Screening Target-Protein Gene Of Artemisinin Antimalarials Using Drug-Western Methoed From CDNA Expression Library

The cDNA expression library that contains no intron theoretically include all expressed genes and conserve resource genes permanently.lt can be used to find out and clone some genes which can express particular protein with modern molecular biological techniques,such as immunological screening, drug-prob screening, southern et al.lt is very important to study the life nature of plasmodium falciparum in molecular level. With the developments of these studies,the drug-resistant mechanism of the plasmodium falciparum and the genes of some specific medicine binding protein can be made well-known. At the same time, the researches will do good to explain the mechanism of some specific medicines in order to design and screen new anti-malaria drugs.Artemisinin was originally isolated from Artemisia annua ,a herb used as the ancient Chinese herbal remedy.Artemisinin and its derivatives are new types of Antimalarial with stable endoperoxide bridges differing from others, for instance chloroquine. Lots of pharmacological and clinical experiments indicate that artemisinin is high effective with low toxicity.Evidences from many laboratories suggest that the antimalarial function of artemisinin depends on the cleavage of the endoperoxide by binding to intraparasitic heme. The ultimate product of the endoperoxide cleavage is a carbon-centered free radical which has functions as an alkylating agent,reacting with both heme and parasite proteins.Tanaka reported a novel method to isolate genes of drug-binding proteins directly from a cDNA expression library by using the combination of drug and marker molecule. It is clearly demonstrated that this method is useful for direct identification and cloning of genes encoding cellular drug-binding proteins without purification.This excellent feature of drug-western method allows more rapid and convenient identification of cellular drug-binding proteins than the other methods described before.Translationally controlled tumor protein(TCTP) was first identified as a tumor protein in ascitic tumor and erythroleukemia in mouse. Subsequently.it has been found in all normal cells examined so far except the kidney cells.TCTP homologues among the species are highly conserved, which suggest that the protein might have an essential function in cell.lt was reported that TCTP caused the release of histamine from IgE+ basophils and was capable of various functions including calcium binding.metal homeostasis,intracellular signaling and reacting with antimalarial drugs.Basing on these backgrounds and motives,the cONA expressing library of Plasmodium falciparum from Hainan was constructed. We screened the cDNA expression library by using 12 B -deoxoartemisinyl -(4'-oxyacetic acid) Phenyl ether conjugated with BSA.One of the screened positive clones was inserted TCTP of plasmodium falciparum from Hainan.The TCTP was cloned to express protein in order to provide materials for further study.Constructing cDNA expressing library of erythrocytic plasmodium falciparum from Hainan :The total RNA was obtained by using . Triplix Kit. A modified oligo (dT)primer(CDS ffi PCR primer)was used in the single-stranded(ss)DNA synthesis reaction.The ss-DNA was reversely transcripted from total RNA.Double-stranded (ds) cDNA was amplified by Long-Distance (LD)PCRAfter the digestion with Proteinase K and Sfi I,the cDNA with no less than 200bp was collected and purified by Glass-milk kitThe library was constructed after the ligation of cDNA to TiplEx2 phage particlepackaged with the packaging extract system in vitro.A high titer and high recombinant ratio of cDNA library was constructed. The constructed cDNA library is suitable to r further study.Screening Target'Gene of Artemisinin Antimalarials Using Drug-Western from cDNA Expressing Library :12 B -deoxoartemisinyl -(4'-oxyacetic acid) Phenyl ether was linked to BSA by using of EDC cross! inker and the product acted as drug-probe. The plaque of bacteriophage was transfer to PVDF membrane after the library amplified. The drug-probe incubated with me...