A robust protein extraction method for two dimensional electrophoresis of silkworm proteins

Silkworm (Bombyx mori) is a species of agricultural importance, as well as a model organism for Lepidoptera insects. Proteomic method has been widely used in silkworm research, and a robust mass spectrometry-compatible protein extraction method is urgently needed. In this study, we adapted phenol extraction method to extract silkworm midgut protein, and coupled this method with pH 5 - 8 gel strip for two dimensional electrophoresis. The phenol extraction method significantly increased the resolution, as well as greatly reduced the background of two dimensional electrophoresis gels. In addition, this method was well compatible with mass spectrometry analysis. This is the first report that phenol extraction method is used for silkworm midgut protein extraction, and may be applied in other researches.

Research Progress of Value, Extraction and Applicationof Silkworm Chrysalis Protein

Silkworm chrysalis protein is a high quality pure natural complete animal protein with high utilization value, and it has been widely usedin many industries in recent years. The nutrition, health care and medicinal value of silkworm chrysalis protein, the degreasing, deodorization anddecoloration extraction process, and its application in food, medicine, feed, etc. are summarized, in order to provide the reference for further development and industrial production of silkworm chrysalis protein.

Effects of silkworm pupa protein on apoptosis and energy metabolism in human colon cancer DLD-1 cells

Objective:To investigate the effects of silkworm pupa(Bombyx mori) protein(SPP) on cell proliferation,apoptosis and energy metabolism in human colon cancer cells DLD-1.Methods:CCK-8 was used to detect cell proliferation rate after 72 h of cell culture for the control group(normal cultured DLD-1 cells) and SPP dose groups;Annexin-V/PI was applied to observe cell apoptosis;XFe24 Extracellular Flux Analyzer was used to detect cell mitochondrial respiratory function and glycolytic function.Results:Comparing with the control,SPP significantly inhibited the proliferation of DLD-1 cells with all the dosage tested(P <0.01);flow cytometry showed that SPP significantly promoted apoptosis(P<0.05).Additionally,SPP could significantly inhibited mitochondrial metabolism and glycolysis of DLD-1 cells and decreased cell energy metabolism in all groups treated with different doses.Conclusion:SPP can cause oxidative damage,promote apoptosis,and reduce mitochondrial respiratory and glycolysis rate in colon cancer DLD-1 cells,which reveals that SPP has the potential to serve as the anti-cancer drugs in the future,but further experimental evidence is needed.

Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm (Bombyx mori) on Day 1 and Day 4 in the 5th Instar Stage

The posterior silk gland （PSG） of silkworm is an important organ where fibroin is synthesized and secreted exclusively. Because fibroin constitutes 75-80% of the silk filament, the mechanism governing fibroin secretion, quality and yield of cocoon can be elucidated by the study on the PSG. Using two-dimensional gel electrophoresis （2-DE） and image analysis system, the changes in the protein composition in the PSG cell were investigated on the day 1 （D1） and day 4 （D4） in the 5th instar stage from five different strains of silkworm （Bombyx mori）. While differences at protein level between days and strains were far less than those observed at the gene level using EST analysis. The change trends in protein composition from D1 to D4 were diverse among the different strains. The results suggest that the secretion of fibroin is regulated by multiple proteins. The site of regulation and the proteins responsible for the regulation vary with the strain, which leads to differences between strains in the capacity of fibroin secretion in the PSG cell.

Molecular cloning of a phosphotriesterase-related protein gene of silkworm and its expression analysis in the silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus is one of the major viral pathogens for the silkworm. The immune response of silkworm to the virus infection is obscure. A phosphotriesterase-related protein gene of silkworm, Bombyx mori (BmPTERP) was found in our previous microarry analysis of the midgut infected with the virus. In the present study, we cloned and analyzed the full-length cDNA of BmPTERP gene by means of rapid amplification of complementary DNA ends (RACE) and bioinformatic analysis for exploring its functions in interaction between the silkworm and the virus. The nucleotide sequence of the gene is 1349-bp and contains a 131 bp 5’UTR and a 165 bp 3’UTR. The 1053 bp open reading frame encodes a 350 amino acid protein. The deduced protein contains specific hits of phosphotriesterase-related proteins and belongs to the amidohydrolase superfamily. RTPCR analysis revealed that BmPTERP gene was expressed in all the tissues tested, including midgut, hemocyte, gonad, fat body and silk gland. Real-time quantitative polymerase chain reaction analysis indicated that the relative transcript of BmPTERP gene in the infected midgut was 19.32 fold lower than that in normal midgut at 72 hours post inoculation.

