Fusion Expression Of Six Temporin-1CEa Genes From The Skin Of Chinese Brown Frogs, Rana Chensinensis Repeat In Tandem

Antibacterial peptides (ABPs),small molecule peptides encoded by the specific gene, have broad-spectrum antimicrobial activity. Amphibian skin peptides are a kind of ABPs considered to be a main element of innate immunity, which are also the weapon for fighting back the pathogenic bacteria. For the emergence of multiple-antibiotic resistant strains, ABPs from amphibian skin play an important role in the development of novel antibiotics because their broad-spectrum antimicrobial activities and specific antimicrobial mechanism. However, the isolation and purification of natural bioactive peptides from amphibian skin is too difficult to meet the needs of large-scale production. So, it is an inevitable choice to produce bioactive peptides by the gene expression in prokaryotes or eukaryotes. Amphibian skin recombination peptides can not be expressed directly in prokaryotic expression system because the high antimicrobial activity. Therefore, the fusion expression system, or expression in eukaryotic cells for recombination anti-bacterial peptides expression often is used.In this reseach ,we use recombinant technology and the fusion expression system to express six temporin-1CEa genes from the skin of Chinese brown frogs. Based on the amino acid sequence of temporin-1CEa,We synthesize temporin-1CEa gene sequence .We use PCR method to connect the six of temporin-1CEa genes, of which each gene is preceded by ATG codon. Lastly, at the 5 'and 3' end, two restriction sites of Sma I / Hind III were added, respectively. The 339bp fragment was constructed into the expressive vector pET-42a(+) for the expression of GST-temporin-1CEa fusion protein after six of temporin-1CE gene were synthesized in tandem.The sequencing analysis confirmed that the six of temporin-1CEa genes had been correctly inserted into vector pET-42a(+). The recombinant vectors were transformed into E.coIi BL21.The GST-temporin-1CEa fusion proteins expressed in the host bacteria under the IPTG induction,,and analyzed with SDS-PAGE. Different induced temperature, pH, IPTG concentrations and the induction time were screened to determine the optimum inducing conditions. At last, we choose the condition as following: after bacteria growing in LB medium of pH7.2 to saturation, induced recombinant protein for 4h at 30℃with 0.8mM IPTG. The experimental results showed that the recombinant protein molecular weight is 43KD, which occupy 10.8% content of total bacterial protein and present in the form of soluble protein in bacterial lysates. Fusion protein was purified with GSTrap FF affinity chromatography and HisTrap HP affinity chromatography. The purity of fusion protein is about 60% after purifying. After the fusion tag was cleaved by factor Xa, the antibacterial activity was tested. The construction of prokaryotic expressive plasmid of temporin-1CEa gene establishes a solid basis for further studying the amphibian skin antibacterial.