Cloning, Expression and Polyclonal Antibody Preparation of Transcription Factors from Plasmodium Falciparum

Abstract: Malaria is a major threat to human life and health, in spread of more than ninetycountries and regions. Roughly forty percent of the world’s population is at risk ofmalaria, which is a mosquito-brone disease that causes upwards of one millionfatalities each year, mostly in infants, young children and pregnant women.P.falciparum, which brings cerebral malaria and pregnant associated malaria, is themajor causes of deadly malaria cases. The complicated life-cycle of p.falciparumcomprised of three major developmental stages, the mosquito, liver andintraerythrocytic stages. A rigorously regulated gene expression pattern controls thedifferentiation of the parasite from one stage to another during its life cycle.Regulation at the transcriptional level is important for the temporal expression of generequired at each stage of p.falciparum development. However, the molecularmechanisms that control gene expression in this parasite remain poorly understood.The identification of cis-sequence elements and their corresponding trans-actingfactors has been a difficult task because intergenic regions in p.falciparum areparticularly A/T-rich sequences.So far,only several studies have identified upstreamcis-acting sequence elements in P.falciparum promoters that specifically interact withnuclear factors.Eukaryotic transcription requires the assembly of a large, multisubunitpre-initiation complex at the promoter. RNA polymerase II, the polymerase thattranscribes genes of coding proteins, itself unable to locate and bind promoters. Just asothers eukaryote, the general transcription factors TFIIA,B,D,E,F and H mustcombine with the promotors to form the special structure, provide binding site forrecognition of RNA polymerase II. Therefor, transcription required numeroustranscription factors and accessory factors to form complicate transcription apparatusto start transcription process. To date, the studies of the p.falciparum genome sequence having revealed fewcis-regulatory elements and associated transcription factors, and little known abouttheir’s function. Genomic center of Novatis obtained 153 transcription factors byusing bioinformatics methods analyzed datas that come from computationalannotation of p.falciparum. The purpose of this study is to prepare polycloneantibodies of five transcription factors, providing foundation for the study oftranscription factors and DNA binding sites.Five specific primer pairs were designed and synthesized according to thetranscription fanctor sequneces that publiced in plasmoDB. The genome DNA ofp.falciparum3D7, was used as template to amplify transcription factor sequences bypolymerase chain reaction(PCR).Sequence verification of the amplicons by 1%agarose gel electrophoresis shown that amplification fragments size coincided withthe prediction fragment size. Five DNA fragments were cloned into pMD18-T vector,then the fragments were confirmed transcription factors by DNA sequencing. Thesequence analysis by DNAMAM showed that they were consistent with the sequencesof transcription factors publised in plasmoDB, the homology were 100% innucleotide.Recombination plasmid pMD-459、pMD-516、pMD-984,pMD-963,pMD-981 byrestriction endonuclease digestion with BamHI and XhoI,BamH I and EcoR I,EcoRI and Xho I,respectively, and were correctly inserted into sites cut with the samerestriction endonucleases of procaryotic expression vector pGEX-4T-1,then ligation、transformation、extracting plasmid. After confirmation by PCR and restrictionendonuclease digestion,DNA sequencing. The results of identification were allcorrect. The prokaryotic expression plasmid pGEX-984、pGEX-516、pGEX-459、pGEX-963、pGEX-981 were transformed into E.coli BL21 cell and to shakeculture.Then they were inducted by IPTG, optimized cultural condition and analyzedexistence and solubility of the recombination protein by SD-SPAGE and Western blot.By induction with IPTG, the E.coli BL21 with pGEX-984、pGEX-516、pGEX-459、pGEX-963 expressed the recombination protein,which stranded at the 64kD、45kD、 43kD、64kD by SDS-PAGE and Western blot analysis comparing to E.coli BL21uninduced, and proteins existed in soluble and inclusion body forms. The E.coli BL21with pGEX-981 also expressed recombination protein, but its molecular weight lowerthan prediction of bioinformatics method, and the protein existed only in solubleforms.The optimized condition for induction was OD600=0.8~1.0,0.1mmol/l IPTG,25℃, 5h~9h.E.coli BL21 with pGEX-984、pGEX-516、pGEX-459、pGEX-963、pGEX-981were amplificated by shaking culture under optimized conditions and collected bycentrifugating, then the bacterium was clearaged. We purified soluble protein insupernatant of E.coli BL21 lysate by Gluthathione-Sepharose 4B affinity purification,and we found the proteins were puried by SDS-PAGE and Western blot analysis.Recombination proteins as antigen, emulsifying with Freund’s adjuvant, the emulsionwas immuned Wistar Rats by intramuscular injection.Antiserum was obtained afterfour times immunity,pured IgG of polyclonal antibodies through affinitychromatography column and assessed the titer of polyclonal antibodies by ELISA.The specificity of polyclonal antibodies were shown by Western blot , then thepolyclonal antibodies were used in localizing transcription factors in parasite byimmunofluore-scence.Parasites were cultivated in candle-jar technique at 37℃,Malaria culture medium was RPMI-1640 culture medium(10% human serum)including 0.03% glutamine and 0.025mg/ml gentamicin. Observation morphous anddevelopment of parasite every 24h by Giema-stained and AO-stained(acridineorange).When the rate of infected RBC(red blood cell) up to 5%, harvested parasites bycentrifugalization,the sediment of parasites were lysed by 0.15% saponin, then adding5×SDS-PAGE loading buffer and boiled at 100℃for 10 min to clearage proteins ofparasite. The denatured proteins were separated by 12% SDS-PAGE.Proteins weresubjected to Western blot and immunologically screened using polyclonal antibodies,alkaline phosphatase conjugated rabbit anti-Rat IgG was used as the second antibody.The results of Western blot shown polyclonal antibodies can recognition primal transcription factors of p.falciparum 3D7. The polyclonal antibodies were used inlocalizing transcription factors in parasites by immunofluorescence, and nucleus wascounterstain with PI. The result indicated that transcription factors were distributed inboth cytoplasm and nuclear of parasites. Furthermore, polyclonal antibodies canconfirmed expression of transcription factors in cell nuclear.We got five purified recombination proteins of transcription factor and preparedhigh titer and specific Rat anti-p.falciparum transcription factor polyclonal antibodies,localizing transcription factors by immunofluorescence; which would providefundamental for further research regulatory sequence of transcription factors andanalyze their’s functions…
Key words: P.falciparum; transcription factor; recombination protein; polyclonal antibody

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