prepared subunit vaccine has certain limitations

Method used

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  • Preparation and application of baculovirus expression system-based duck tembusu virus subunit vaccine
  • Preparation and application of baculovirus expression system-based duck tembusu virus subunit vaccine
  • Preparation and application of baculovirus expression system-based duck tembusu virus subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1: Construction of recombinant baculovirus AC-prME expressing duck Tembusu PrM-E gene

[0079] 1. Construction of recombinant baculovirus AC-prME

[0080] 1. Acquisition of prME gene

[0081] A 2100bp prME gene and its signal peptide sequence (GenBank ID: KJ489355) were artificially synthesized with reference to the prME gene and its signal peptide sequence of Duck Tembusu virus DF2 strain (Duck Tembusu virus DF2), and the sequence was cloned into the vector pUC-18 (Invitrogen Company) The recombinant plasmid pUC-prME was obtained. Use the plasmid as a template with a primer pair

[0082] Forward: 5'-TAGGCGGCCGCATGCAGATGCTCGACGGACTGAAT-3',

[0083] Reverse: 5'-AGCTGCAGTTAGGCATTGACATTTACTGC-3';

[0084] PCR amplification was carried out, and a prME gene fragment with a size of 2100 bp was recovered.

[0085] 2. Construction of baculovirus transfer vector pFastBac1-prME

[0086] Baculovirus universal vector pFastBac1 TM (Invitrogen Company) as the backbone,...

Embodiment 2

[0118] Embodiment 2: the subunit vaccine prepared by recombinant baculovirus Ac-prME

[0119] 1. A subunit vaccine prepared by recombinant baculovirus Ac-prME.

[0120] The recombinant baculovirus Ac-prME obtained in Example 1 was used to inoculate suspension-cultured insect cells sf9 at a multiplicity of infection dose of 2.0 MOI, and the cells and supernatant were collected after 72 hours, and the cells were purified according to the method described in 4 of Example 1 DTMUV-VLPs in the medium, after measuring the protein concentration, the water phase is composed of protein and Tween 80 according to the volume ratio of 97:3, and the water phase and the oil phase are mixed and emulsified according to the volume ratio of 1:2 to prepare a sub-vaccine, so that each ml The total protein content is 0.5 mg / ml, which is used in the following examples 3 and 4, and stored at 4 degrees.

[0121] 2. Subunit vaccine prepared by recombinant baculovirus Ac-prME.

Embodiment 3

[0123] Example 3: Subunit vaccine components and routine testing

[0124] Dosage form: water-in-oil (W / O)

[0125] Adjuvant composition: white mineral oil (Marcol52)

[0126] Tween 80 (CRILLET4)

[0127] Vaccine emulsification: the water phase is composed of protein and Tween 80 at a volume ratio of 97:3, and the water phase and oil phase are mixed and emulsified at a volume ratio of 1:2 to prepare a sub-vaccine.

[0128] Vaccine property test: after emulsification, the vaccine is dripped into clear water, the first drop will disperse, and the second drop will be in the form of oil beads, and the oil beads on the liquid surface will not break the emulsification.

[0129] Sterility test: Take a small amount of the vaccine and coat it on a TSA plate, and incubate in a 37°C incubator for 18 hours without colonies growing.

[0130] Stability test: Centrifuge at 3000rpm / min for 15 minutes, the emulsified vaccine is not separated after centrifugation.

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Abstract

The invention discloses preparation and application of a baculovirus expression system-based duck tembusu virus subunit vaccine. Tembusu prME protein is expressed by adopting a baculovirus expression system; WB shows that the prME protein is successfully expressed in an sf9 cell and a supernatant, and is partially cut into M protein and E protein after being maturely processed; existence of tembusu virus-like particles of which the diameters are about 30-50nm in the sf9 cell and secreted supernatant is observed through an electron microscope; and the virus-like particles are formed through self-assembly of the protein in the sf9 cell and have relatively high immunogenicity. The protein or the virus-like particles can be purified through two different modes and mixed with an adjuvant at the ratio to prepare the safe, stable and efficient duck tembusu virus subunit vaccine. The vaccine is capable of immunizing cherry valley ducks and egg-laying sheldrakes and then inducing the organism to generate a specific immune response, and 60% of egg-laying sheldrakes can be prevented from being attacked by a duck tembusu virus.

Description

technical field [0001] The invention relates to the field of livestock genetic engineering vaccines, in particular to the preparation and application of a duck Tembusu virus subunit vaccine based on a baculovirus expression system. Background technique [0002] Duck tembusu virus disease is an infectious disease caused by duck tembusu virus (DTMUV). The virus belongs to the Flaviviridae family. It first broke out in my country in 2010 and quickly spread throughout duck-intensive areas such as Fujian, Zhejiang, Anhui and other places. Only in 2010, it caused up to 5 billion economic losses to my country's duck industry. The disease occurs suddenly and spreads rapidly. Both meat and egg poultry are susceptible. Waterfowl are more susceptible to infection than chickens. The incidence rate of infected eggs / breeding birds is as high as 100%, and egg production drops from 90-95% in the peak period to 5-10. %. The death rate of waterfowl is 5-15%, and the mortality rate can reac...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N15/66C12N7/01A61K39/12A61P31/14C12R1/93
CPCA61K39/12A61K2039/5256A61K2039/5258A61K2039/552C07K14/005C12N7/00C12N15/66C12N15/86C12N2710/14021C12N2710/14043C12N2710/14051C12N2770/24122C12N2770/24134
Inventor 金梅林邓雪霞张丹康超姚蓉杨应立李淑云魏燕鸣张强孙小美马季
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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