Patent ID: 11879154
Assignee: nan
Field: Biotechnology (Chemistry)
Classification: CPC C  Y | IPC C

Claim 0:
1. A method of preparing a superoxide dismutase with a transmembrane capability, comprising the following steps:
(1) extracting mRNA from Geobacillus stearothermophilus, synthesizing cDNA by a reverse transcription method, designing a specific primer sequence,
amplifying a large number of coding regions of the cDNA with the specific primer sequence to obtain a sequence encoding the superoxide dismutase from natural Geobacillus stearothermophilus, ligating the coding regions to an E. coli expression vector, and transforming the E. coli expression vector into engineering bacteria BL21 (DE3) to clone a sequence of the superoxide dismutase;
(2) ligating the sequence of the superoxide dismutase cloned in (1) to a prokaryotic expression vector and designing a primer for identifying a positive clone;
(3) obtaining an expression strain, wherein the superoxide dismutase cloned from the Geobacillus stearothermophilus has the amino acid sequence as follows (SEQ ID NO: 1):, MPFELPALPYPYDALEPHIDKETMNIHHTKHHNTYVTNLNAALEGHPDL

 

QNKSLEELLSNLEALPESIRTAVRNNGGGHANHSLFWTILSPNGGGEPT

 

GELADAINKKFGSFTAFKDEFSKAAAGRFGSGWAWLVVNNGELEITSTP

 

NQDSPIMEGKTPILGLDVWEHAYYLKYQNRRPEYIAAFWNVVNWDEVAK

 

RYSEAKAK;

(4) mutating aspartic acid at position 20 to glycine and mutating leucine at position 141 to asparagine; and
(5) coupling a cell penetrating peptide (CPP) sequence YGRKKRRQRRR (SEQ ID NO: 10) with a transmembrane capability on an HIV-1 peptide segment with the superoxide dismutase for expression to enable the superoxide dismutase to have the transmembrane capability, wherein the CPP sequence is a trans-activating transcriptional activator sequence.