Extraction Of Total Proteins From Demodex And Qualification Of Their Molecular Weights

Demodex Humanis, also known as Hair Follicle Mite, belong to Arthropoda, Demodicidae, which includes Demodex folliculorum and Demodex brevis. These two kinds of Demodex are respectively parasite in hair follicles and sebaceous glands of humans, causing demodicidosis which is one of the most common dermatosis, and leading to folliculitis, acne, rosacea, blepharitis, sebaceous cyst, alopecia, etc. Babies can get the infection of demodex mites from their mothers’ skin during lactation. Demodex Humanis is prevalent all over the world. Humans get demodex infection by direct or inderect contact transmission. In last decade, the study on demodicidosis gets the focus of research, to which parasitologists and dermatologists have payed great attention. Searching the literature, a few researches of human Demodex mites were published and mostly were domestic, which mainly refer to aspects of morphology and clinical therapy. The domestic researches were more indepth and general. Although the theory of mechanical pathogenesis was approved by seeing mites’sharp structures of body surface, their chemical and immunopathological mechanisms have not been known. Dermatological doctors have to deal with demodicidosis using symptomatic therapy. Primary researches of human demodex in the field of molecular biology are just at the beginning stage. The extraction of DNA, RNA as well as construction of cDNA library of demodex mites were reported. However, we have not seen the experimental investigation of demodex gene expression, gene function. The main reasons for lag of the molecular biology study on human demodex are that demodex samples are collected and preprocessed difficult due to there was no special instrument and technique. Because of the microsize of mites, they have to be cleaned under the microscope. There is also no mature method to extract demodex DNA, RNA and protein. These technical problems were solved by our research team, and we also proved that the genetic distance between Demodex caprae and Demodex brevis is more close than that between Demodex folliculorum and Demodex brevis. In theory, Demodex caprae is the alternative model of human demodex. Proteomics is the feature of post-genomic era in the life science, which not only provides the material basis of life, but also provides theoretical basis and solutions to clarify the mechanism of many diseases. Proteomics is a new and fundamental way to explore demodex physiological metabolism and pathogenic mechanism.Objective Owing to the report of demodex proteomics is rare, we try to explore the optimal extraction method of demodex protein, extract the whole proteins of Demodex caprae and preliminarily analyze its compositions, which provide the demodex protien database of NCBI with datas, lay the foundation for the further study on demodex physiological metabolism and pathogenic materials and direct the treatment and research on demodecidosis.Methods Demodex caprae was designated as the experimental samples, which were collected from the contents of Boer Goat’s skin nodules. Experimental apparatus and reagents were prepared and sterilized. Nodules were cut open from inside the skin of Demodex caprae. The caseous materials (0.5g) were fetched from the nodules and transfered to a micro mortars to which drop equivalent detergent and fully ground them. The mixed emulsion was transferred to a glass tubes and then diluted with 8 ml PBS. The diluted emulsion was fully mixed by glass syringe and then precipitated 1 h at 20℃, and then the supernatant was removed. The above-mentioned processes repeated 3 times. The precipitate was transferred to 1.5 ml Eppendorf tubes and centrifuged 20 minutes at speed of 8000 rpm, the supernatant remove followed, and the rest is clean simple. The treated mite samples were equally divided into Eppendorf tubes and then cryopreserved at -80℃ Two methods, mechanical homogenization and liquid nitrogen grinding, were used to smash mites to pieces, and then these two groups were respectively cracked by RIPA lysate and self-made lysate. Four groups of cracked samples were produced. These samples were centrifuged 60 minutes at speed of 13000 rpm and then the supernatants were respectively collected into a Eppendorf tube to store in a refrigerator at-20℃. The protien concentrations of samples extracted respectively by RIPA lysate and self-made lysate were measured using "Non-interference-type protein concentration assay kit". The extraction effects were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Parts of protein bands were identified by Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrorometry (MALDI-TOF-MS). The NCBI database was searched with Mascot software to analyze the protein components of Demodex caprae.Results The total proteins of Demodex caprae was extracted successfully. The effect of smashing mites with liquid nitrogen grinding was better than that with mechanical homogenization method. The effect of extracting mite protein using self-made lysate was better than that using conventional RIPA lysate. The combination of liquid nitrogen grinding and self-made lysate is the best way to extract Demodex protein. Through analysis and identify parts of protein bands by the method of MALDI-TOF-MS and the search of the NCBI database, the natures of 21 mite proteins were confirmed. Among these, the bands of 170 kD were actin and cytoplasmic actin, bands of 130 kD were glutamine ligase and adenosine acyltransferase, and most of bands below 15 kD are histone. The Demodex protein was first extracted so there was no its exclusive database. The proteins lying in 70 kD and 15 kD were not be found in the Arachnida protein database, which might be the specific protein of Demodex.Conclusion The method which is composed of liquid nitrogen grinding and self-made lysate is the best way to extract Demodex caprae proteins, with this method we successfully extract the total proteins of Demodex mites. Our study utilizes SDS-PAGE which is based on different molecular weights of proteins, and 2-DE which depends on different molecular weights and protein isoelectric points, to sort out Demodex proteins. Then we apply MALDI-TOF-MS to analysis parts of protein bands. Our studies lay the foundation for the study of Demodex mites in the area of Proteomics and provide references to the study of functional protein and chemical pathogenic mechanisms of Demodex mites.