Artificial optimization and synthesis method and application of beta-casein
A casein and protein technology, applied in the field of artificial milk, can solve the problems of high allergenicity of beta-casein, and achieve the effect of high application value
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Embodiment 1
[0044] Example 1 Deletion and structural optimization of milk protein allergens
[0045] The database obtains β-casein sequences of different species such as yak, buffalo, goat, and camel. The surface accessibility of milk proteins was analyzed by the Emini-SurfaceProbability method; the antigenic index amino acid regions were calculated by the Jameson-Wolf method.
[0046] The sequence similarity between isoallergenic milk proteins and allergens was compared by the whole sequence alignment method, and the structural motif composition and location of the allergens and conformational epitopes of yak, buffalo, goat and camel β-casein were compared , to obtain the conserved domain of β-casein allergens.
[0047] Structural analysis was performed in combination with the crystal data of β-casein in the Swissprot database of molecular biology. Based on multiple sequence alignment and phylogenetic analysis, the cross-recombination strategy of Schema homologous structural elements i...
Embodiment 2
[0049] Example 2 Efficient biosynthesis of high-quality β-casein in yeast
[0050] Codon-optimized DNA coding sequences for optimized milk proteins for yeast cell factory expression characteristics. The optimized milk protein encoding gene AF-CS2 was synthesized by chemical synthesis method.
[0051]The artificially optimized milk protein gene was added to both ends of the restriction endonuclease EcoRI and NotI sequences to connect the yeast expression vector pPIC9K-His multi-cloning site. The ligation products were heat-shocked into E. coli competent cells and coated with ampicillin-resistant LB plates to screen strains containing recombinant expression plasmids.
[0052] Use restriction endonuclease SacI to cut the correct positive recombinant expression vector, prepare linearized plasmid DNA, and transform it into Pichia GS115 by electroporation. Positive recombinant yeast strains were screened by Geneticin resistance plate and identified by PCR. The results showed that...
Embodiment 3
[0055] Example 3 ELISA sensitization analysis of optimized β-casein
[0056] Dilute milk protein samples to 5 μg mL with CBS solution -1 , 100 μL of each sample was placed in a 96-well plate and coated overnight at 4 °C.
[0057] After washing 4 times with Tween 20 / PBS (PBS-T), 350 μL of 5% nonfat dry milk was added and incubated at 37° C. for 1 h. Washed again, then BSA diluted allergen serum (1:500) was added and incubated for 3 h at room temperature. After washing, 100 μL of peroxidase-labeled goat anti-human / rat IgE antibody (1:5000) was added to the wells, and the plate was incubated for 1 h at room temperature.
[0058] After 5 washes with PBS-T and 3 washes with PBS, 200 μL of TMB solution was added to the wells for 30 min, followed by 50 μL of 2M H 2 SO 4 Terminate the reaction.
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