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.
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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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