Recombinant sheep prion protein oPrP and preparation method and application thereof

A technology of sheep prion protein and protease, applied in the field of recombinant sheep prion protein oPrP and its coding gene and preparation, recombinant expression vector and recombinant sheep prion protein oPrP in the detection of prion diseases, which can solve the problem that cannot meet the requirements of precise amino acid positions Research work and other issues to achieve good specificity and immunoreactivity

Inactive Publication Date: 2014-11-05
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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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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  • Recombinant sheep prion protein oPrP and preparation method and application thereof
  • Recombinant sheep prion protein oPrP and preparation method and application thereof
  • Recombinant sheep prion protein oPrP and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of recombinant expression vector pPROEX-htb-oPrP

[0055] 1. PCR amplification of sheep prion protein polypeptide gene (oPrPc gene)

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

[0057] Template: sheep whole blood (collected from a healthy flock in Inner Mongolia Autonomous Region) 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: pre-denaturation at 98°C...

Embodiment 2

[0077]Example 2 Expression, purification and renaturation of recombinant ovine prion protein oPrP

[0078] 1. Induced expression of sheep prion protein

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

[0080] Inoculate the constructed ovine 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 37°C and 160rpm, 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 load...

Embodiment 3

[0086] Example 3 Identification of recombinant ovine prion protein oPrP

[0087] After successful expression of recombinant ovine prion protein oPrP, 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 the repurified sheep prion protein oPrP 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 sample band mov...

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Abstract

The invention provides a recombinant sheep prion protein oPrP and a preparation method and application thereof. Amino acid sequences of the protein oPrP are shown in SEQ ID No.2. The preparation method includes: using a whole-blood sheep gene as a template, obtaining a sheep prion protein DNA (deoxyribonucleic acid) sequence with enzyme cutting sites by using nucleotide sequences shown in SEQ ID No 3 and 4 as primers and by a PCR (polymerase chain reaction) method, connecting the DNA sequence onto pPROEX-htb plasmids, and removing redundant enzyme cutting sites before a target gene in an expression vector by site-directed mutagenesis, so that an oPrP entire expression vector is obtained; and expressing the vector in escherichia coli, and purifying and renaturing by an overhigh affinity chromatographic column so that the recombinant sheep prion protein is obtained. The amino acid sequences of the obtained recombinant sheep prion protein are highly similar to those of a natural sheep prion protein (only an N terminal comprises one more glycine) and can be used for preparing sheep prion protein monoclonal antibodies and research and development of scrapie diagnostic reagent kits.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a recombinant ovine prion protein oPrP and its encoding gene and preparation method, as well as the application of the recombinant expression vector containing the encoding gene and the recombinant ovine prion protein oPrP in the detection of prion diseases . 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, expres...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C07K1/16C12N15/12C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/66G01N33/68
Inventor 杨利峰赵德明杨秀进王伊琴宋志琦周向梅尹晓敏李丽好
Owner CHINA AGRI UNIV
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