Expression preparation method and application of novel anticoagulant Aedes albopictus salivary gland aegyptin-like protein ALP

A technology of Aedes albopictus and salivary glands, which is applied in the field of expression, preparation and application of the new anticoagulant Aedes albopictus salivary gland aegyptin-like protein ALP, which can solve the problem of inability to perform correct processing, folding and translation, and prolong prothrombin time Thrombin time Thromboplastin time, lack of anticoagulant activity, etc.

Inactive Publication Date: 2015-06-17
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0020] The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a method for preparing the expression of the aegyptin-like protein ALP from the salivary gland of Aedes albopictus, a novel anticoagulant. Correct processing, folding and post-translational modification, without defects such as anticoagulant activity, the method uses the pPICZαA vector Pichia

Method used

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  • Expression preparation method and application of novel anticoagulant Aedes albopictus salivary gland aegyptin-like protein ALP
  • Expression preparation method and application of novel anticoagulant Aedes albopictus salivary gland aegyptin-like protein ALP
  • Expression preparation method and application of novel anticoagulant Aedes albopictus salivary gland aegyptin-like protein ALP

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Experimental program
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Embodiment 1

[0112] Materials and methods used in this implementation can be as follows:

[0113] Material:

[0114] Plasmids and strains:

[0115] Yeast expression plasmid pPICZαA was purchased from Invitrogen Company of the United States, Pichia pastoris (pichia pastoris) X-33 strain, for example, can be obtained from the Department of Parasitology, Wenzhou Medical University.

[0116] Main reagents:

[0117] Bleomycin (Cat. No.: R25001) was purchased from Invitrogen, USA; Ni-NTA Agarose (Cat. No.: 30210) and corresponding purification column, Anti-6X His Antibody (Product No.: ab5000) and corresponding secondary antibodies were purchased from QIAGEN, USA; Yeast Genome Extraction Kit (Product No.: DP307) was purchased from Tiangen Biotechnology Co., Ltd.; APTT, PT, TT kits were purchased from Shanghai Sun Biotechnology Co., Ltd. Company); EcoR I, Xba I and Sca I enzymes were purchased from Fermantas, and heparin tablets: produced by Shanghai Xinyi Jiufu Pharmaceutical Co., Ltd. Othe...

Embodiment 2

[0175] A method for the expression and preparation of a novel anticoagulant Aedes albopictus salivary gland aegyptin-like protein ALP, the steps are as follows:

[0176] (1) Optimized synthesis of ALP mature peptide DNA

[0177] The synthesis was optimized according to the codon preference of Pichia pastoris, and the optimized synthetic ALP mature peptide gene sequence was:

[0178] gaattc AGGCCAATGCCAGAGGGTGAAGAGGGTGAAGGCGAGGAAGAATCCCCAGATGATGCCTCTGGAGACGAAACGGAAGGCGGAGAGGAAAAGACGGATGATGGTGCCAGTGAGGACGGTGGAGAAGAAGAATCCAAAGCAGACGAGGAAGATGGTGGTGAGAATGCTGATGGCGAAGACGCCGGGGGAGATGACGCCGGTGGCGAAAATGCAGATGGAGAAAACACCGATGGGGAAAACACTGACGGTGAGAACACCGACGGGGAGAACGCTGATGGAGAAGACGCCGAGGGTTCTAAGGACGAGGGAGACGATTCCGAAGGGGATGGAAGTAAGGAGGAATCCACAGGCGGTGATGAAGGAGGAGATAATGCCGGTGGCGGTGAAGGTGGGGGGGAAAATGACCCAGTTAATACTTACCACAAAGTAGTGGCTATTCTgGACAAAGATACAAAGGTTGATAACATTCAATCCGAGTATCTGAGATCTGCACTGAACAATGATTTACAGTCCGAAGTACGAAATCCAGTTGTGGAGGCTATTAGTCGATTGGGGTCCTTCAGTAAGATTGAAGGCTGTTTCAAGTCTATGGGATCTGAT...

Embodiment 3

[0225] A novel anticoagulant Aedes albopictus salivary gland aegyptin-like protein ALP expression preparation method, characterized in that: the steps are as follows:

[0226] (1) Optimized synthesis of ALP mature peptide DNA

[0227] The synthesis was optimized according to the codon preference of Pichia pastoris, and the optimized synthetic ALP mature peptide gene sequence was:

[0228] gaattc AGGCCAATGCCAGAGGGTGAAGAGGGTGAAGGCGAGGAAGAATCCCCAGATGATGCCTCTGGAGACGAAACGGAAGGCGGAGAGGAAAAGACGGATGATGGTGCCAGTGAGGACGGTGGAGAAGAAGAATCCAAAGCAGACGAGGAAGATGGTGGTGAGAATGCTGATGGCGAAGACGCCGGGGGAGATGACGCCGGTGGCGAAAATGCAGATGGAGAAAACACCGATGGGGAAAACACTGACGGTGAGAACACCGACGGGGAGAACGCTGATGGAGAAGACGCCGAGGGTTCTAAGGACGAGGGAGACGATTCCGAAGGGGATGGAAGTAAGGAGGAATCCACAGGCGGTGATGAAGGAGGAGATAATGCCGGTGGCGGTGAAGGTGGGGGGGAAAATGACCCAGTTAATACTTACCACAAAGTAGTGGCTATTCTgGACAAAGATACAAAGGTTGATAACATTCAATCCGAGTATCTGAGATCTGCACTGAACAATGATTTACAGTCCGAAGTACGAAATCCAGTTGTGGAGGCTATTAGTCGATTGGGGTCCTTCAGTAAGATTGAAGGCTGTTTCAAGTCTATGGGA...

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Abstract

The invention relates to an expression preparation method and application of a novel anticoagulant Aedes albopictus salivary gland aegyptin-like protein ALP. The method comprises the following step: 1, carrying out optimized synthesis of ALP mature peptide DNA; 2, constructing a recombinant plasmid pPICZalphaA-ALP; and 3, expressing and purifying a recombinant protein ALP. The method uses the ALP protein expressed by a pichia yeast expression system (pPICZalphaA-ALP X33) of a pPICZalphaA vector in order to realize correct secretion and expression, and the purified ALP protein has anticoagulation activity, can substantially prolong the prothrombin time (PT), the thrombin time (TT) and the activated partial thromboplastin time (APTT) in vitro, can be used as a novel anticoagulant, and lays a research foundation for research of the feasibility of the purified ALP protein as a novel anticoagulant for preventing and treating thrombotic diseases and other vascular diseases, so the method has positive social benefit and substantial economic benefit if the method is widely proved and clinically used.

Description

technical field [0001] The invention belongs to the field of biomedical technology, and relates to the optimized synthesis of ALP gene, the construction of recombinant expression plasmid pPICZαA-ALP, the construction of recombinant expression strain pPICZαA-ALP X33, the induced expression and purification of target protein ALP, and the anticoagulation of ALP protein in vitro In particular, the expression preparation method and application of a new type of anticoagulant Aedes albopictus salivary gland aegyptin-like protein ALP. Background technique [0002] ALP protein is Aedes albopictus salivary gland aegyptin-like protein (Aedes albopictus aegyptin-like protein, alALP), hereinafter referred to as ALP. [0003] 1. The treatment and prevention of vascular diseases such as thromboembolic diseases require new anticoagulants [0004] Anticoagulant drugs are widely used in the prevention and treatment of thromboembolic diseases, such as stroke, atrial fibrillation (AF), acute c...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/12C07K14/435C12R1/84
Inventor 梁韶晖李星潘刘文权张亮亮
Owner WENZHOU MEDICAL UNIV
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