Whitening Effect of Storage Protein 2 from Silkworm Hemolymph

This study investigated the effects of silkworm hemolymph-derived storage protein 2 (SP2) on the whitening process in mouse B16F1 melanoma cells. After the cells were treated with various concentrations of SP2 (0.1 - 1.0 mg/mL), cytotoxicity, melanin contents, and differences in mRNA and protein expression associated with melanogenesis were observed. No cytotoxicity was observed when cells were treated with SP2, even with increased SP2 concentrations of up to 2.0 mg/mL. When treated with various SP2 concentrations in the cells, the protein and mRNA expression of tyrosinase were dose-dependently decreased, respectively, and inhibition of tyrosinase was further increased by 50.0% with increasing SP2 concentration of 1.0 mg/mL. Expression mRNAs coding tyrosinase related protein-1 and protein-2 (TRP-1 and TRP-2) was also significantly decreased in a dose dependent manner. When measuring the melanin content in melanoma cells, SP2 at 1 mg/mL inhibited melanin synthesis by 73.5% compared with non-treated cells. The inhibitory effect was 2.8-fold higher than that obtained using arbutin as a positive control. This study demonstrates that SP2, as a whitening material, is capable of suppressing melanin synthesis through the downregulation of proteins and genes in the melanin biosynthesis pathway.

交配前后雌雄桑蚕蛾营养价值评价

为深入了解桑蚕蛾的营养物质成分,检测了交配前后9芙雌雄蚕蛾蛋白质、脂肪酸和氨基酸的组成和含量。通过粗蛋白、粗脂肪、不饱和脂肪酸含量、动脉粥样硬化指数(indices of atherogenicity,IA)、血栓形成指数(thrombogenicity,IT)以及氨基酸评分、化学评分、必需氨基酸指数(essential amino acid index,EAAI)、营养指数(nutrition index,NI)和生物价(biological value,BV)等指标评价了蚕蛾的营养价值。结果表明,蚕蛾氨基酸和不饱和脂肪酸含量丰富,交配前雌蛾蛋白质(21.6%,鲜重)、总氨基酸(46.94 g/100 g)、必需氨基酸含量(15.86%)以及EAAI(99.33)、NI(21.45)和BV(96.57)最高,同时含有丰富的甜味、药效和支链氨基酸,粗脂肪含量(7.11%,鲜重)低,多不饱和脂肪酸占比(78.18%)高,IA(0.21)和IT(0.14)低,是理想的蛋白源。交配后的雄蛾粗脂肪(24.46%,鲜重)、不饱和脂肪酸(44.31 g/100 g)、亚麻酸含量(19.37 g/100 g)最高,IA(0.28)和IT(0.22)指数较低,是提取不饱和脂肪酸的优质原料。该研究为桑蚕蛾在食品领域的开发应用提供了理论依据。

两个家蚕CPFL表皮蛋白基因的鉴定与表达分析

昆虫CPFL表皮蛋白是一类重要的结构蛋白。本研究通过生物信息学分析从家蚕基因组中鉴定出2个CPFL基因,命名为BmHCP1和BmHCP2。氨基酸序列分析发现,2个蛋白在羧基末端均具有CPFL表皮蛋白典型的保守结构序列YGW以及重复序列AAH、AAPA、AAPV。进化树分析显示,2个蛋白与鳞翅目昆虫艺神袖蝶(Heliconius erato)和柑橘凤蝶(Papilio xuthus)具有较高同源性。荧光定量PCR分析显示,BmHCP1和BmHCP2存在组织、时相特异性表达,幼虫期在头部、中肠、表皮等组织中表达量较高;蛹期除翅原基、血淋巴、触角和足等组织外均有较高表达量;成虫期在足、触角、翅原基、表皮等组织中表达量较高;蛹期在大多数组织的表达量明显高于幼虫期和成虫期。研究结果为进一步探明家蚕表皮蛋白基因的功能奠定了基础。

