본 연구에서는 인체에 보다 안전한 천연 화장품보존제 발굴을 목표로 하여 인간유래 항균펩타 이드 LL-37의 짧은 유사체인 FK-13을 화장품보존제로 사용하고자 연구를 진행하였다. 이를 위해 13개의 아미노산으로 구성된 FK-13 펩타이드를 고상 펩타이드 합성법로 합성하였고 reversed phase-high performance liquid chromatography (RP-HPLC)로 정제 후 liquid chromatography-mass spectrometry (LC-MS)를 이용하여 순도와 분자량을 확인하였다. 합성된 FK-13을 이용하여 미생물에 대한 방부력을 검 정하였고 화장품에 적용 가능성을 살펴보았다. FK-13은 3개의 그람-양성균 (Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis)과 3개의 그람-음성균 (Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa), 그리고 진균인 Candida glabrata 에서도 높은 항균 활성을 보였 으며 넓은 항균활성 스펙트럼을 나타내었다. 이를 바탕으로 화장품보존제로서의 적합성을 확인하였다. 또 한 FK-13은 고온에서 분자의 열안정성을 보였으며 기존의 천연 한방화장품 방부제 및 화학보존제들과의 항균활성 비교 실험에서도 보다 월등한 항균활성 효능을 나타내었다. 따라서 FK-13 펩타이드는 인체 친화 적이고 효과적인 항균활성을 나타내므로, 기존 화학보존제를 대체할 새로운 천연 화장품보존제로 적용이 가능할 것으로 판단된다.

Insect peptides have been extensively studied due to beneficial effects in the treatment of infectious diseases. Melittin, a fundamental component of honeybee venom produced by European honeybee Apis mellifera, has applied to prevent various inflammatory disease and bacterial infections in human. However, the therapeutic application of melittin is limited due to its low stability, hemolytic activity and expensive manufacturing costs. In this study, we aimed to discovery unknown peptides from the Apis mellifera and evaluate its antibacterial activity against Escherichia coli KACC 10005. A total 15,853 peptide sequences were diciphered using Illumina HiSeq 2500 next-generation sequencing (NGS) platform and analyzed based on the Apis mellifera official Gene Set Version 3.2 (amel_OGSv3.2) and the Collection of Anti-Microbial Peptides (CAMPR3) database. All the peptide sequences and annotation data sets were combined and sorted by physicochemical features of antimicrobial peptides (AMPs), such as short peptide length <=50, positive charge, isoelectric point (8.0<=pl<=12), and aggregation propensity (in-vitro: <=500, in-vivo: –40<= Na4vSS <=60). Among the screened peptides, four unknown peptide candidates, named AMP1-4, were chemically synthesized and tested for antimicrobial activity in comparison with a reference peptide, melittin. Inhibition of bacterial growth was observed in the AMP4 treated group from 6 hours to 48 hours post-treatment against E. coli. These results suggest that honeybee-derived peptide sequences can be applied as natural resources to acquire novel AMPs and the peptide sequences derived parameters are enough to recognize antibacterial peptides. In addition, the selected novel peptide candidate, AMP4, has antibacterial activity.

Several Mites are currently the most serious threat to the world bee industry. The ectoparasitic honey bee mites was originally confined to the Asian honey bee(Apis cerana etc.). Varroa destructor and Tropilaelaps clareae has plagued European honey bees, Apis mellifera. Differences in mite tolerance are reported between two honey bee species A. mellifera and A. cerana. We were amplified antimicrobial peptide cDNA genes (Defencin, Abaecin, Royalisin, Apidaecin and Hymenoptaecin) by RT-PCR. We explored the transcriptional response to mite parasitism in A. mellifera 4th instars larvae which differ in susceptibility to V. destructor and T. clareae, comparing parasitized and non-parasitized 4th instars larvae (worker and Drone) from same hive. Differential gene expression of worker bees and Drone bees induced by mites (V. destructor and T. clareae) infection was investigated by northern blot. Mites (V. destructor and T. clareae) parasitism caused changes in the expression of genes related to sex distinction. Bees tolerant to mites (V. destructor and T. clareae) were mainly characterized by differences in the expression of genes regulating antimicrobial gene expression. It provides a first step toward better understanding molecular expression involved in this differential sex distinction host-parasite relationship. We were detected bee virus in A. mellifera, comparing parasitized and non-parasitized 4th instars larvae (worker and Drone). Therefore, this result was demonstrated that mites were another possible route of horizontal transmission, as several viruses were detected in mites and their hosts.

