Study On Differentially Expressed Genes From Microsporum Canis Incubated In Different Induced Medium By Using SSH

Objective: Microsporum canis,the most important dermatophyte of domestic animals, infect human's scalp, hair, glabrous skin and nail, causing tinea corporis,tinea cruris,tinea manuum and pedis, onychomycosis, tinea capitis and kerion.Nowadays,as living level improving and the way of life changing,people get in touch with house pet more closely, the frequency of Microsporum canis infection especially kerion is going up year by year.Several proteases produced by M.canis are considered to play a role in the virulence of this fungus,but the role of proteases in kerion remains unclear. Because nearly children's scalp was invaded by microsporum canis and caused kerion.It was doubted that the special age and part of tissue can be easily invaded. Mybe the children's scalp can produced some special pathogenic factors and they were proteases reported.In this study,M.canis was incubated on mineral medium with child's glabrous skin,scalp tissue or adult's scalp tissue to study gene expression on these proteins as sole substrates.Differential library were constructed by using SSH technique .Differential cDNA genes were screened and analyzed through genebank, then the genes'function were identified.On the basis of these results, we will study the pathogenic mechanism of kerion caused by microsporum canis, which may be helpful for kerion prevention and cure and development of new drugs'target site.Methods:1.One clinical isolated microsporum canis was incubated on rice medium (rm) for 2 weeks and the spores were parted.2.The same number of spores were cultivated on mineral medium with child's glabrous skin, scalp tissue , adult's scalp tissue and no any tissue as sole substrates for 21-28 days,and the frist three mycelium masses were collected.3.The forward and reverse cDNA library from microsporum canis incubated on mineral medium with child's scalp tissue(tester) and child's glabrous skin(driver1) was constructed by using SSH.4.The forward and reverse cDNA library from microsporum canis incubated on mineral medium with child's scalp tissue(tester) and adult's scalp skin(driver2) was constructed by using SSH.5.The four subtractive library were screened firstly by PCR.6.Five positive clones screened after PCR with different bases from each four cDNA library were randomly chosen and sequenced and analysed.7.The differentialy expressed clones screened by PCR from the forward cDNA library of microsporum canis incubated on mineral medium with child's scalp tissue(tester) and adult's scalp (driver2)were screened secondly by dot blotting.8.The second screening differentialy expressed clones were sequenced and analysed.Result:1.Microsporum canis was cultivated well on mineral medium with child's glabrous skin, scalp tissue and adult's scalp tissue and formed mycelium mass,but cultivated badly on mineral medium with no tissue.2. The forward and reverse cDNA library from microsporum canis incubated on mineral medium with child's scalp tissue(tester) and child's glabrous skin(driver1)and the forward and reverse cDNA library from microsporum canis incubated on mineral medium with child's scalp tissue(tester) and adult's scalp skin(driver2) were constructed successfully by using SSH.The PCR products showed that the base of the smear is between 250-750bp.3.Two hundred and fourty clones from each four cDNA library were selected firstly by PCR method, about 85%of them showed single band. And the most single bands were about 250-750bp .4.Four of the twenty clones were not sequenced ,and fifty of the twenty clones were unknown in fungus.One of the twenty clones was high homologous with Trichophyton rubrum mitochondrial cytb gene and NADH1 to NADH5 genes.5.Six differently expressed genes were screened by dotting blotting from the forward cDNA library of microsporum canis incubated on mineral medium with child's scalp tissue(tester) and adult's scalp skin(driver2). The sequencing results revealed that all the 6 ESTs were unknown in fungus,but 5 of the 6 ESTs had high homology and 1 had not from NCBI.Conclusion:1.It is the first time to consruct the cDNA library from microsporum canis incubated in different proteases -induced medium successfully by using suppression subtractive hybridization (SSH)at home and in the world.2.One sceened clone was high homologous with Trichophyton rubrum mitochondrial cytb gene and NADH1 to NADH5 genes.It was a base of further screening other protease genes .