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Construction method, expression method and purification method of eukaryotic expression vector of human PAK1 protein

A technology for eukaryotic expression vectors and construction methods, which is applied in the field of expression and purification, and the construction of eukaryotic expression vectors. It can solve the problems of poor antibody specificity, low sensitivity, and poor specificity in recognizing natural PAK1 proteins, and achieve high specificity and sensitivity. High, high protein purity effect

Pending Publication Date: 2022-03-01
浙江格物致知生物科技有限公司
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Problems solved by technology

[0003] At present, there are many methods for the recombination and expression of human PAK1 protein, but all of them are expressed in the E. coli system. Another fact is that most of the PAK1 protein monoclonal antibodies currently available on the market have poor specificity when establishing a PAK1 protein detection method. , the disadvantage of low sensitivity
We believe that in the process of protein expression using the prokaryotic system, due to the lack of the same expression environment as the natural PAK1 protein, the structure of the recombinant PAK1 protein is different from the natural PAK1 protein, resulting in poor specificity and poor specificity of the prepared monoclonal antibody in recognizing the natural PAK1 protein. The disadvantage of low sensitivity

Method used

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Examples

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Embodiment 1

[0038] Construction of embodiment 1 PAK1 protein eukaryotic expression vector and its expression and purification

[0039] 1. Construction of PAK1 protein eukaryotic expression vector

[0040] Design amplification primers according to the sequence of PAK1 protein:

[0041] Upstream primer PAK1-F (SEQ ID NO.1): 5'-AGCTCAACTATGATCTTTTT-3',

[0042] Downstream primer PAK1-R (SEQ ID NO.2): 5'-GAATATTTTTCATCTCCTTTT-3'.

[0043] Among them, a BamHI restriction site was introduced into the upstream primer, and an EcoRI restriction site and a 6×histidine tag were introduced into the downstream primer.

[0044] Using HELA cells as a template, PAK1-F / PAK1-R was amplified by PCR with primers. The reaction system was: PCR reaction MIX 25 μl, forward and reverse primers with a concentration of 10 μM each 1 μl, DNA template 2 μl, and ddH 2 0 to 50 μl. The PCR amplification program is: 95°C for 180 seconds; 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, 35 cycles; finally...

Embodiment 2

[0061] 1. Molecular cloning of PAK1 gene

[0062] Using HELA cells as a template, PAK1-F / PAK1-R was amplified by PCR using the primers in Example 1. The reaction system was: 25 μl of PCR reaction MIX, 1 μl of forward and reverse primers with a concentration of 10 μM, 2 μl of DNA template, ddH 2 0 to 50 μl. The PCR amplification program is: 95°C for 180 seconds; 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, 35 cycles; finally 72°C for 300 seconds.

[0063] After PCR amplification, perform agarose gel electrophoresis detection, use the gel recovery kit to recover and purify the PCR amplification products from the gel, and establish the following ligation reaction according to the pGEM-TEasy vector system product manual: 10×T4 DNA ligation reaction buffer 1 μl, 1μl (50ng) of pGEM-T vector, 3μl of purified PCR amplified fragment, 1μl (3U) of T4 DNA ligase, add ddH 2 O to a total volume of 10 μl, ligated at 16°C for 2 hours to obtain ligation reactions.

[0064...

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Abstract

The invention discloses a construction method of an eukaryotic expression vector of human PAK1 protein, which comprises the following steps: 1) taking a gene sequence for expressing the PAK1 protein as a template, and adopting PCR (Polymerase Chain Reaction) amplification to obtain a PAK1 gene segment; 2) connecting the obtained PAK1 gene segment to an eukaryotic expression vector to obtain a recombinant plasmid; and (3) carrying out transfection, transformation and duplication on the obtained recombinant plasmid to obtain the eukaryotic expression vector of the human PAK1 protein. The invention also discloses a method for expressing the recombinant human PAK1 protein by using the eukaryotic expression system. The method also comprises the step (4) of transfecting plasmids obtained by amplifying and purifying the eukaryotic expression vector of the human PAK1 protein obtained in the step (3) into eukaryotic cells for expression. The method further comprises the following steps: (5) culturing the positive clone obtained in the step (4) to enlarge a system, collecting cultured cells, centrifugally collecting supernatant, and purifying through a chromatographic column to obtain the recombinant human PAK1 protein.

Description

technical field [0001] The invention relates to the technical field of molecular biology, relates to recombinant protein eukaryotic expression technology, and in particular relates to a construction method, expression and purification method of a eukaryotic expression vector of human PAK1 protein. Background technique [0002] Protein kinases (PK for short) are enzymes that catalyze the phosphorylation of proteins. The phosphorylation process of proteins is the last link in the transmission of nerve information in cells, leading to state changes of ion channel proteins and channel gates. It is an extremely important part of signaling pathways in the body, and there are many types in nerve cells. Therefore, the activity of protein kinases is regulated in many ways, one of which is the activation of autophosphorylation. The PAK (p21-activated kinase) protein family plays an important role in a variety of cellular processes and is also subject to very fine regulation; PAK1 is ...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/54C12N9/12C07K1/22C12N15/85C12N5/10
CPCC12N15/66C12N9/1205C12Y207/01037C12N15/85C12N5/0682C12N2510/02
Inventor 张浩洋石坚徐爱华朱国方
Owner 浙江格物致知生物科技有限公司
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