Method for preparing human laryngeal squamous cell carcinoma related gene SKA3 eukaryotic expression protein monomer

A squamous cell carcinoma, eukaryotic expression vector technology, applied in the fields of biotechnology and molecular biology, can solve problems such as unfavorable protein purification

Inactive Publication Date: 2019-05-17
FIRST HOSPITAL OF SHANXI MEDICAL UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the protein expressed by the E.coli expression system needs to be removed from the endot...

Method used

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  • Method for preparing human laryngeal squamous cell carcinoma related gene SKA3 eukaryotic expression protein monomer
  • Method for preparing human laryngeal squamous cell carcinoma related gene SKA3 eukaryotic expression protein monomer
  • Method for preparing human laryngeal squamous cell carcinoma related gene SKA3 eukaryotic expression protein monomer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] A method for preparing a human laryngeal squamous cell carcinoma related gene SKA3 eukaryotic expression protein monomer, comprising the following steps:

[0062] First provide the source or preparation method of the plasmid and main reagents:

[0063] PGEX-4T-1 vector, pCMV-HA vector and cDNA of laryngeal squamous cell carcinoma Hep-2.

[0064] DH5α competent cells (Beijing Quanshijin Biotechnology Company), Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific Company), EcoR I, Xho I and Not I restriction endonucleases (NEB Company), EasyPfu DNA Polymerase (Beijing Quanshijin Shijin Biotechnology Co., Ltd.), Solution I Ligase Reagent (TaKaRa, Gel Recovery Kit (Sangon Bioengineering (Shanghai) Co., Ltd.), Plasmid Extraction Kit (Shanghai Gemma Pharmaceutical Technology Co., Ltd.), GST Fusion Protein Purification kit (Gentry Biotechnology), etc.

[0065] (1) Construction and verification of vector PGEX-4T-1-SKA3

[0066] (1) Design and synthesize primers ...

Embodiment 2

[0094] A method for preparing a human laryngeal squamous cell carcinoma related gene SKA3 eukaryotic expression protein monomer, comprising the following steps:

[0095] The sources or preparation methods of the plasmids and main reagents provided in this example are the same as those in Example 1.

[0096] (1) Construction and verification of vector PGEX-4T-1-SKA3

[0097] (1) Design and synthesize primers according to the sequence of human laryngeal squamous cell carcinoma related gene SKA3

[0098] (a) According to the sequence information of the human laryngeal squamous cell carcinoma-related gene SKA3 in the NCBI database, such as SEQ.ID.NO.1, the sequence information of SEQ.ID.NO.1 is shown in the sequence information table, designed and synthesized using the software Primer Premier 5 Primers, synthetic primers GEX-4T-SKA3-F-EcoRI:5'-CCGGAATTCATGGACCCTATCCGGAGCTT-3', GEX-4T(CMV-HA)-SKA3-R-NotI:5'-CGAGCGGCCGCTCAGTTTTCTTTGTTGCTGA-3'.

[0099] (b) In order to facilitate i...

Embodiment 3

[0125] A method for preparing a human laryngeal squamous cell carcinoma related gene SKA3 eukaryotic expression protein monomer, comprising the following steps:

[0126] The sources or preparation methods of the plasmids and main reagents provided in this example are the same as those in Example 1.

[0127] (1) Construction and verification of vector PGEX-4T-1-SKA3

[0128] (1) Design and synthesize primers according to the sequence of human laryngeal squamous cell carcinoma related gene SKA3

[0129] (a) According to the sequence information of the human laryngeal squamous cell carcinoma-related gene SKA3 in the NCBI database, such as SEQ.ID.NO.1, the sequence information of SEQ.ID.NO.1 is shown in the sequence information table, designed and synthesized using the software Primer Premier 5 Primers, synthetic primers GEX-4T-SKA3-F-EcoRI:5'-CCGGAATTCATGGACCCTATCCGGAGCTT-3', GEX-4T(CMV-HA)-SKA3-R-NotI:5'-CGAGCGGCCGCTCAGTTTTCTTTGTTGCTGA-3'.

[0130] (b) In order to facilitate i...

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Abstract

The invention provides a method for preparing a human laryngeal squamous cell carcinoma related gene SKA3 eukaryotic expression protein monomer. The method includes inserting a human laryngeal squamous cell carcinoma related gene SKA3 into a PGEX-4T-1 carrier to obtain a PGEX-4T-1-SKA3 prokaryotic expression carrier; obtaining a fusion fragment GST-SKA3 PCR and inserting the segment into a pCMV-HAcarrier to obtain an SKA3 eukaryotic expression carrier pCMV-HA-GST-SKA3 with HA and GST labels; cultivating a laryngeal squamous carcinoma cell Hep-2 transfected with the eukaryotic expression and then conducting purification to obtain a recombinant GST fusion protein. The method specifically includes the following steps: (1) construction and verification of the prokaryotic expression carrier PGEX-4T-1-SKA3; (2) construction and verification of the eukaryotic expression carrier pCMV-HA-GST-SKA3; (3) eukaryotic expression and purification in laryngeal squamous cell carcinoma Hep-2 cells. TheGST label can increase the solubility and the expression quantity of the protein expression. The eukaryotic expression carrier pCMV-HA is utilized to expresses the protein and forms a stable disulfidebond during the expression to correctly fold the expressed protein and perform complex glycosylation modification on the expressed protein. The protein activity is close to the native protein, and the endotoxin is not required to be removed.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and molecular biology, in particular to a method for preparing a human laryngeal squamous cell carcinoma related gene SKA3 eukaryotic expression protein monomer. Background technique [0002] Laryngeal cancer is a common malignant tumor of the head and neck, among which squamous cell carcinoma of the larynx ranks second in the malignant tumors of the head and neck, and is highly prevalent in northern my country. Laryngeal squamous cell carcinoma is prone to cervical lymph node metastasis in the early stage, and has the malignant characteristics of invasion, which is the main risk factor for postoperative recurrence and poor prognosis. The up-regulation of human laryngeal squamous cell carcinoma-related gene SKA3 expression is closely related to the occurrence and development of laryngeal squamous cell carcinoma, and has different effects on tumor cell proliferation, invasion and metastasis....

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/85C07K19/00
Inventor 刘红亮吴勇延高伟王斌全张宇良牛敏薛绪亭郑希望
Owner FIRST HOSPITAL OF SHANXI MEDICAL UNIV
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