桑蚕蛋白复合粉抗运动疲劳功效研究

为了研究桑蚕蛋白复合粉的抗运动疲劳功效与作用机制,C57BL/6雄性小鼠分为对照组、低剂量组(50 mg/kg·d)、中剂量组(100 mg/kg·d)和高剂量组(200 mg/kg·d),灌胃30天后检测力竭游泳时间和运动疲劳标志性生化指标(血尿素氮、血乳酸、肌乳酸、肝糖原、肌糖原和骨骼肌中抗氧化相关酶活性)。与对照组相比,各剂量蚕蛋白复合粉组小鼠力竭游泳时间显著延长,血尿素氮、血乳酸、肌乳酸水平显著降低,肝糖原、肌糖原含量显著升高,骨骼肌中总抗氧化能力、谷胱甘肽过氧化物酶、超氧化物歧化酶和过氧化氢酶的活性显著升高,脂质过氧化产物丙二醛水平显著降低。桑蚕蛋白复合粉可以通过减少代谢废物乳酸与尿素氮的积累、增加肝糖原和肌糖原的储量、降低骨骼肌的氧化应激水平来发挥抗运动疲劳的功效。

Analysis on the Variation Expression of Difficult-Dissolved-Proteins of Diapause Eggs After Activating by Acid Treatment

Using HCl to activate the diapause eggs is a traditional technique of artificial hatching applied in silkworm egg production. Its mechanism has not yet been clarified. This experiment explored the effect of HCl on the termination of diapause of silkworm eggs cold-stored for 45 days from the point of proteomes. Two-dimensional electrophoresis techniques and ESI- MS-MS were used to compare and analyze the variation expression of difficult-dissolved-proteins of diapause eggs coldstored for 45 days before and after acid treatment. Through analysis on the two-dimensional electrophoretogram, there were 296 dots before acid treatment and 302 after the treatment, respectively. Amongst them 265 dots were matchable. The matchability reached 88.6%. There were 31 specific protein dots before acid treatment and 37 after acid treatment, respectively. ESI-MS-MS analysis was conducted for two specific protein-rich dots which disappeared after acid treatment. The results indicated that the sequence of No. 1 protein dot had 55 amino acids＇ peptide matched with those of chorion protein （Bombyx mori）. While the sequence of No. 2 protein dot had only 15 amino acids＇ peptide matched with those of heat shock protein hsp 19.9 （Bombyx mori）, and it was presumed to be an unknown protein. The difficult-dissolvedproteins of diapause eggs have variation expression after acid treatment. Some proteins before and after acid treatment are changed in MW.

牛角瓜纤维/蚕蛹蛋白纤维/改性聚酯纤维混纺抗菌机织小提花面料

为了开发新面料,提高产品附加值,开拓新的市场,设计研发了牛角瓜纤维/蚕蛹蛋白纤维改性聚酯(Aircell纤维)混纺抗菌机织小提花面料。介绍了选用材料的特点,阐述了创作灵感,绘制了包括组织图、穿筘图、穿综图和纹版图的上机图;概述了面料制作工艺流程,着重总结了整经、浆纱及织造使用的设备和具体的工艺方案,并制定了优化工艺技术措施;为验证面料的功能性,对面料进行抗菌性能测试。所得成品纬密大于经密,直条纹明显,滑爽挺括,具有麻织物的粗犷特点,质感明显。成品一等品率99%,对金黄色葡萄球菌、大肠杆菌及白色念珠球菌抑菌率分别为78%、78%、71%,产品质量和抗菌性较好。

胃肠道环境对蚕蛹蛋白稳定Pickering高内相乳液及其负载虾青素的影响

以蚕蛹蛋白稳定的Pickering高内相乳液(high internal phase emulsions,HIPEs)为研究对象,评价其在体外胃肠道环境中的稳定特性及负载虾青素的能力,阐明其靶向肠道控释的机理。通过测定粒径分布、Zeta电位以及利用激光粒度分布仪、激光共聚焦扫描电子显微镜、反相高效液相色谱和电泳技术等分析蚕蛹蛋白颗粒及其所稳定的Pickering HIPEs在模拟胃肠道环境中的变化,以及乳液负载虾青素的能力和生物可给性。结果表明:蚕蛹蛋白颗粒可稳定油相体积分数为78%的Pickering HIPEs,其对虾青素的负载率约为88%;该乳液在胃环境中90 min内保持稳定,在肠环境中随消化时间延长逐渐失稳,显著提高了虾青素在肠道的生物可给性;界面蛋白组成表明分子质量为70 kDa的蚕蛹蛋白是稳定乳液的主要蛋白,肠消化酶的水解作用是乳液在肠道失稳并释放虾青素的主要原因。本研究为蚕蛹蛋白颗粒稳定的Pickering HIPEs用于口服靶向肠道递送疏水性生物活性物质提供了理论基础,同时为虾青素在食品和保健品领域中的应用提供了新的可能。

