BLyS antagonistic peptide SS12, fusion protein SS12-Fc containing antagonistic peptide and gene
A fusion protein, SS12 technology, applied in the field of biomedicine, can solve the problems of short half-life, low efficiency, unstable polypeptide and so on
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Embodiment 1
[0057] The structural information of the interaction between BLyS and its receptors and the design of new targeted BLyS antagonistic peptides
[0058] Computer-aided molecular docking and dynamics simulation were used to explore the interaction mode of BLyS and its receptor, and the key amino acid residues that BLyS and its receptor recognize each other were analyzed with the aid of computer graphics and distance geometry.
[0059] Through computer-aided molecular design and high-throughput virtual screening technology, a short peptide that can simulate the key amino acid conformation of the receptor is designed from scratch, and then the role of BLyS and antagonistic peptide can be theoretically evaluated with the help of de novo construction, molecular docking, and dynamic simulation; computer graphics To evaluate the potential biological effects of antagonistic peptides by scientific technology, distance geometry and intermolecular hydrogen bonding. After theoretical screening, ...
Embodiment 2
[0061] The plasmids SS12-Fc-pET30a and 3SS12-Fc-pET30a containing SS12-Fc and 3SS12-Fc fusion protein genes were constructed respectively ( figure 1 ).
[0062] 2.1 The gene encoding the BLyS antagonistic peptide SS12 was constructed using the following method: According to the amino acid sequence of the antagonistic peptide SS12, we designed two DNA primers encoding short peptides and ordered them to synthesize them through the company.
[0063] The upstream primer of SS12 is shown in SEQ ID NO.5, and the downstream primer of SS12 is shown in SEQ ID NO.6
[0064] The synthesized DNA primers of SS12 were dissolved by adding 200μl of 10mM Tris-Hcl (pH8.5) (final concentration of 16pmol / μl). Take 20μl of the positive and negative strands of the SS12 gene, mix them in an EP tube, boil in a water bath at 100°C for 3 minutes, and gradually cool to room temperature. Use 2% agarose gel electrophoresis to observe the annealing results. The result of electrophoresis showed that there is a c...
Embodiment 3
[0078] Induction and expression of SS12-Fc and 3SS12-Fc fusion proteins
[0079] The correctly sequenced recombinant plasmids SS12-Fc-pET30a and 3SS12-Fc-pET30a were respectively transformed into the expression host strain E. Coli. BL21 for high expression strain screening, and it was found that the concentration of IPTG was 0.5 mM, and it was shaken slowly at 16°C. Under the conditions of induction, the target protein can be induced. Highly expressing strains were selected for large-scale expression, purified by protein A gel affinity chromatography column, collected the eluate, dialyzed in 1×PBS, and measured with a UV spectrophotometer to calculate the protein concentration of SS12-Fc as 700ng / μl, 3SS12 -Fc protein concentration is 1400ng / μl. After dialysis, the target protein was subjected to SDS-PAGE electrophoresis. It was found that the sizes of SS12-Fc and 3SS12-Fc proteins were 25.4kDa and 28.6kDa, respectively, which were the same size as SS12-Fc and 3SS12-Fc, and had ...
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