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Duck Tembusu virus genetically engineered subunit vaccine and its preparation method and application

A duck Tembusu virus and the technology of encoding genes are applied in genetic engineering, botany equipment and methods, biochemical equipment and methods, etc., and can solve the problems of strong virulence, further research, and poor protein biological activity. , to achieve strong immunogenicity, lower production costs, and high expression levels

Active Publication Date: 2020-04-24
苏州沃美生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing duck Tembusu virus commercial vaccines are mainly attenuated vaccines. The attenuated vaccines have the possibility of returning to strong virulence, which poses a great risk to the immune duck population. The immune effect is easily affected by many factors, and the use of The biological activity of the protein expressed by the prokaryotic protein expression system is not good
[0005] Since this pathogen is a newly emerged pathogen, relevant research needs to be in-depth

Method used

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  • Duck Tembusu virus genetically engineered subunit vaccine and its preparation method and application
  • Duck Tembusu virus genetically engineered subunit vaccine and its preparation method and application
  • Duck Tembusu virus genetically engineered subunit vaccine and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Example 1 Construction of recombinant eukaryotic expression vector pCI-E2-GS

[0110] 1. The codon-optimized DTMUV E2 gene was obtained from Nanjing GenScript Biotechnology Co., Ltd., and cloned into the pUC-57 vector to construct the pUC-E2 plasmid vector. The optimized E2 gene sequence is shown in SEQ ID NO:1.

[0111] 2. E2 gene amplification uses pUC-E2 as a template, and E2-F and E2-R as primers for PCR amplification (the gene sequences of E2-F and E2-R are shown in SEQ ID NO: 3 and 4), and the amplified See Table 1 for the augmentation system. The reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 45 seconds, renaturation at 60°C for 45 seconds, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 10 minutes, and storage at 4°C.

[0112] Table 1 E2 gene amplification system

[0113]

[0114] Perform gel electrophoresis on the PCR product to identify the size of the target gene, such as figure 1 As shown, ...

Embodiment 2

[0126] Example 2 Construction and screening of recombinant CHO cells expressing E2 protein

[0127] 1. Cell Transfection

[0128] 1.1 Prepare cells Take CHO cells in the logarithmic growth phase, sample and count, and use 1×10 6 The cell density of cells / ml continues to be subcultured, maintain the seeds, centrifuge the remaining cells, centrifuge at 1000rpm for 4 minutes, discard the supernatant, resuspend with about 20ml of fresh CHO-WM medium, centrifuge again, centrifuge at 1000rpm for 4 minutes, discard the supernatant After resuspending with a small amount of medium for counting, the final cell density was adjusted to 1.43×10 7 cells / ml.

[0129] 1.2 Plasmid and cell mixing Take 5ug of the pCI-E2-GS plasmid vector in Example 1, add it to the EP tube, add 0.7ml cells, mix well, and let stand for 15 minutes.

[0130] 1.3 Electroporation 280V 20ms electric shock for 2 pulses. Immediately after the electric shock is completed, the cells are transferred to a shaker flask a...

Embodiment 3

[0140] Example 3 SDS-PAGE detection

[0141] The cell culture supernatant harvested in Example 2 was subjected to SDS-PAGE detection, and empty CHO cells were used as a negative control. The specific operation is as follows: take 40 μl of the harvested cell culture, add 10 μl of 5× sample buffer, bathe in boiling water for 5 minutes, centrifuge at 12000 r / min for 1 minute, and take the supernatant for SDS-PAGE gel (12% concentration gel) Electrophoresis. After electrophoresis, the gel was stained and decolorized to observe the target band.

[0142] The detection result of culture in embodiment 2 is as Figure 4 As shown, the target band appears around the molecular weight of about 53kDa, and the negative control has no band at the corresponding position.

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Abstract

The present invention provides a duck tambosu virus genetic engineering subunit vaccine and a preparation method and an application thereof. A nucleic acid molecule having a sequence shown in SEQ ID NO:1 or a nucleic acid molecule identical to 95% or more of the nucleotide sequence shown in the SEQ ID NO:1 encodes a duck tambosu virus E2 protein, an immune composition comprising the duck tambosu virus E2 protein can be used to prepare the duck tambosu virus genetic engineering subunit vaccine, antigenicity, immunogenicity and functions of the vaccine are similar to natural proteins, and the vaccine is relatively high in expression level, strong in the immunogenicity, free of pathogenicity to ducks, and can also be prepared by large-scale serum-free suspension culture in a bioreactor, which greatly reduces a production cost of the vaccine.

Description

technical field [0001] The invention relates to a genetically engineered subunit vaccine, in particular to a duck Tembusu virus genetically engineered subunit vaccine and its preparation method and application, belonging to the technical field of animal immune medicines. Background technique [0002] Duck Tembusu virus disease, also known as duck yellow virus disease, or duck egg drop syndrome, is an infectious disease caused by duck Tembusu virus (Duck Tembusu Virus, DTMUV). The disease was first discovered in southeastern China in 2010. It is mainly caused by laying ducks, but also occurs in breeding geese and meat ducks. The clinical manifestations of the disease of laying ducks are unstable limbs, difficulty in walking, decreased feed intake, and a sudden decrease in egg production. In a short period of time, the egg production can be reduced by more than 90%. Some sick ducks die within a few days, and the mortality rate is 5%. -10%. Visceral lesions include ovarian he...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/12A61P31/14C12N15/85C12N15/40C07K14/18
CPCA61K39/12A61K2039/5256A61K2039/552A61P31/14C07K14/005C12N15/85C12N2770/24022C12N2770/24134
Inventor 曹文龙孔迪滕小锘易小萍张大鹤
Owner 苏州沃美生物有限公司
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