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Porcine pseudorabies virus gE-gI protein and expression plasmid, preparing method and application thereof

A porcine pseudorabies virus and protein expression technology, applied in the biological field, can solve problems such as differences in titer and value, and achieve the effects of easy purification, large-scale production, and improved specificity and sensitivity

Active Publication Date: 2019-08-23
北京标驰泽惠生物科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the gE antibody of pseudorabies acquired by piglets through breast milk and the gE antibody produced by wild virus infection are both positive, but the titers are different

Method used

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  • Porcine pseudorabies virus gE-gI protein and expression plasmid, preparing method and application thereof
  • Porcine pseudorabies virus gE-gI protein and expression plasmid, preparing method and application thereof
  • Porcine pseudorabies virus gE-gI protein and expression plasmid, preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Expression and purification of porcine pseudorabies virus gE-gI protein

[0060] 1.1. Codon optimized synthesis of porcine pseudorabies virus gE and gI protein expression genes

[0061] According to the published porcine pseudorabies virus gE and gI gene sequences (GenBank: KJ675640), in order to make the gE and gI genes more stable and efficient expression in the baculovirus system, they were optimized and synthesized to obtain two new Sequence, named OPTI-gE (shown in SEQ ID NO: 1) and OPTI-gI sequence (shown in SEQ ID NO: 2), and cloned into the dual expression vector pFastBac-Dual (Gibco, catalog number: 10712024), and named It is a recombinant double expression plasmid of pFastbac-gE-gI. The work was entrusted to Nanjing KingScript Biotechnology Co., Ltd. to complete. figure 1 Shows the spectrum of the recombinant dual expression plasmid pFastbac-gE-gI; figure 2 Shows the results of double restriction digestion of pFastbac-gE-gI plasmid, where M represents ...

Embodiment 2

[0098] Example 2: Colloidal gold rapid quantitative detection test strip or test card for detecting porcine pseudorabies virus gE-gI antibody (referring to gE antibody and gI antibody)

[0099] Such as Figure 5 Shown is a schematic diagram of the structure of a colloidal gold test strip, where Figure 5 Panel A is a schematic diagram of the front structure of the colloidal gold test strip. Figure 5 Panel B is a schematic diagram of the side structure of the colloidal gold test strip. Figure 5 Panel C is the chromatography direction of the colloidal gold test strip, according to Figure 5 , The colloidal gold rapid quantitative detection test strip used to detect porcine pseudorabies virus gE-gI antibody consists of conjugate release pad 3, absorbent pad 1, nitrocellulose membrane (NC membrane) 2, glass cellulose membrane sample pad 4 and The PVC backplane 7 is composed of a nitrocellulose membrane 2 with a detection line 6 (T line) and a quality control line 5 (C line). In this...

Embodiment 3

[0114] Example 3: Kit for rapid and quantitative detection of porcine pseudorabies virus gE-gI antibody

[0115] The colloidal gold test card in Example 2 is used to quantitatively detect the porcine pseudorabies virus gE-gI antibody, which can be used to determine the porcine pseudorabies virus infection status, guide the porcine pseudorabies virus purification and the elimination of infected pigs. Among them, the immunochromatographic quantitative analyzer was purchased from Beijing Kuai Rui Read Technology Co., Ltd., and the model is KRD-105.

[0116] 3.1. The kit includes:

[0117] 1) Colloidal gold test cards: several (20 in this embodiment), prepared according to the method described in Example 2, and individually packaged in aluminum foil bags. Each aluminum foil bag contains 1 test card and a bag of desiccant;

[0118] 2) Instructions for use: 1 copy, refer to the description of this Example 3.2;

[0119] 3) Standard curve QR code:

[0120] The preparation method of the standar...

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Abstract

The invention discloses a porcine pseudorabies virus gE-gI protein and an expression plasmid, preparing method and application thereof, and belongs to the field of the biotechnology. The porcine pseudorabies virus gE-gI protein with the purity of 90% or above can be obtained after expression and purification are conducted on recombinant baculovirus based on an established, screened and codon-optimized porcine pseudorabies virus gE-gI protein expression gene, the spatial conformation of gE protein or gI protein is closer to that of natural protein by means of the gE-gI protein, and the specificity and sensitivity of detecting a gE or gI antibody in porcine serum are higher. A colloidal gold test card or kit prepared from the gE-gI protein is used in cooperation with an immunochromatographyanalyzer, the antibody total titer of the gE-gI in the porcine serum can be rapidly and quantitatively detected, and the gE-gI protein can be used for evaluating the wild virus infection state and guiding the purification of the pseudorabies virus, the elimination of live pigs and the optimization of the immune procedure.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a porcine pseudorabies virus gE-gI protein and its expression plasmid, preparation method and application. Background technique [0002] Pseudorabies virus (PRV) belongs to the Herpesviridae family (Herpesviridae), the genus of porcine herpesvirus, the virus particle is round, the diameter is 150-180nm, the nucleocapsid diameter is 105-110nm, the virus genome is linear double-stranded DNA The molecule is about 150kb in size. The virus can cause an acute infectious disease in pigs-pseudorabies in pigs. The disease presents an outbreak in pigs. It can cause abortions, stillbirths, mummified fetuses in pregnant sows, sterile boars, massive deaths of newborn piglets, and fattening Breathing difficulties and growth stagnation in pigs are one of the major infectious diseases that endanger the global pig industry. [0003] For the control of porcine pseudorabies, vaccine immunization ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/03C12N15/866C12N5/10G01N33/569
CPCC07K14/005C12N15/86C12N2710/14043C12N2710/16722G01N33/56994
Inventor 郑金来吴园园钱泓刘建兴张强李东辉
Owner 北京标驰泽惠生物科技有限公司
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