RESUMO
The effects of melittin on the activities of Na+-K+-ATPase and glucose-6-phosphate dehydrogenase (G-6-PD) which are on the membrane of red blood cell (RBC) are chosen as the index of this study. The possible target sites of these effects through enzyme activity determination by spectrophotometry are investigated, and the hemolytic process and the activity change of these two types of enzymes on the RBC membrane are discussed. The results show that the main mode of melittin inhibition to the activity of enzymes on the RBC membrane is the coexistence of adhesion/insertion form and free-state form, and the effect of the former is more stronger than the latter. The K+ binding site of Na+-K+-ATPase is one of the target sites of melittin. The membrane-insertion process of melittin synchronizes with the action of melittin on this enzyme. Melittin slowly inhibits the catalysis of G-6-PD through the action on G-6-P and NADP, and the extent in which melittin forms tetramers isclosely related to the enzyme activity. EDTA inhibits the aggregation of melittin, and interferes with its action on G-6-P. The biochemical mechanisms of melittin effects on the substrate G-6-P and the coenzyme NADP are similar, and the inhibition of melittin is not related to the structure of G-6-PD.
RESUMO
Objective To achieve the fusion expression of the entire human beta-defensin-3(hBD-3) gene. Method We synthesized two oligonucleotide primers accor ding to the codon preference of Escherichia coli. The gene was cloned into p GEX -4T-2 to establish the pGEX-4T-2-hBD-3 as the fusion expression vector by PCR. Transformed into E.coli strain DH5α, the express vector was induced an d ex pressed by IPTG. The fusion protein GST-hBD-3 was obtained by repeated cycles of freezing and thawing, cut by thrombin to attain the recombinant hBD-3 protei n. Result The result of the antibacterial peptide agarose diffu sion assay shows the antibacterial activity of the rhBD-3 against the S.aureu s exists, and it reaches 0.843U. Conclusion The fusion expr ession of the hBD-3 gene is successful.
RESUMO
Objective To induce the the 17th condon antonymous mutagenesis of salivary histatin 5(HRP5) cDNA,express the mutant and hrp5 in Pichia pastoris,and to study the effects of mutation on expression.Methods According to the Pichia pastoris' codon bias,two pairs of primers(H1 and H2,H3 and H4) were designed.H1 and H2,H3 and H4 have complementary 3' end,and the EcoR I site was added to the 5' end of H1 and H3,Sal I site to H2,H4.The cDNA of hrp5 and hrp5' was generated with PCR by H1 and H2,H3 and H4,respectively.The secrete vector pPICZ?-A,hrp5 and hrp5' were digested with EcoR I +Sal I,linkede by T4 DNA ligase and transformed to E.coli TOP10 comptetent cell,positive colonies were screened on LB plates with Zeocin.The recombinant plasmids pPICZ?-A-hrp5 and pPICZ?-A-hrp5' identified by digestion and DNA sequencing were amplified largely,linearized by Sac I and transformed to GS115 comptetent cell by electroporation,positive colonies were screened on YEPD plates with Zeocin,the recombinant GS115 were confirmed by PCR,cultured and induced expression by methanol.The amount and anticandidal activity of the expressed products was compared with synthetic HRP5.Results Both hrp5 and hrp5' were integrated into the genome of GS115 and expressed successfully,the anticandidal activity of the recombinant HRP5 and HRP5' was identical with synthetic HRP5,the amount of expressed HRP5 and HRP5' was 4?mol/L and 5?mol/L,respectively.Conclusion Both the recombinant HRP5 and HRP5' showed better anticandidal activity.The amount of expressed prodcts increased 25% by substituting Asn for Lys17 without changing anticandidal activity.