To Construct DNA Subtractive Library Of Kidney Yin Deficiency Syndrome Of Lupus Nephropathy

OBJECTIVE: To build up the method of SSH and test it's power. To construct DNA subtractive library of kidney Yin deficiency of lupus nephropathy.METHODS: suppression subtractive hybridization(SSH) was used to obtain differentially genes associated with kidney yin deficiency syndrome of LN , comparing blood DNA of patients with kidney yin deficiency of LN (as Tester or Driver) with normal blood DNA (as Tester or Driver). After Rsa I enzyme restriction, Tester DNA were divided two groups to ligate to the specific adaptor 1 and adaptor 2.Then Tester DNA were hybridized with Driver DNA twice and underwent nested PCR twice. The PCR product was ligated with U plasmid vectors, transformed into E. coli TOP10, screened through the blue-white screening system, and then positive recombinant clones were confirmed by PCR method. After that, we farther screened the positive clones using the dot blot technology. We select the positive clones from the DNA subtractive library to go to sequencing and homology analysis.RESULTS: This study successfully founded the way to screening the differential genes of complex genome — SSH. For the first time, this study successfully constructed the DNA subtractive library of the kidney yin deficiency syndrome of Chinese Han LN. After the blue-white screening system, 295 clones were got in the norientatial subtractive library, and 286 clones were got in the reverse subtractive library. This study constructed the norientatial subtractive library of the kidney yindeficiency syndrome in LN. After that, we screened the positive clones using the dot blot technology. We respectively selected 5 positive clones form the norientatial and reverse subtractive library, to go to sequencing and homology analysis. 1 clone of norientatial subtractive library sequencing failed, 1 clone is the DNA repeated sequence, and the other 3 clones are the genes related with immunology (interleukin 28 receptor interleukin 10), and 1 is unknown. 1 clone of reverse subtractive library sequencing failed, 1 clone is the DNA repeated sequence, 1 clone is not found homologous genes, and the other 1 clone is p40 and 1 is unknown.CONCLUSIONrSSH technology can be used to clone the related genes of Chinese syndrome, but it needs some improvements. We had successfully constructed the DNA subtractive library of the kidney yin deficiency syndrome of LN, and We had confirmed there were some different genes fragments. But we can't confirm whether these genes are the related genes of the kidney yin deficiency syndrome of LN. Only when we confirm that they would exert some effect to the kidney yin deficiency syndrome of LN, can the gene be the related gene of the kidney yin deficiency syndrome of LN.We'U construct more subtractive libraries of kidney Yin deficiency of different disease, so can we find the related genes of yin deficiency.