| In the privious experiments we detected and analysed the difference of the gene cluster expression in AQP4-knockout mice brain tissues by using the genechip technology, in which three kinds of mouse, including the normal phenotype of wide-type CD1 mice; the normal phenotype of AQP4-knockout mice and the spontaneous hydrocephalus of AQP4-knockout mice were studied. We screened out some pertinent possible genes, which provided new clues for studying further the pathogenesis of hydrocephalus. The results displayed mRNA level of Glia maturation factor beta(GMFβ) decreased notably in the hydrocephalus pathological conditions. It may indicate GMFβas a possibly relevant protein factor participating in the hydrocephalus pathological process and need to be further studied.GMFβcoding sequence was amplified by RT-PCR from total RNA of CD1 mouse brain tissue. Then it was subcloned into the prokaryotic expression vector of pGEX-2T. We successfully obtained the recombinant plasmid pGEX-2T- GMFβand transformed it into E.coli BL21/DE3. The fusion protein of GST-GMFβwere expressed via the induction of IPTG and analyzed by SDS-PAGE. In order to produced the large amount of proteins, firstly by optimizing the expression conditions and extraction conditions, we raised the output of the soluble fusion proteins in the supernatant after bacteria lysis. Then we used Glutathione Sepharose 4B for the affinity purification of GST control proteins or GST- GMFβfusion proteins. Subsequently, the rabbit polyclonal antibody against GMFβwere generated and the antiserum with high titer and specificity were detected in ELISA and Immunohistochemistry methods. Finally, the declined GMFβin the brain tissue of AQP4-/- spontaneous hydrocephalus mice were detected in Semiquantitative analysis of RT-PCR and Western blotting methods, compared with the normal mouse of wild type or AQP4-/-. The results accorded with gene-chip testing on the whole. |
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