Azurin as bacterial protein with wide-spectrum antitumor function and its use and medicinal composition
An anti-tumor effect, azurin technology, applied in the field of preparing anti-tumor drugs
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Embodiment 1
[0054] The separation and purification of the azurin of embodiment 1 natural bacterial source
[0055] Adopt saturated ammonium sulfate salting-out technology known in the art and SDS-polyacrylamide gel electrophoresis technology (Wang Jiazheng etc., editor-in-chief, "Protein Technology Handbook", Science Press), from the culture of Pseudomonas aeruginosa Isolation and purification of the natural bacterial protein azurin.
[0056] a) Inoculate Pseudomonas aeruginosa into 200 ml of LB culture solution according to the conventional method, and cultivate overnight (about 16 hours) at 37° C. with shaking.
[0057] b) Centrifuge at 10,000 rpm for 15 minutes, take the supernatant, add solid ammonium sulfate to 40% saturation, and ice-bath for 1 hour.
[0058] c) Centrifuge at 10,000 rpm for 15 minutes, take the supernatant, add solid ammonium sulfate to 80% saturation, and ice-bath for 1 hour.
[0059] d) Centrifuge at 10,000 rpm for 15 minutes, and discard the supernatant. Disso...
Embodiment 2
[0063] Example 2 Using the PCR method to prepare the polynucleotide sequence encoding the azurin protein of the present invention
[0064] The PCR technology known in the application field is used to isolate and clone the full-length sequence encoding the azurin protein, and the specific operation steps are as follows:
[0065] Design PCR primers:
[0066] Upstream primer: 5'-GCC CAA GCT TAC CTA GGA GGC TGCTCC ATG CTA-3' (SEQ ID NO: 4),
[0067] Downstream primer: 5'-TGA GCC CCT GCA GGC GCC CAT GAAAAA GCC CGG C-3' (SEQ ID NO: 5).
[0068] Genomic DNA was extracted from Pseudomonas aeruginosa by conventional methods and purified. Take 1 μg of purified genomic DNA as a template and add it to 50 μl of PCR reaction system. Add the above primers, dNTP, Taq enzyme, MgCl2, and PCR buffer in sequence.
[0069] PCR cycle parameters: 94°C 2'; 94°C 20" / 62°C 30" / 72°C 10'×35 cycles; 72°C 7'; 4°C.
[0070] 1% agarose gel electrophoresis was used to separate the DNA band with a size of ab...
Embodiment 3
[0073] Example 3 Preparation of broad-spectrum anti-tumor bacterial protein azurin by recombinant method
[0074] The full-length nucleotide sequence encoding azurin obtained in Example 2 above was constructed into a commercially available expression vector to express and purify the recombinant protein.
[0075] To facilitate purification, azurin was prokaryotically expressed in Escherichia coli in the form of 6XHIS fusion protein or GST fusion protein.
[0076] Construction of prokaryotic expression vector
[0077] According to the sequence provided by SEQ ID NO.1, PCR primers for amplifying the nucleic acid sequence encoding the complete bacterial protein azurin were designed, and after PCR amplification, the amplified DNA fragment was cloned into pQE30 or pGEX-T vector. Then, the calcium chloride method is used to transfer into suitable Escherichia coli, and the recombinants containing the bacterial protein azurin are screened and identified.
[0078] Isolation and Purifi...
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