Tandem Expression Of LNK-16 Like Bovine Antimicrobial Peptide In E.coli

Antimicrobial peptides are a kind of polypeptides with antimicrobial activity produced by microorganisms, plants, invertebrates, and various animal cells and tissues, which are important component of innate immune system of biosystem in vivo. Due to their distinct antimicrobial mechanism of antibiosis, antimicrobial peptides are expected to replace antibiotics and become the next generation of anti-microbial drug. However, to obtain antibacterial peptides is always the bottleneck of their research and utilization. In order to solve the problem, this research successfully expressed antimicrobial peptides LNK-16 with antimicrobial activity using bio-engineering techniques and methods.With the methods of bioinformation, the bovine antimicrobial peptide Indolicidin was found with high antibacterial activity, broad antibacterial spectrum, low molecular weight and linear structure in the database NCBI, which containing 13 amino acid peptides. In order to achieve the expression of Indolicidin in E.coli, amino acid recognition sites of kallikrein (PFR) was added to the N terminal of indolicidin. The new peptide was named LNK-16 and artificially synthesized. The result of test of antibacterial activity detection revealed that LNK-16 still preserved activity,it makes the foundation for proteinase digestion ofconnected expressed production. Three LNK-16 peptides were connected by using codon preference of E.coli and the target nuclear acid sequence was translated reversely according to the linked amino acid sequence. After adding restriction enzyme sites EcoR I and Xho I and its protective bases at both ends of the target gene, the 178bp target gene form. The target gene was divided into four complementary overlapping nuclear acid sequences through the method of SOEing-PCR.Then these four sequences were synthesized, amplified and connected by TD-PCR. After double digestion of the target gene and plasmid vector pET-30a (+), the recombination plasmid vector pET-30a(+)-LNK-16 was obtained by the help of T4 DNA ligase. The result of double digestion and PCR identification manifested that the vector pET-30a(+)-LNK-16 was successfully constructed. Sequencing revealed that it was the same as the target gene we designed before.Positive plasmid was identified and transformed into the competent cell BL21(DE3)which was induced by IPTG. The optimal conditions were determined that the induction time was ten hours, the temperature was 37℃, the pH was 7.2 and the IPTG concentration was 500μm/L. Under the conditions, the expressed recombinant protein existed in the form of inclusion bodies, amounting to 32.6% of the induced recombinant bacteria at the presence of IPTG. The inclusion bodies was rinsed with TrionX-100, and finally was dissolved in 8 mol/L of urea to refold through dialysis.The purified protein was then digested by kallikrein in triethanolamine buffer, the total protein content of Enzymatic hydrolyzate is 0.307 mg/mL and LNK-16 accounts for 1/2. Using agar gel diffusion to detect the antimicrobial activity,the antimicrobial activity of production with 1h digestion to E.coli is higest and the inhibition zone is 1.06 cm. It means the expression production has antimicrobial activity and then provides the new way for resolving the resource of antibacterial peptide.