Cloning And Expression Of Antibacterial Peptide Spinigerin α In Pichia Pastoris

Antimicrobial Peptide is a kind of small size molecular peptides that are encoded by a special gene, still is important molecular protective screen of that host defend pathogenic bacteria invaders. AMP has the advantages in thermal stability, high water-solubility, wide-spectrum antibacteria, etc, and has a relatively higher resistance in extreme conditions like high ionic strength or environment of broad spectrum of pH. AMP effects on prokaryotic cells or pathological eukaryotic cells but hardly on normal eukaryotic cells. Unlike antibiotic, which effects by means of repressing macromolecular synthesis, AMP has a totally different mechanism, thus leads to a lower tolerance of its pathogenic bacteria. So far, there are three sourees for Antimicrobial Peptide:isolating form natural resourees, manual synthesis and expressing through gene engineering techniques. According to character of every methods, the expressing of gene engineering is a effective way to obtain a lot of AMP.Spinigerin is a linear antibacterial peptide derived from a termite insect (Pseudacanthotermes Spiniger).It consists of 25 amino acids and is devoid of cysteines. Spinigerin displays good lytic activities against Gram-positive and Gram-negative bacteria,but has no hemolytic activities against human erythrocytes. Spinigerin contents of a-helical structure from Lys4和Leu21,and this a-helical retain antibacterial activity,too. Mechanism of action is that a-helical structure and the strong electrostatic attraction play important roles in the negatively charged polr head groups of the phospholipids.In this study, according to GenBank (ID:GI:13959577) registered Spinigerin peptide amino acid sequence, it selects with the a-helix structure of 18 amino acid fragment, as a fragment. Using the principles of Pichia pastoris' preferred codon, the fragment of the nucleotide sequence and its primers were designed and synthesised. Recombination expression vector pPICZa A-Spinigerin a was constructed, and successfully cloned target gene. By comparing the size of vector, double digestion and the PCR amplification of identification, It is confirmed that the expression vectors are the recombinant plasmids.In the course of the expression, Protein expression system is Pichia pastoris GS115. Yeast transformation requires a lot of DNA, so preparation of expression vector requires a lot of pPICZa A-Spinigerin a. Expression vector pPICZa A-S a is linearlized by Sac I, and is transformed into Pichia pastoris GS115 though electroporation. The high resistance of transformants were screened by using a gradient of concentration of Zeocin.28-30℃cultured 2-7d, the yeast genome for PCR was extracted from recombinant strain GS115-S a single colony. By UV observations, the purpose of bands have been successful obtained. Transformant GS115-S was going methanol induction and the bacteriostasis experment was done by the fermentation supernatant. The expression product purified have good Antibacterial effect to Gram-positive and Gram-negative by determining the antimicrobial diameter.In summary, the expression vector was constructed successfully pPICZa A-Spinigerin a and was transformed into Pichia host cell GS115, the recombinant strain GS115-S a. In the course of methanol induced the expression of the supernatant obtained has been shown to have some antibacterial activity. The studying of Spinigerin a Peptide lay a certain foundation for further study.