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TMPRSS2 mutant protein, expression carrier and expression engineering bacterium thereof, preparation method of expression carrier of TMPRSS2 mutant protein, and preparation method of expression engineering bacterium of TMPRSS2 mutant protein

A protein expression and expression carrier technology, applied in biochemical equipment and methods, carriers, nucleic acid carriers, etc., can solve the problems of high protein difficulty and low protein purity

Pending Publication Date: 2021-03-23
CUSABIO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, it is relatively difficult to obtain high-purity and high-activity proteins, so the wild-type protein has self-cleavage, and the protein purity is not high

Method used

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  • TMPRSS2 mutant protein, expression carrier and expression engineering bacterium thereof, preparation method of expression carrier of TMPRSS2 mutant protein, and preparation method of expression engineering bacterium of TMPRSS2 mutant protein
  • TMPRSS2 mutant protein, expression carrier and expression engineering bacterium thereof, preparation method of expression carrier of TMPRSS2 mutant protein, and preparation method of expression engineering bacterium of TMPRSS2 mutant protein
  • TMPRSS2 mutant protein, expression carrier and expression engineering bacterium thereof, preparation method of expression carrier of TMPRSS2 mutant protein, and preparation method of expression engineering bacterium of TMPRSS2 mutant protein

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preparation example Construction

[0060] According to another typical implementation of the embodiments of the present invention, a method for preparing the expression vector of the TMPRSS2 mutant protein is provided, the method comprising:

[0061] Obtaining the optimized codon fragment of the TMPRSS2 mutant protein (specifically including: synthesizing the carrier containing the optimized codon fragment of the TMPRSS2 mutant protein, using the pSecTag2A vector as a template, using the primer pair shown in SEQ ID NO.4-5 Perform PCR to obtain an optimized codon fragment of the TMPRSS2 mutant protein), the nucleotide sequence of the optimized codon fragment is shown in SEQ ID NO.3;

[0062] Obtain an expression vector, insert the optimized codon fragment of the TMPRSS2 mutant protein into the expression region of the expression vector, and obtain the TMPRSS2 mutant protein expression vector; specifically include:

[0063] The pSecTag2A vector is double-digested with Hind III / Not I to obtain the restriction vect...

Embodiment 1

[0075] Embodiment 1TMPRSS2 plasmid preparation

[0076] 1. TMPRSS2 codon optimization

[0077] According to NCBI code number (O15393), the nucleic acid sequence of TMPRSS2 is selected as shown in SEQ ID NO.1, and the encoded amino acid is shown in SEQ ID NO.2.

[0078] Considering that different expression systems have different preferences for codons when expressing proteins, the applicant optimized the above codons, and the nucleotide sequence of the optimized gene fragment is shown in SEQ ID NO.3. The amino acid sequence encoded by the nucleotide sequence of the optimized gene fragment is shown in SEQ ID NO.2.

[0079] 2. Obtaining the TMPRSS2 gene fragment

[0080] The optimized gene fragments were synthesized by Wuhan Jinkairui Bioengineering Co., Ltd. Primers were designed according to the optimized nucleic acid sequence of TMPRSS2, and the primers are shown in Table 1.

[0081] Table 1

[0082]

[0083]

[0084] Using the synthesized optimized gene fragment a...

Embodiment 2

[0106] Example 2 Expression of TMPRSS2 mutant protein in mammalian cells and protein nickel column purification

[0107] 1. Mammalian cell expression

[0108] (1) Transfer the positive monoclonal bacterial liquid to 300ml LB medium to cultivate the large-scale plasmid overnight;

[0109] (2) Prepare two 15ml sterile centrifuge tubes, add 5ml transfection reagent and 100μg sterile plasmid DNA to one of them, and gently blow and mix;

[0110] (3) Standing at room temperature for 10 minutes to prepare the plasmid-carrier complex;

[0111] (4) Take out the 293T cells from the constant temperature shaker, add the prepared plasmid-carrier complex while shaking, and return to 5% CO 2 , Shaking culture in a constant temperature shaker at 37°C.

[0112] (5) Shake the flask for 5 to 7 days, harvest the supernatant, and take the secretory supernatant and intracellular supernatant for WB his tag detection to determine whether the protein is expressed. The results are shown in Figure ...

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Abstract

The embodiment of the invention provides TMPRSS2 mutant protein, an expression carrier and an expression engineering bacterium thereof, and a preparation method of the expression carrier of the TMPRSS2 mutant protein, and the preparation method of the expression engineering bacterium of the TMPRSS2 mutant protein. The amino acid sequence of the above TMPRSS2 mutant protein is disclosed by SEQ ID NO.7; the nucleotide sequence of the expression zone of the expression carrier comprises a nucleotide sequence disclosed by SEQ ID NO.3; and the engineering bacterium comprises the expression carrier of the TMPRSS2 mutant protein. Wild TMPRSS2 protein can carry out self cutting between Arg255 and Ile256 so as to carry out active form conversion, the wild TMPRSS2 protein has self shearing, and protein purity is low. By use of the embodiment of the invention, through point mutation, the TMPRSS2 protein mutant capable of carrying out lactation cell expression is realized, expression and purification are successfully carried out, and the TMPRSS2 high-purity and high-activity protein is obtained.

Description

technical field [0001] The embodiments of the present invention belong to the field of biotechnology, and relate to a TMPRSS2 mutant protein and its expression vector, expression engineering bacteria and preparation method. Background technique [0002] TMPRSS2 is one of the members of type II transmembrane serine proteases, a family of proteins with conserved serine protease domains located on the cell membrane. The basic structure of this family is highly similar, and they all contain four parts. From N-terminal to C-terminal, they are short cytoplasmic domain, transmembrane domain, backbone region and serine protease domain. The protease catalytic domain includes His296, Asp345 and Ser441 amino acid residues. It has a single transmembrane domain, in which the backbone region and the serine protease domain are located outside the cell, and the differences between different members are mainly concentrated in the backbone region. The C-terminal protease domain is extracell...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12N9/64C12N1/21C12Q1/37G01N21/64C12R1/19
CPCC12N15/70C12N15/66C12N9/6424C12Y304/21109C12Q1/37G01N21/6428C12N2800/22
Inventor 殷婷子张振易汪雪于倩王静廖怡辉王营袁文文
Owner CUSABIO TECH LLC
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