an antibacterial protein
An antibacterial protein and strain technology, applied in the field of biochemistry, can solve the problems of inconsistent raw material quality and chemical residues.
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Embodiment 1
[0053] Example 1: LYS-ABP gene acquisition
[0054] Design the polypeptide sequence of LYS-ABP gene according to Genbank, and then design the LYS-ABP gene sequence according to the principle of optimal password, synthesize the LYS-ABP fusion protein gene sequence through the whole gene, and add EcoRI-HindIII enzyme digestion at both ends site, and the gene sequence is shown in SEQ ID NO:4.
Embodiment 2
[0055] Embodiment 2: the preparation of antimicrobial protein MBP-LYS-ABP
[0056] 1. Construction and transformation of recombinant plasmid pMAL-p2x / LYS-ABP
[0057] The LYS-ABP fusion protein gene fragment synthesized by the whole gene is double-cut with EcoRI-HindIII, and recovered as the fragment to be inserted.
[0058] The expression vector pMAL-p2x was purchased from NEB Company. The pMAL-p2x plasmid was double-cut with EcoRI-HindIII and the large fragment was recovered as a vector. It was ligated with the above-mentioned LYS-ABP fragment with T4 ligase, and then CaCl 2 Transformed into E.ColiDH5a, the specific steps were carried out according to the method in the "Molecular Cloning" manual. The positive clones were screened by enzyme digestion, and those with a band of about 490 bp were plasmids inserted into the target gene, named pMAL-p2x / LYS-ABP.
[0059] The screened recombinant plasmid was transformed into expression strain BL21(DE3)pLysS to obtain recombinant e...
Embodiment 3
[0067] Embodiment 3: the preparation of antibacterial protein LYS-ABP
[0068]Prepare 25mMpH8.0 Tris-HCl buffer solution, add Lys-ABP pure product containing MBP into the solution, the final protein concentration is 1 mg per ml, add appropriate amount of thrombin, place at 25°C for 5 hours, and cut to obtain Lys-ABP and MBP The mixture was stored in a refrigerator at 4°C for later use.
[0069] Ion exchange chromatography purification: chromatography medium: QSepharoseFastFlow; chromatography column specifications: inner diameter 50cm, height 40cm, medium height about 10cm, medium volume about 200mL; monitoring: 280nm; equilibrium liquid is 25mM pH8.0 Tris-HCl buffer. First, equilibrate the chromatographic column with more than 5 column volumes with the balance solution, and the flow rate is 20mL / min, then dilute the DNA-digested bacterial liquid sample 3 times with the balance solution, and then load the sample, the flow rate is 20mL / min, after loading the sample, continue E...
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