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Objective To reversing methicillin-resistant Staphylococcus(MRS) to methicillin-susceptible Staphylococcus(MSS) by changing nutritional conditions and continuous transfer of culture .Methods MRS trains separating from clinical specimens were cultured in different conditions ,continuous cultural transfer ,and drug sensitive test were proceeded periodically to observe the phe-notypic and chemical reaction change of MRS .The mecA gene were detected of the original and mutant strains by polymerase chain reaction(PCR) ,then the gene sequenced and compared .Results 53 MRS strains were studied .6 strains were phenotype successful-ly converted to MSS in different cultural conditions ,among them mecA gene was undetected in 2 strains ,and down expressed in 4 strains .Conclusion The MRS strains separated from clinical specimens may revert to MSS by culture under different nutritional conditions .The mecA gene of MRS may be lost or lower expressed and the MRS and mutant strains may be different in genomics .
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AIM:To generate monoclonal antibodies against human augmenter of liver regeneration (rhALR). METHODS:After BALB/C mice were immunized by the purified rhALR, the cells of spleen were fused with the cells of SP2/0; The titer and speciality were respectively fathomed from ascites or foster fluid by ELISA and Western-blot test. RESULTS:2 hybridoma cell lines were successfully obtained. The McAbs titer from ascites and foster fluid are respectively about 10-3-10-5 and 10-2-10-3. It is evident that the two McAbs were directed at different epitopes. CONCLUSIONS:The McAbs have higher speciality. It is significantly useful of the value that how hALR distribute in tissue organs, how the hALR signals the metabolism in the body and the control distribution of the hALR on cell growth on the translational level and so on is researched.
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AIM:To check the physical interaction between GST-Na+-K+-ATPase domain and recombinant human augmenter of liver regeneration (rhALR) by GST pull down assay. METHODS: With PCR and genetic recombinant techniques, the coding region of ? subunit of Na+-K+-ATPase was cloned into expressing plasmid pGEX-4T and identified by endonuclease digestion and sequencing methods. Under the inducing of 0.1 mmol/L IPTG, the fusion protein GST-Na+-K+-ATPase domain was highly expressed by E.coli DH-5?. After hypersound quassating, the GST-Na+-K+-ATPase domain was purified by glutathione agarose beads and the physical interaction with rhALR was checked by GST pull down assay. RESULTS: Analysis by SDS-PAGE showed the rhALRs of monomer and dimmer in GST-Na+-K+-ATPase domain lane. The Western blotting of the GST-pull down assay showed the same results as well. CONCLUSION: The Na+-K+-ATPase domain is associated with rhALR specifically in vitro.
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AIM: To screen the proteins interacting with human augmenter of liver regeneration (hALR) by yeast two-hybrid system and to study the mechanism of hALR action. METHODS: hALR bait plasmid was constructed by ligating the gene of hALR into pGBKT7, then transformed into yeast AH109. The yeast strain AH109 containing pGBKT7-hALR was mated with yeast Y187 containing human liver cDNA library plasmid. Diploid yeast was plated on SD/-trp-leu-his-ade (QDO) for screening and on QDO containing X-?-gal for further selection.The AD/library inserts were amplified by PCR and the PCR products were characterized by digesting with Sau3AⅠ and HaeⅢ restriction enzyme to eliminate the duplicates. After sequencing, the positive clones were analysed by bioinformatics. RESULTS: Several positive clones were obtaind. The sequencing and analysis shown that one of them is 669 bp DNA fragment encoding ? subunit of Na +,K +-ATPase. The 224 bp 3′terminal DNA fragment is non-encoder region, and the 445 bp 5′terminal DNA encodes C-terminal 147 amino acid residues of Na +, K +-ATPase ? subunit. CONCLUSION: The results of screening proteins using yeast two-hybrid system showed that hALR could interact directly with Na +, K +-ATPase in the yeast cell.
