Insect Molecular Biology Laboratory, Department of Environmental Biotechnology, School of Environmental Sciences, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India.
Rapid Commun Mass Spectrom. 2011 Nov 15;25(21):3203-6. doi: 10.1002/rcm.5213.
Silk from the domesticated silk worm Bombyx mori procures foreign body response naturally, so it has been utilized as a biomaterial for decades. In India the prime focus of the sericulture industry is to improve silk production with high quality silk. Naturally, the silk worm builds its cocoon not only with silk proteins, but also with antimicrobial proteins to avoid infection since the cocoon is non-motile and non-feeding. The aim of the present study is to elucidate the antimicrobial proteins that persist in the cocoon of the silk worm Bombyx mori. At the pupal stage, the silk worm cocoon shell extract was prepared from the day of pupation (P0) to the day of natural rupture of the cocoon for the eclosion of moth (NR). Using the cocoon shell extract a microbial susceptibility test was performed by the disc diffusion method against the microbes Escherchia coli, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The development of a zone of inhibition against the microbes confirmed the presence of antimicrobial/immunogenic activity of the cocoon shell extract. For further analysis, the cocoon shell extract was subjected to 7-15% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The protein profile of the cocoon extract revealed the coomassie blue stained bands resolved from the 150-15 kDa molecular range. Interestingly, a polypeptide localized at around 29 kDa showed remarkable expressional changes during the development of pupa. To characterize the 29 kDa protein, it was eluted from the gel, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The trypsin-digested peptide peaks were analyzed through MASCOT and peptides were matched with the NCBI nr database. The peptides were very well matched with the 18 wheeler protein, which is reported to be responsible for innate immunity, belonging to the Toll family in insects and responsible for cellular mediated immunity.
家蚕的丝自然引发异物反应，因此几十年来一直被用作生物材料。在印度，丝绸行业的主要关注点是提高丝绸的产量和质量。由于蚕茧是无运动和非摄食的，蚕不仅用丝蛋白，还用抗菌蛋白来建造它的茧，以避免感染。本研究的目的是阐明存在于家蚕茧中的抗菌蛋白。在蛹期，从化蛹日（P0）到茧自然破裂日（NR），从蚕茧壳提取物中制备蚕茧壳提取物。采用圆盘扩散法，用大肠埃希菌、蜡状芽孢杆菌、金黄色葡萄球菌、铜绿假单胞菌和肺炎克雷伯菌对微生物进行敏感性试验。抑菌圈的发展证实了茧壳提取物具有抗菌/免疫原性活性。为了进一步分析，将茧壳提取物进行 7-15%十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS-PAGE）。茧提取物的蛋白质图谱显示，从 150-15 kDa 分子量范围内分离出考马斯亮蓝染色带。有趣的是，一条定位于约 29 kDa 的多肽在蛹发育过程中表现出显著的表达变化。为了表征 29 kDa 蛋白，将其从凝胶中洗脱，用胰蛋白酶消化，并通过基质辅助激光解吸/电离质谱（MALDI-MS）进行分析。用胰蛋白酶消化的肽峰通过 MASCOT 进行分析，并将肽与 NCBI nr 数据库进行匹配。肽与 18 轮蛋白非常匹配，18 轮蛋白被报道负责先天免疫，属于昆虫中的 Toll 家族，负责细胞介导的免疫。
