Cloning, Expression Of Cdc42Gene From Hainan Yellow Cattle And Tissue Expression Analysis

Cdc42(Cell division cycle42) is an important functions gene and be related to developm-ment.Cdc42cDNA was obtained from the Hainan Yellow Cattle Peripheral Blood leukocyte cDNA library, and the pET28a-Cdc42recombinant plasmid were constructed. Protein expre-ssion in E.coli BL21(DE3) was induced by IPTG and analyzed by SDS-PAGE and Western blot. Real-time fluorescence quantitative PCR was used to examined the mRNA expression levels of Cdc42and Western blot was used to protein expression levels of Cdc42in heart, liver, spleen tissues.Immunohistochemical were performed to analysis of characteristics of Cdc42protein in a various tissues.The alignment results showed that the Cdc42was composed of576base pairs, encoding191amino acids. The protein about30kD with His-tag as expected was expressed after induction with IPTG in E.coli BL21(DE3) and detected by Western Blot, The mainly forms of protein is inclusion bodies and indicated that prokaryotic expression vector of Cdc42was constructed and expressed successfully.The results of Real-Time quantitative PCR indicated that Cdc42mRNA has the highest expression level in lung, then spleen, kidney, intestine and heart decline one by one,the lowest level in liver.There are satistically significant differences of Cdc42mRNA express.The results of Western blot indicated that the protein expression level in lung, spleen, kidney, intestine were identical with the mRNA expression level, but liver and heart are opposite. Immunohistochemical analysis showed that positive reaction products of the Cdc42protein present in the cytoplas.We detected gene Cdc42all expressed in each tissues. Although Cdc42express in different tissues widely, it has not tissue specificity.