高低温催青温度敏感期家蚕卵的差异蛋白筛查

滞育(Diapause)是昆虫发育停滞或减缓的生理状态,家蚕Bombyx mori作为卵滞育的代表,其滞育过程已得到广泛研究,但诱导滞育发生的分子机制尚不清楚。二化性家蚕滞育性由遗传和母系在胚胎期所处的环境条件决定,25℃催青蚕卵孵化后的家蚕产滞育卵,15℃低温催青则诱导家蚕产下非滞育卵。本研究分别用25℃和15℃催青蚕卵,在发生滞育诱导的温度敏感期取样,抽提蛋白质通过非标(Label-free)蛋白质组定量技术进行质谱测序。筛选出具有明显表达差异的蛋白104个,其中56个蛋白上调,48个蛋白下调。通过生物信息学对差异蛋白进行GO分类和KEGG功能富集分析,结果显示差异蛋白主要参与生长发育、物质代谢和胁迫应答等生物过程;同时差异蛋白主要参与胰岛素信号通路、乙醛酸和二羧酸代谢等相关途径。分别选取上调基因和下调基因进行qRT-PCR验证,其趋势与蛋白组学结果一致。该研究将为进一步解析家蚕滞育诱导发生机制提供靶标蛋白和数据参考。

pH值对柞蚕蛹粉凝胶特性及蛋白质热聚集行为的影响

柞蚕蛹富含优质蛋白质,是一种具有很高开发利用价值的食品原料。采用质构仪、流变仪和激光扫描共聚焦显微镜等分析不同pH值对柞蚕蛹粉凝胶特性及蛋白质分子热聚集行为的影响。结果表明:pH值为7时形成的柞蚕蛹粉凝胶网络较为连续和均匀,凝胶保水性最高(51.67%±0.79%,P<0.05);pH值为9时的柞蚕蛹粉有最低的临界凝胶质量分数(8%)、最高的凝胶硬度(41.67 N±3.37 N,P<0.05)和弹性(2.81 mm±0.07 mm,P<0.05)。柞蚕蛹粉凝胶的储能模量与蛋白质的溶解度呈显著正相关(R=0.92,P<0.05),而与蛋白质的凝固率(R=-0.52)和表面疏水性(R=-0.77)呈显著负相关(P<0.05)。pH值为9时,蛋白质溶解度最高(62.13%±0.63%,P<0.05),表面疏水性最低(70.06μg±9.32μg,P<0.05),形成了更多的小尺寸热聚集物并增强了凝胶基质;而在pH值为5时,蛋白质溶解度最低(28.96%±1.13%,P<0.05),表面疏水性最高(195.66μg±1.30μg,P<0.05),促使更多的大尺寸热聚集物形成并无序堆叠而削弱凝胶基质。研究旨在通过调节pH值控制柞蚕蛹粉中蛋白质的热聚集行为,进而获得理想质地和保水性的柞蚕蛹粉凝胶。

2019-nCoV N蛋白在家蚕杆状病毒表达系统中的表达及纯化

核衣壳(nucleocapsid,N)蛋白是新型冠状病毒的结构蛋白,与病毒RNA的转录和复制有关。目前,N蛋白已被广泛应用于新型冠状病毒肺炎的检测诊断及治疗。本研究克隆的2019-nCoV-n基因CDS由1257个碱基构成,编码419个氨基酸。将该基因连接到昆虫表达载体pFastBacTMHTB构建重组Bacmid,转染至BmN细胞中获得重组病毒,将重组病毒注射家蚕蛹体内,收集发病家蚕蛹血淋巴,通过镍柱亲和纯化,SDS-PAGE和Western blot检测表明获得一定纯度具有活性的重组2019-nCoV-N蛋白,为后期进一步开发抗体检测试剂盒鉴定了基础。

