Chicken Infectious Bursal Disease Virus Structural Protein vp2 Suitable for Expression in the Middle Silk Gland of Bombyx mori and Its Expression Vector and Application

A chicken infectious and bursal disease technology, applied in the field of genetic engineering, can solve the problem that there is no transgenic silkworm expressing the structural protein of chicken infectious bursal disease virus, and achieve the effect of consistent structure and good thermal stability

Active Publication Date: 2016-08-24
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far there is no method and related reports for expressing chicken infectious bursal disease virus structural protein VP2 by using transgenic silkworm

Method used

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  • Chicken Infectious Bursal Disease Virus Structural Protein vp2 Suitable for Expression in the Middle Silk Gland of Bombyx mori and Its Expression Vector and Application
  • Chicken Infectious Bursal Disease Virus Structural Protein vp2 Suitable for Expression in the Middle Silk Gland of Bombyx mori and Its Expression Vector and Application
  • Chicken Infectious Bursal Disease Virus Structural Protein vp2 Suitable for Expression in the Middle Silk Gland of Bombyx mori and Its Expression Vector and Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Construction of transgene expression vector pBac[3×P3EGFP, hr3CQSer1-vp2-Ser1PA]

[0028] According to the reported IBDV super-virulent strain virus structural protein VP2 gene sequence (GenBank.JF907703.1) and the codon preference of the silkworm, the codon was optimized to obtain the optimized vp2 gene sequence, and then 6 His was added to the C-terminal The nucleotides of the tag form vp2-His6, and BamHI and NotI restriction sites are attached to both ends of the nucleotide sequence of vp2-His6, and then Shenzhen Huada Gene Company is commissioned to synthesize the nucleotide sequence as SEQ ID NO.1 For the sequence shown, the amino acid sequence encoded by the coding region is shown in SEQ ID NO.2. In the sequence of SEQ ID NO. 1, positions 1 to 6 are BamHI restriction sites, positions 7 to 1329 are the coding sequence of vp2 gene, positions 1330 to 1350 are C-terminally added 6 A His tag, positions 1351 to 1358 are NotI restriction sites. Then the nucleot...

Embodiment 2

[0030] Example 2. Microinjection of transgene and screening and succession of positive individuals

[0031] The diversified silkworm species "D9L" was raised in a standard environment with mulberry leaves (temperature: 25°C, humidity: 90%). After the moths, the female and male silkworm moths were mated in pairs for about 4 hours. The eggs are used for the next microinjection.

[0032] Arrange the laid silkworm eggs neatly on a clean glass slide, and mix the pBhSer1vp2 transgene vector and the auxiliary plasmid pHA3PIG at an equal molar ratio with the Eppendorf microinjector within 1h to 4h after the silkworm eggs are laid, and then inject them into 400 capsules. D9L" silkworm eggs were sealed with non-toxic glue and incubated in an environment of 25℃ and 90% relative humidity for about 10 days. The hatched 239 heads of G0 ant silkworms were collected and raised with mulberry leaves to turn moths, G0 The generation of silkworm moths obtained 110 moth circle G1 silkworm eggs through...

Embodiment 3

[0033] Example 3. Detecting the copy number of vp2 gene in pBhSer1vp2 transgenic system

[0034] The pBhSer1vp2 transgenic system prepared in Example 2 and the normal "D9L" were reared under standard conditions, and 50 μg of silkworm moth genomic DNA was extracted for Southern Blot detection. During detection, digest with restriction enzymes Xbal I and BamHI overnight. After separation by 0.8% agarose gel electrophoresis, the DNA in the agarose gel was transferred to the pretreated nylon membrane with a vacuum transfer device. After the membrane is over, the pre-hybridization, hybridization, membrane washing and color development are carried out. image 3 Shown. The results showed that the vp2 gene in the pBhSer1vp2 transgenic system was mainly inserted into the silkworm genome as a single copy.

[0035] The above-mentioned hybridization probe is the digoxigenin-labeled EGFP coding sequence, and the digoxigenin-labeled EGFP coding sequence is prepared by the following method: desi...

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Abstract

The invention discloses a chicken infectious bursal disease virus structural protein VP2 applicable to silkworm middle silk gland expression as well as an expression vector and application thereof. A gene sequence of the chicken infectious bursal disease virus structural protein VP2 is as shown in SEQ ID NO.1; a coded amino acid sequence of the chicken infectious bursal disease virus structural protein VP2 is as shown in SEQ ID NO.2; the sequence for coding the chicken infectious bursal disease virus structural protein VP2, and a promoter and a terminator of an excretion sericin 1 gene form an expression frame, and expression is enhanced by an enhancer hr3; meanwhile, a piggyback transposition arm and a fluorescence-screening marker gene constitute the expression vector; the expression vector can express the chicken infectious bursal disease virus structural protein VP2 in the silkworm middle silk gland, has immunogenicity, and can stimulate a mouse to generate antibodies, so that a foundation is laid up for expressing an animal virus subunit vaccine by utilizing a silkworm middle silk gland biological reactor.

Description

Technical field [0001] The present invention belongs to the field of genetic engineering, and particularly relates to the chicken infectious bursal disease virus structural protein VP2 suitable for expression in the central silk gland of the silkworm, and also relates to an expression vector for expressing the chicken infectious bursal disease virus structural protein VP2 and the use of the central silkworm Method for expressing the structural protein VP2 of chicken infectious bursal disease virus in silk glands. Background technique [0002] Infectious Bursal Disease (IBD) is an infectious disease caused by Infectious Bursal Disease virus (IBDV) that mainly affects the central immune organs of chicks-the bursal disease. The disease can lead to immune suppression and even individual death, and increase the susceptibility of chickens to other pathogens and immune failure, and bring huge economic losses to the poultry industry. In recent years, more virulent IBDV variants that can...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/40C07K14/08C12N15/85C12R1/93
Inventor 赵萍袁林王峰徐汉福夏庆友
Owner SOUTHWEST UNIV
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