1.1 Plasmid transformation
For the purpose of delivering the expressed urate oxidase into the blood, four secretion signal peptide sequences (PhoA ,which is an exocrine signal of the outer membrane protein alkaline phosphatase of E. coli; OpmA, which is an exocrine signal of the outer membrane protein of E. coli; STiI, which is an exocrine signal of the outer membrane protein heat stable enterotoxin II Stb of E. coli; PeLB, which can directs the protein to the bacterial periplasm), and four cell penetrating peptide sequences (DNP, which is a cyclic heptapeptide DNP that can be used as a permeation signal for Caco-2 cells; TAT peptide, which is derived from the transactivator of transcription of human immunodeficiency virus and is a cell-penetrating peptides; R8 (Octaarginine) , which is a representative cell-penetrating peptide which is a synthetic sequence; Pep-1, called HIV-reverse transcriptase/SV40 T-antigen) were chosen and fused expression with the target gene, respectively, to evaluate their ability.
Five DNA fragments we designed and constructed, which include four signal peptide with four cell penetrating peptidefused GFP encoded genes and urate oxidase encoded gene, were inserted into the multiple cloning site of the pET-22b plasmid, and then were introduced into the BL21 strain, and were selected on the basis of Amp resistance . To ensure that we introduced the plasmid into the BL21 strain, we performed colony PCR on colonies grown on solid media.
Figure 1 Results of agarose gel electrophoresis of colony PCR
Figure 2 Results of agarose gel electrophoresis of enzyme digestion
1.2 Testing Signal Peptide
For the purpose of delivering the expressed urate oxidase into the blood, four secretion signal peptide sequences (PhoA ,which is an exocrine signal of the outer membrane protein alkaline phosphatase of E. coli; OpmA, which is an exocrine signal of the outer membrane protein of E. coli; STiI, which is an exocrine signal of the outer membrane protein heat stable enterotoxin II Stb of E. coli; PeLB, which can directs the protein to the bacterial periplasm), and four cell penetrating peptide sequences (DNP, which is a cyclic heptapeptide DNP that can be used as a permeation signal for Caco-2 cells; TAT peptide, which is derived from the transactivator of transcription of human immunodeficiency virus and is a cell-penetrating peptides; R8 (Octaarginine) , which is a representative cell-penetrating peptide which is a synthetic sequence; Pep-1, called HIV-reverse transcriptase/SV40 T-antigen) were chosen and fused expression with the target gene, respectively, to evaluate their ability.
Five DNA fragments we designed and constructed, which include four signal peptide with four cell penetrating peptidefused GFP encoded genes and urate oxidase encoded gene, were inserted into the multiple cloning site of the pET-22b plasmid, and then were introduced into the BL21 strain, and were selected on the basis of Amp resistance . To ensure that we introduced the plasmid into the BL21 strain, we performed colony PCR on colonies grown on solid media.
Figure 1 Results of agarose gel electrophoresis of colony PCR
M: Marker DL2000; O: OpmA signal peptide sequence; P: PeLB signal peptide sequence; A: phoA signal peptide sequence; U: urate oxidase sequence; S: STiI signal peptide sequence.
Figure 2 Results of agarose gel electrophoresis of enzyme digestion
DL: DL2000 marker;
PCR: A fragment obtained by PCR of a urate oxidase-encoding gene;
PD: A target fragment obtained by PCR of a urate oxidase-encoding gene and performing enzyme digestion;
A: PhoA's target fragment;
AD: Target fragment of the phoA after digestion;
O: OpmA's target fragment;
OD: Target fragment of the OpmA after digestion;
B: PoLB's target fragment;
BD: Target fragment of the PoLB after digestion;
I: STiI's target fragment;
ID: Target fragment of the STiI after digestion;
ER: λEcoT14.
1.2 Testing Signal Peptide
Table 1 Group Description
The recombinant molecules were expressed induced by IPTG. The supernatant were collected to measure the absorbance at 525 nm using a wavelength of 395 nm in a microplate reader. By one-way analysis of variance (Table 2) and multiple comparisons (Table 3/Figure 3), the GFP fluorescence intensity of the four experimental groups was significantly different from the blank control, but the difference between the four signal peptides was not significant. So we concluded that the four signal peptides all function, but there is no one more effective.
Table 2 ANOVA of four signal peptide functions
Table 3 Significant difference of five group