Study on ionic liquids based novel method for separation and purification of silkworm pupa protein

Silkworm pupa protein(SPP)that obtained by traditional method usually had a high fat content,which would impose restrictions on the further use of SPP.Herein,various functionalized ionic liquids(ILs)were used to extract SPP from silkworm pupae,the structure-performance relationship of ILs with their SPP separation performance were explored at the same time.The research showed that the maximum extraction yield of SPP was up to 62.6%with less than 0.5%low fat content by using 1-ethyl-3-methylimidazolium chloride([Emim]Cl),when the dissolution experiment was conducted at 90°C for 24 h with ethanol bath as the regeneration solvent.Comparing with the structure of raw material,the regenerated SPP maintained the native protein backbone.Meanwhile,all regenerated SPP showed a decreased crystallinity,which also exhibited decreased fraction of theα-helix comparing to thatβ-sheet united with coil random structures.

蚕蛹蛋白的生物活性和过敏特性的研究进展

具有高营养价值的昆虫一直以来都是我国优质的食物来源之一。蚕蛹是家蚕业的重要副产物之一,其在东亚各国作为食物和饲料已有十分悠久的历史。蚕蛹含有丰富的蛋白质、脂质、矿物质和维生素,具有十分重要的营养价值和经济价值。蚕蛹的应用范围十分广泛,我国有许多保健品、药物还有食物添加剂中都有蚕蛹的成分。蚕蛹蛋白及其水解物具有多种生物活性,具备一定的开发潜力。然而,蚕蛹也能导致轻度甚至严重的过敏反应,这些不良反应极大的限制了蚕蛹在全国范围内的普及程度。因此该研究综述了蚕蛹蛋白的生物活性和过敏特性,以期为蚕蛹的开发与利用提供一定的参考。

丝素缺失突变型丝胶资源在生物医药领域的开发与利用

丝胶是由家蚕分泌的天然活性蛋白,具有良好的生物相容性、无(低)免疫原性、无人畜共患病以及多种有益的生物活性,成为近年来备受青睐的天然生物材料。丝素缺失突变型丝胶蛋白因便于人们提取、纯化、加工和制备高品质丝胶蛋白及性能优异的生物活性材料而成为丝胶蛋白开发和利用的首选。本文基于丝素缺失突变型丝胶蛋白资源在生物医药领域的应用角度从品种培育,家蚕饲养与原材料生产,丝胶蛋白的提取与纯化,以及丝胶蛋白新型活性材料的开发与应用等方面进行了概述。

我国食品工业副产物资源化利用现状

我国是农业生产大国,农产品及食品工业的副产物数量巨大。为推进可持续化发展,这些副产物的资源化利用越来越受到人们的重视,并且很多副产物的深度开发与利用在研究与实践层面均取得了长足的进步,显示出良好的发展前景。以小麦、豆制品、油脂、蚕桑、石榴、葡萄等6大类食品加工业的副产物为例,系统阐述了我国小麦麸皮、小麦次粉、豆渣、豆粕、豆皮、油脚、脱臭馏出物、蚕蛹蛋白、蚕蛹油、石榴皮、石榴籽、葡萄皮、葡萄籽等食品工业废弃物的资源化利用现状。并以前期工作为基础,分别介绍了豆制品副产物及排放的黄浆水的资源化利用,石榴皮及石榴籽多酚提取物,石榴籽油及葡萄籽油的产品开发,桑蚕副产物制成的蚕蛹蛋白、活性肽、蚕蛹油、蚕蛹甲壳素的开发利用情况等。希望为我国食品工业副产物的深度开发与高值化利用提供借鉴和参考。

蚕蛹蛋白过敏与综合利用的研究进展

蚕蛹蛋白是一种营养价值很高的天然动物蛋白,富含有18种氨基酸,其中八种人体必需氨基酸含量超过总氨基酸的40%,且比例均衡,具有非常高的利用价值,近年来被广泛利用于多个行业中。本文介绍了蚕蛹蛋白的种类和含量,进一步汇总了过敏原的相关信息,在此基础上总结了蚕蛹蛋白在食品、饲料、医药、纺织、生物等行业中的开发利用。