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A kind of prokaryotic expression preparation method of btv1 VP2 protein

A prokaryotic expression, VP2U-F technology, applied in the field of animal vaccine preparation, can solve the problems of difficult to achieve industrial production application, purification consumes a lot of manpower, material resources, financial resources, unsatisfactory VP2 protein expression, etc., to achieve increased specific productivity and increased solubility , highly active effect

Active Publication Date: 2022-06-24
ZHENGZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the VP2 protein expression of engineering bacteria in the existing research is not satisfactory, and purification and concentration require a lot of manpower, material resources, and financial resources, making it difficult to achieve industrial production applications

Method used

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  • A kind of prokaryotic expression preparation method of btv1 VP2 protein
  • A kind of prokaryotic expression preparation method of btv1 VP2 protein
  • A kind of prokaryotic expression preparation method of btv1 VP2 protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] It should be noted that the existing BTV1 VP2 gene sequence is limited by the difference between the E. coli genome sequence and the viral gene sequence during prokaryotic expression, resulting in the fact that the VP2 protein obtained from the actual expression mostly exists in the form of inclusion bodies, thus causing a relatively high expression efficiency. Low, in order to overcome this defect, the inventors carried out a detailed bioinformatics analysis of the existing BTV1 VP2 gene (GenBank: JX101695.1) sequence, and adopted the most frequently used codons for all amino acids. The secondary structure of mRNA, combined with the modification of the optimal codon frequency and the avoidance of the restriction enzyme cleavage site in the subsequent expression process, have designed and obtained a brand-new BTV1 VP2 DNA sequence with a full length of 2886bp, as shown in SEQ ID NO. 1 shown.

[0062] The designed gene sequence was further synthesized by Sangon Bioengine...

Embodiment 2

[0066] On the basis of Example 1, the inventors further used plasmid pET28 as a carrier, and recombined the optimized VP2U into plasmid pET28 to construct a recombinant plasmid expression vector pET28-VP2U, thereby facilitating subsequent prokaryotic expression. The specific recombinant plasmid expression vector pET28 - The construction process of VP2U is briefly described below.

[0067] It should be noted that, in order to prove the expression effect of the optimized gene sequence of the present application, with the existing BTV1 VP2 gene (GenBank: JX101695.1) as a control, the inventor also entrusted Sangon Bioengineering (Shanghai) Co., Ltd. The recombinant plasmid was named: pUC-VP2. On this basis, the inventors also prepared a recombinant plasmid expression vector pET28-VP2 as a control.

[0068] (1) PCR amplification

[0069] When the optimized VP2U gene sequence was amplified by PCR (the primers were synthesized and provided by Sangon Bioengineering (Shanghai) Co., ...

Embodiment 3

[0093] On the basis of Example 2, the inventors transformed the constructed recombinant plasmid I (pET28-VP2U) and recombinant plasmid II (pET28-VP2) into E. coli BL21 (DE3) competent cells, respectively, and obtained large intestine expressing BTV1 VP2 protein. Bacillus expresses strains pET28-VP2U-BL21 and pET28-VP2-BL21, and then carried out preliminary expression verification. The specific experimental process is briefly described as follows.

[0094] (1) Strain culture and induction of expression

[0095] The E. coli expression strains pET28-VP2U-BL21 and pET28-VP2-BL21 were respectively inoculated into LB liquid medium containing 50 μg / mL Kana+ and cultured to OD 600 When the value was about 0.8, IPTG was added to a final concentration of 0.3 mM, and then expression was induced for 10 h at 25°C.

[0096] (2) Protein purification and identification

[0097] Take 5 mL of the induced expression bacterial solution, centrifuge at 12000 r / min for 10 min, discard the supernat...

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Abstract

This application belongs to the technical field of animal vaccine preparation, and specifically relates to a patent application for a preparation method of BTV1 (Bluetongue Virus Type 1) VP2 protein using genetic engineering technology. The coding sequence VP2U of the bluetongue type 1 virus VP2 protein gene consists of 2886 bp bases, and the specific base sequence is shown in SEQ ID NO.1. The preparation of BTV1 VP2 protein by prokaryotic expression includes: PCR amplification, enzyme digestion, connection with plasmid pET28 to construct recombinant plasmid pET28-VP2U, transformation, and induction of expression. This application has laid a certain technical foundation for the fermentation and preparation of BTV1 VP2 protein by optimizing the relevant coding genes and further relying on the technical advantages of the prokaryotic expression system. Preliminary experimental results show that the expression level of the target protein in the supernatant of the fermentation broth can reach 58.4 μg / ml, which can lay a good technical foundation for industrial production.

Description

technical field [0001] The present application belongs to the technical field of animal vaccine preparation, and specifically relates to a patent application for a preparation method of BTV1 (Bluetongue Type 1 virus) VP2 protein using genetic engineering technology. Background technique [0002] Bluetongue (BT) is a serious infectious ruminant disease caused by bluetongue virus (BTV) using Culicoides, Aedes mosquitoes and other insects as vectors. This virus mainly affects ruminant livestock. It can cause symptoms such as high fever, depression, and ulcerative inflammatory changes in the oral, nasal and gastrointestinal mucosa. It has been defined as a statutory reportable infectious disease by the International Organization for Animal Health (OIE), and the Ministry of Agriculture of my country has also designated the disease as a Animal diseases. Based on the above mentioned causes, it can be seen that the occurrence and distribution of bluetongue is closely related to the d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/46C12N15/70C07K14/14C07K1/36C07K1/34C07K1/22
CPCC07K14/005C12N15/70C12N2720/12122
Inventor 陈玉梅王爱萍刘运超刘燕凯冯景
Owner ZHENGZHOU UNIV
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