Human alpha-defensin 5 antiviral active mutant polypeptide and preparation method and application
A technology of antiviral activity and mutants, which is applied to the preparation method of peptides, antiviral agents, botanical equipment and methods, etc., can solve the problems of defensin antibacterial and antiviral activity decline, and achieve the goal of improving antiviral ability Effect
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Embodiment 1
[0071] Embodiment 1 Solid-phase chemical synthesis of HD5 mutant polypeptide:
[0072] 1. According to the amino acid sequence of the HD5 mutant, use a fully automatic peptide synthesizer (433A, Applied Biosystem) to entrust Shanghai Sangon Bioengineering Company to synthesize the HD5 mutant polypeptide.
[0073] 2. Purify the synthesized HD5 mutant polypeptide by HPLC reverse phase C18 column chromatography and desalting.
[0074] 3. Use the same method to synthesize and purify the maternal HD5 polypeptide reference substance.
[0075] 4. For the purified HD5 mutant polypeptide, use high pressure liquid chromatography (HPLC) method for purity identification, apply matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) to determine its molecular weight, and determine its molecular weight by isoelectric focusing electrophoresis. The isoelectric point was determined by an automatic amino acid sequencer.
[0076] 5. The results of HPLC showed t...
Embodiment 2
[0077] Example 2 Expression and purification of recombinant HD5 mutant polypeptide in Pichia pastoris
[0078] 1. Synthesize the full-length gene encoding the HD5 mutant. Entrusted Shanghai Sangon Bioengineering Company to synthesize the full-length gene encoding the HD5 mutant, the gene sequence is as follows:
[0079] GCC ACC TGC TAT TGC CGA ACC GGC CGT TGT GCT ACC CGT GAG TCC CTC TCCGGG GTG TGT ATC AGT GGC CGC CTC TAC AGA CTC TGC TGT CGC
[0080] Note: The amino acid encoded by the three bases in bold is the mutated arginine, and the three bases in the box are encoded by the stop codon.
[0081] 2. Construction of recombinant HD5 mutant yeast expression vector
[0082] (1) PCR primer design: In order to ensure that there is no redundant amino acid at the N-terminal of the recombinant HD5 mutant expressed in yeast, the gene sequence encoding the HD5 mutant was directly inserted into the STE of the yeast expression vector pPIC9K using Red / ET recombination technology 13...
Embodiment 3
[0091] Example 3 Cytotoxicity assay of HD5 mutant polypeptide
[0092] Inoculate vero cells (African green monkey kidney cells) in a 96-well plate with a seeding density of 2000 cells / well and a seeding volume of 100 μl. The vero cells were cultured in 1640 culture medium containing 10% fetal bovine serum for 24 hours until the cells were completely attached. The culture medium was discarded, and the culture medium containing different concentrations of HD5 mutant polypeptides was replaced to continue the culture. Each concentration was repeated for 5 wells, and a maternal HD5 polypeptide control group and a blank control group were set at the same time. After culturing for 72 hours, add 10 μl of CCK-8 reaction solution to each well, continue culturing at 37°C for 4 hours, discard the supernatant gently, shake and mix well, and measure the absorbance value (A value) at 490 nm wavelength with a microplate reader. Cell survival rate (%)=A value of the drug group / A value of the b...
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