Full-length CDNA Cloning, Prokaryotic Expression, And Antimicrobial Activity Analysis Of F-I Hemoglobin In Urechis Unicinctus

Hemoglobins (Hb) is a kind of protein with functions of carrying oxygen, opioidpeptide activity, immunoregulatory activity, antimicrobial activity, analgesic effect,and so on. The present studies show that Hb’s fractions (hemocidins) are importantfactors of killing exogenous microorganism in congenital immunity.Urechis unicinctus(Uu) belonging to Echiurioidea, is one of the most importantvaluable invertebrate animal. Being a filter feeder, it lives in mud substrate ofintertidal belt. Although exposed to various kinds of pathogenic bacteria and parasites,it can avoid invasions successfully with its non-specific immune system. Hb may playan impotant role in its innate immune defending system. However, no reports aboutUu’s Hbs are available.According to Urechis caupo F-I complete cDNA sequences (GI:945055), weobtained Urechis unicinctus F-I complete cDNA sequences. The full-length of cDNAwas780bp with an open-reading frame (ORF) of429bp encoding142aminoacids,the mature peptide was named as UuHb-F-I. Its estimated molecular mass was15120.67Da, and theoretical isoelectric point was9.02. The protein also containedplenty of α-helix.To obtain UuHb-F-I, A prokaryotic expression system named BL21(DE3)/pET-22b-UuHb-F-I had been bulided for expressing protein. IPTG and lactose weresuccessfully adopted to induce expression, respectively. Of which the lactose couldinduce a maximum expression dose under the condition of37℃and180r/min for16h,with fewer other proteins. After large expression,we dissolved the inclusion bodywith urea, flowed through Ni2+affinity chromatography to purify recombinantprotein,we gained objective protein with few other proteins.To remove urea and other saline ions gradually, repeatedly dialysis were needed. Finally, solid recombinationprotein was obtained by freeze drying.Antimicrobial activity was tested for recombination protein through liquid phaseassay against8microoganisms including gram-negative, gram-positive bacteria andfungi. Our research found UuHb-F-I had a stronger antimicrobial activity againstsome gram-positive bacteria than that of gram-negative bacteria,the minimum MICagainst Micrococcusluteus was2.78～4.63μM. These results may be helpful inunderstanding invertebrates (Urechis unicinctus) immune defense mechanism andgiving insights into new AMPs in drug discovery.