Purification And Identifiedcation Of Proteins With Antibacterial Activity From The Serum Of Pinctada Martensii

Pinctada martensii is one of the most important pearl oyster in pearl culture industry. Because the coastal water quality deteriorations and worse in recent years, a variety of diseases are frequently happened, leading to large number of deaths and caused huge economic losses on the pearl industry in recent years. Therefore, improving the resistance of P.martensii is an urgent task to solve the problem. Antimicrobial peptide is information one of the most important immue molecules for shellfish, however, little sequence information and characterization was reported from P. martensii. In this study, multi-step high performance liquid chromatography(HPLC) combined with Mass Spectrometry(MS) were performed to identify the peptides with antimicrobial activity. Three components with significant anti-bacteria activity were isolated from the serum of P. martensii, which named PmECSOD, PmC LEC-1 and PmAMP-1, respectivly. Partial N-terminal sequences determined by Mass spectrometry were searched against the geneome o f P. martensii and dbEST database. The matched EST sequences were used as the templates to design specific primers for PC R amplification. cDN A sequences of these three proteins were successfully cloned by the rapid amplification of cDN A ends(RACE). The or ganazatin expression of three genes and at different time interval after bacterial challenge were analyzed by quantitative real-time PC R(qPCR). Results are as follows:The full length c DNA sequence of P. martensii Superoxide Dismutase(PmECSOD) is comprised of 1864 bp, with an open reading frame(ORF) of 1422 bp, which encoded 473 amino acids. The N-terminal 20 amino acids in the signal peptide and 453 amino acids in the rest mature peptide. Amino acid sequence analysis revealed that the peptide has the typical SOD domain in its N-terminal end and also has FRI&IGc2 domains. PmECSOD shared low sequence similarity with the long term Pacific oyster ECSOD and bay scallops PmECSOD, which is 36.42% and 28.32% respectively. mRNA of PmECSOD can be detected with q RT-PCR in all tested tissues, including adductor muscle, mantle, hemocytes, gonad, hepatopancreas and gill. Highest expression level was observed in gill, the lowest level was observed in adductor muscle. The expression level in the hemolymph was the highest at 48 h after stimulation, with a significant statistically difference.The full length lectin cDNA(PmC LEC-1) sequence of P. martensii is comprised of636bp, with an open reading frame(ORF) of 450 bp, it encoded a 149 amino acids, perfom with the N-terminal signal peptide of 19 amino acids and mature peptide of 130 amino acids contain C-type lectin domain-sugar-binding recognition site(CDR). Blast alalysis revealed that PmCLEC-1 shared low sequence similarity with the C-type lectin PoLEC-1 and Po LEC-2 of P. martensii.Spatial structure display the compact structure in PmC LEC-1,in addition,there are two cysteines in N-terminal. Translations of PmCLEC-1 can be detected by qRT-PCR in all tested tissues, The highest expression level was observed in mantle and hepatopancreas and the lowest level was observed in hemolymph. The expression level reached the highest value at 12 h after stimulation in the hemolymph(p<0.05).The full length antimicrobial peptides(PmAMP-1) c DNA sequence of P. martensii is comprised of 700 bp, with an open reading frame(ORF) of 306 bp, which encoded a 101 amino acids. PmAMP-1 is a α- helical structure. Furthermore, the components caused significant morphological alterations in E. coli as shown by transmission electron microscopy(TEM). Translations of PmAMP-1 can be detected with q RT-PCR in all tested tissues. The highest expression level was observed in adductor muscle, the lowest level was observed in hemolymph. the expression level reached the highest value at 8 h after stimulation in the hemolymph(p<0.05).In conclusion, three protein components with antibacterial activity was seperated in serum of P. martensii for the first time. The study are valuable for further exploring the immune response and may help us better understand molluscan innate immunity.