Dissertation > Medicine, health > Basic Medical > Human biochemistry, molecular biology

Cloning, Expression and Purification of PDF Gene of Helicobacter Pylori and Studies on the Activity of the Recombinant Protein

Author ZuoShanShan
Tutor ZhangJinGang
School PLA Military Academy of Medical Sciences
Course Immunology
Keywords Helicobacter pylori Peptide deformylase enzyme Peptide deformylase gene Drug targets
CLC R346
Type Master's thesis
Year 2005
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Helicobacter pylori ( Helicobacter pylori , Hp ) , gastritis, peptic ulcer disease-causing factor , closely related to the incidence of gastric cancer , due to the high rate of people infected with harm , the World Health Organization has listed it as the first class carcinogen . The traditional treatment with antibiotics there is a low cure rate , recurrence rate and strain resistance , the development of new antimicrobial agents become problems to be solved . Looking for specific , accurate target screening of new antimicrobial agents . Compared with mammalian , bacterial protein synthesis has its own characteristics. Bacterial protein synthesis , the polypeptide chain N-terminal formylated methionine , the peptide deformylase enzymes ( the peptide deformylase PDF ) needs to begin by in order to become mature protein after removal of methyl , or can not complete the process , the bacteria can not grow . Therefore , PDF has become a new type of potential targets for drug screening . In this experiment Helicobacter pylori , a standard strain , strain an Australian and China's different regions ( north, east, southwest, south ) of 12 isolates , its def gene cloning and sequence comparison and tried in a different expression vector . GenBank published international standard H. pylori strains 26695 , J99 peptide demethyl- acylase gene ( the deformylase def ) the nucleotide sequence in the culture and identification on the basis of extraction of bacterial DNA , primers were designed to PCR methods expansion increase def, and cloned into pGEM - T easy vector . Sequence analysis showed that the cloned strain def gene was 525bp, achieve consistency of the nucleotide sequence of the 97.50% up to 98.13% , the consistency of the amino acid sequence of function-related motifs point does not mutate . This result suggests that the same with the other bacteria , Hp has a highly conserved peptide demethyl- acylase gene (def) . This work laid the foundation for the expression of PDF , provides a theoretical basis for inhibitor screening target in PDF . With other strains of high homology DM strain , the def and cloned into pET - 32a ( ) expression vector for expression in the host cells BL21 (DE3) . The results show that the The def gene fusion protein highly expressed , but most of the product form of inclusion bodies , lower levels of soluble fraction . By metal chelate and pro-

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