To find some antibacterial peptides responsible for bacterial resistance, we performed differential hybridization with total cDNA probes which synthesized from normal and immunized larvae. Thirteen individual cDNA transcripts were expressed differentially in a total 1,862 random cDNA clones. One of upregulated genes is a novel member of the insect defensin-like peptide(Coprisin), a family of antibacterial peptide. Northern blot analysis showed that Coprisin was up-regulated at 4h and reached the highest point level at 16h after injection of E.coli. The deduced amino acid sequence of Coprisin was composed of 80 amino acids with predicted molecular weight of 8.6 kDa and PI of 8.72. Comparison of the deduced amino acid mature portion of Coprisin with defensin-like peptide of other insect indicated that it has 79.1% and 67.4% identity with Anomala cuprea and Allomyrina dichotoma, respectively. To find antibacterial active region of Coprisin, we synthesized four peptides corresponding to amino acid residues 1V-43N-NH2(CopN1), 5-16(CopN2), 19-30(CopN3) and 31-43(CopN4) of coprisin having amidated amino acid residues at their Cterminal. A 12-mer amidated at its C-terminus, ACALHCIALRKK-NH2 (Ala19-Lys30-NH2) was synthesized based on the deduced amino acid sequence, assumed to be an active site sequence. This peptides showed antibacterial activity against E.coli, Staphylococcus aureus, MRSA, Psedomonas syringae, and Pectobacterium carotovorium. Modified 9-mer peptide, LRCIALRKK-NH2, showed strong antibacterial activity than mellitin peptide used as a positive control against gram-negative and gram-positive bacteria. This peptide showed no haemolytic activity and quite stable at 100℃ for several hours of incubation and in a wide pH range(pH2-12). Therefore, this peptide may be a good candidate for the development of new drug with potent antibacterial activity without cytotoxicity.

누에에서 새로운 항세균성 펩타이드 유전자를 탐색하기 위하여 E. coli K12로 체강 주사한 누에 유충의 cDNA 유전자 은행에서 차별화 선별로, 잠재 항세균성 펩타이드 유전자로 추정되는 BmInc8 클론을 분리하였다. BmInc8은 564bp의 크기를 가지며, 59개 아미노산을 coding하는 open reading frame과 2개의 잠정 폴리아데닐화 부위를 보유하고 있었다. BmInc8은 M. sexta에서 분리, 보고된 bactericidine 유전자와 61.2%의 DNA 상동성을 나타내는 것으로, 그 연역된 펩타이드 구조는 항세균성 펩타이드의 일종인 cecropin과 유사한 2가닥의 -helix가 Lysine-Proline 경첩부위에 의해 포개져 있는 형태를 갖는 것으로 추정되었다. 또한 cDNA 삽입 부위의 기능성 검정을 위해 원핵 발현벡터인 pT7-5를 이용하여 E. coli BL21(DE3) 균주에 형질전환하고 IPTG로 induction한 결과 E. coli BL21(DE3) 균주의 성장이 정지됨을 관찰할 수 있었다. 이상의 결과로 BmInc8은 DNA 상동성 비교, 연역 아미노산의 구조 추정 및 cDNA 삽입 부위를 이용한 transient expression 결과 항세균성 펩타이드를 coding하는 유전자임을 추정할 수 있었다. 또한 Bminc8의 cDNA 유전자 정보를 GenBank에 등록하였으며 등록 번호는 U30289이었다.