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Method for recombinant expression of BCMA protein by E.coli cell-free protein expression system

A cell-free protein and expression system technology, applied in the direction of receptor/cell surface antigen/cell surface determinant, recombinant DNA technology, microbial-based methods, etc., to achieve high solubility, increase expression, and strong controllability

Inactive Publication Date: 2022-03-15
杭州迅耀生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

BCMA is easily detected in delayed and frozen MM samples, study shows

Method used

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  • Method for recombinant expression of BCMA protein by E.coli cell-free protein expression system
  • Method for recombinant expression of BCMA protein by E.coli cell-free protein expression system
  • Method for recombinant expression of BCMA protein by E.coli cell-free protein expression system

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Embodiment 1

[0044] In this embodiment, a method for recombinantly expressing BCMA protein using the E.coli cell-free protein expression system comprises the following steps:

[0045] (1) Amplify the BCMA protein coding gene;

[0046] First, use the pET-11a(+)-BCMA plasmid (provided by Qingke Biotechnology, the full length is 582bp, and the nucleotide sequence is shown in SEQ ID No.1) as a template, and use the Z1 or Z2 high-fidelity DNA polymerase PCR kit ( Purchased from Hangzhou Jinao Biotechnology Co., Ltd.) to amplify the coding gene sequence of BCMA protein; the sequence of the amplification primer is as follows:

[0047] Upstream primer: 5'-TAATACGACTCACTATAGG-3' (as shown in SEQ ID No.2);

[0048] Downstream primer: 5'-CCAAGGGGTTATGCTAG-3' (as shown in SEQ ID No.3).

[0049]The composition of the 50 μL PCR amplification system is: 48 μL PCR reaction master mix, 1 μL Z1 high-fidelity DNA polymerase, and 1 μL plasmid template.

[0050] The PCR reaction program was: pre-denaturatio...

Embodiment 2

[0062] A method for recombinantly expressing BCMA protein using E.coli cell-free protein expression system, comprising the following steps:

[0063] (1) Preparation of DNA / PEI@PLGA nanoparticles by layer-by-layer self-assembly method;

[0064] Specifically, the same method as in Example 1 was used to amplify the coding DNA of the BCMA protein, and then the PCR product was used to prepare DNA / PEI@PLGA nanoparticles. The preparation method of the nanoparticles was as follows:

[0065] (a) Adding the PLGA nano-core to the polyethyleneimine solution, so that the polyethyleneimine layer is self-assembled on the PLGA nano-core, and then centrifuged and freeze-dried to obtain PEI@PLGA nanoparticles;

[0066] Wherein, the concentration of polyethyleneimine solution (polyethyleneimine is purchased from American sigma, Mr 25000) is 1mg / mL, the average particle diameter of PLGA nano-core is 200nm, and the addition amount of PLGA nano-core is 10mg / mL;

[0067] (b) PEI@PLGA nanoparticles ...

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Abstract

The invention discloses a method for recombinant expression of BCMA protein by using an E.coli cell-free protein expression system. The method comprises the following steps: (1) amplifying coding DNA of the BCMA protein; and (2) adding the amplification product into an E.coli cell-free protein expression system to synthesize the BCMA protein. An E.coli cell-free protein expression system is adopted to recombine and express the BCMA protein outside cells, the obtained BCMA is high in solubility, and the controllability of the protein expression process is high.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for recombinantly expressing BCMA protein using an E.coli cell-free protein expression system. Background technique [0002] Multiple myeloma (MM) is the second most common hematologic malignancy after non-Hodgkin's lymphoma, originating in the B-cell lineage due to the abnormal accumulation of immunoglobulin-producing plasma cells that develop into osteogenic lesion. In recent years, the incidence of multiple myeloma has also been increasing, and it is still incurable. [0003] In the past 20 years, due to the continuous progress of traditional therapy and new therapy (single or combined transplantation) in the treatment of MM, the effect of MM treatment has been improved and the survival period of patients has been prolonged. For example, the protease inhibitor bortezomib had a significant effect in relapsed and newly diagnosed MM; the second-generation prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C12N15/70C12P21/02C12R1/19
CPCC07K14/70578C12N15/70C12P21/02
Inventor 王尧姜乐乐林森唐宏峰陈志玲卢彩婷徐涵瑜王芳王再花唐亚杰吴银荣刘夏月蔡海莺
Owner 杭州迅耀生物科技有限公司
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