Construction And Preliminary Analysis Of A CDNA Library For Demodex

Objective Demodex mites, which can cause demodicidosis, are permanent parasitic mites in hair follicles and sebaceous glands of human and mammals. Human demodicidosis is one of the most common skin diseases. The basic theoretical researches of demodex mites have been still at a low level and its molecular studies is just starting. Construction of demodex cDNA library can lay the foundation for the study on genic function. It is convenient to desire target gene from the cDNA library.Methods Demodex caprea was extract from goat skin nodules, under the dissecting microscope, the mite sample was separated from the sebum and cleaned repeatedly. The cleaned mites were collected to a homemade miniature mortar and grinded into fragments at Low temperature. The total RNA was ertracted from the mites using TransZol Up kit. Detect the integrity of the total RNA by gels electrophoresis. First-strand cDNA is synthesized by reverse transcription and double-strand cDNA is synthesized and amplified by LD (long-distance) PCR. After size fractionation with Universal DNA purification Kit, the cDNA fragments longer than500bp are ligated to pGM-T vector. The ligation mixture was transformed into the competent cells of E.coli TOP10to complete the construction of cDNA library for the mites. the content and recombination rate of cDNA library were tested and the length of inserted fragments was analyzed by PCR.Results plenty Demodex mites were collected for the experiment. Demodex total RNA was extracted successfully. The RNA structural integrity and no significant degradation, can meet the needs of the cDNA library construction. Double-stranded cDNA is dispersed and concentrate in the200to3000bp in line with the law of eukaryotic mRNA distribution. The cDNA fragments longer than500bp were ligated to pGM-T vector, and then transform the ligation mixture into the competent cells of E.coli TOP10to complete the construction of cDNA library for the Deomdex. The content of cDNA library is estimated as2.56×106cfu with recombination rate reach to98%. The average length of inserted cDNA fragment was longer than1000bp.Conclusion In this study, using the homemade miniature mortar of our project team, extract the Demodex total RNA successfully, and then construct the cDNA library of goats Demodex, laid the foundation for the future study of specific genes. In addition, The results showed that the quality of cDNA library is good enough to set the basis for future exploring the genes of Demodex and also directly provides a theoretical foundation and technical methods to build human Demodex cDNA library.