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A kind of recombinant bovine prion protein bprp and its preparation method and application

A protein, bovine prion technology, applied to the application of recombinant bovine prion protein bPrP in the detection of prion diseases, recombinant bovine prion protein bPrP and its encoding gene and preparation, the field of recombinant expression vectors, can solve the problem of inability to meet precise amino acid sites Research work and other issues to achieve good specificity and immunoreactivity

Inactive Publication Date: 2015-10-14
CHINA AGRI UNIV
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AI Technical Summary

Problems solved by technology

However, these expression technologies all have a shortcoming in the construction of the vector: after the target fragment is connected to the expression vector by double restriction technology, the upstream restriction site (restriction site for constructing the vector and additional restriction site) If it is not removed, it will be translated together with the target gene, and the N-terminus of the target protein will have a few more amino acids (≥2). Such a protein may have a certain impact in general research, such as the preparation of corresponding antibodies. , and in the process of research on the mechanism of prion protein resistance, the research work on the precise amino acid position cannot be satisfied

Method used

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  • A kind of recombinant bovine prion protein bprp and its preparation method and application
  • A kind of recombinant bovine prion protein bprp and its preparation method and application
  • A kind of recombinant bovine prion protein bprp and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 Construction of recombinant expression vector pPROEX-htb-bPrP

[0055] 1. PCR amplification of bovine prion protein polypeptide gene (bPrPc gene)

[0056] Primer: Upstream: cgcGGATCC cgcGGATCC CTCTGCAAGA AGCGACC (the underlined part is the protected base and BamH I restriction site), downstream: cccAAGCTT ATGCCCCTCGTTGGTAAT (the underlined part is the protected base and Hind III restriction site) primers were synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.

[0057] Template: Bovine whole blood (collected from healthy cattle in Inner Mongolia) gene as a template (the whole blood gene extraction kit used was purchased from Dingguo Biotechnology Co., Ltd.).

[0058] PCR system and conditions:

[0059] A high-fidelity PCR kit (Quanshijin Biotechnology Co., Ltd.) was used for PCR amplification, and the total volume of the reaction system was 50 μL. reaction system:

[0060]

[0061] Reaction conditions:

[0062] Pre-denaturation at 9...

Embodiment 2

[0077] Expression, purification and renaturation of embodiment 2 recombinant bovine prion protein bPrP

[0078] 1. Induced expression of bovine prion protein

[0079]The pPROEX-htb-bPrP prepared in Example 1 was transformed into BL21 competent cells, and after the sequence analysis was correct, the species was preserved to obtain bovine prion protein expression engineering bacteria.

[0080] Inoculate the constructed bovine prion protein engineering bacteria into 20 mL of LB medium containing 50 mg / mL ampicillin for overnight culture (about 18 hours), inoculate 2 L of LB liquid medium containing 50 mg / mL ampicillin at an amount of 1%, Cultivate at 200rpm at 37°C until OD in the logarithmic phase of bacterial growth 600 =0.4~0.6. Add IPTG to make the final concentration 1mmol / L, culture at 160rpm at 37°C, and collect the bacterial liquid after 6h. Take 1 mL of expressing bacteria and centrifuge, add 30 μL of PBS to the pellet for suspension, and then add 30 μL of loading buf...

Embodiment 3

[0086] The identification of embodiment 3 recombinant bovine prion protein bPrP

[0087] After the recombinant bovine prion protein bPrP was successfully expressed, SDS-PAGE and Western-Blot tests were carried out to determine whether the target recombinant protein was obtained.

[0088] 1. SDS-PAGE detection: 20 microliters of repurified bovine prion protein bPrP was mixed evenly with the same volume of 2× loading buffer, boiled in boiling water for 10 minutes to fully denature the protein, and then centrifuged at 10,000 rpm for 1 minute. Put the prepared gel into the vertical electrophoresis tank according to the instructions, and pour an appropriate amount of SDS-PAGE electrophoresis buffer; carefully add protein markers and samples to the SDS-PAGE gel holes in order, 10 microliters / well, protein marker . Electrophoresis at a voltage of 90V for about 30min until the blue BPB in the sample is taken through the stacking gel, then the voltage is adjusted to 120V until the sam...

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Abstract

The invention provides a recombined bovine prion protein (bPrP), and a preparation method and an application thereof. The amino acid sequence of the bPrP protein is shown as SEQ ID NO. 2. The bPrP employs a bovine whole blood as a template, adopts nucleotide sequence shown as SEQ ID No. 3 and 4 as primers via a PCR method to obtain the bovine prion protein DNA sequence having enzyme cleavage sites, connects to plasmids of pPROEX-htb, removes redundant enzyme cleavage sites ahead of target genes in expression vectors via a site-directed mutagenesis to obtain the bPrP eukaryotic expression vectors. The recombined bovine prion protein with identical is obtained conformation with natural prion protein by expressing the vector in escherichia coli, purifying and renaturing through high affinity chromatographic column. The amino acid sequence of the bovine prion protein is remarkably similar to that of the natural bovine prion protein (only one glycine is added in N terminal). The bovine prion protein can be used for exploring a monoclonal antibody of the bovine prion protein, and for a diagnostic kit of the bovine prion diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant bovine prion protein bPrP and its encoding gene and preparation method, as well as the application of the recombinant expression vector containing the encoding gene and the recombinant bovine prion protein bPrP in the detection of prion disease . Background technique [0002] Prion disease, also known as transmissible spongiform encephalopathies (TSEs), is a fatal neurodegenerative disease characterized by brain vacuolation, neuronal death, and microglial proliferation. [0003] Prion protein is closely related to the occurrence of transmissible spongiform encephalopathy. Therefore, recombinant prion protein is essential for the research and development of diagnostic reagents and pathogenesis of this disease. The current method for obtaining recombinant prion protein is to construct a prion protein expression vector through genetic engineering technology, express the t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47
Inventor 杨利峰赵德明杨秀进王伊琴宋志琦周向梅尹晓敏赵化粉
Owner CHINA AGRI UNIV
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