RESUMO
Some effective antiparasitic drugs for chemotherapy schistosomiasis, such as praziquantel and artemether, are proved to act on lactate dehydrogenase (LDH) in vitro, but detailed molecular action mechanisms of these have not yet been elucidated. To this end, the sequence encoding LDH of Schistosoma japonicum (SjLDH) was amplified from the cDNA library of the adult parasite. Sequence analysis revealed an open reading frame (ORF) of 999 bp, encoding 332 amino acids with a molecular weight of 36.120 kDa. It has three transmembrane regions and may be located in the cytoplasm membrane. The encoding ORF was inserted into pET-28a vector and expressed in Escherichia coli (E. coli) induced by IPTG. A 36-kDa protein with 6xHis-tag was purified by metal affinity column and identified by SDS-PAGE and Western blot, which had high LDH activity of 379 U/mg. Characterization such as Michaelis constant (K (m)) and maximum velocity (V (max)), optimum pH, and temperature of the protein were assayed. It provides a model for drug screening on SjLDH.
Assuntos
L-Lactato Desidrogenase/genética , Praziquantel/farmacologia , Schistosoma japonicum/enzimologia , Esquistossomicidas/farmacologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar , DNA de Helmintos , Etiquetas de Sequências Expressas , Expressão Gênica , Biblioteca Gênica , Proteínas de Helminto/química , Proteínas de Helminto/genética , Cinética , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Schistosoma japonicum/efeitos dos fármacosRESUMO
BACKGROUND: Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase II beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E. coli). METHODS: The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3'RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E. coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. RESULTS: A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase II beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E. coli JM109. The recombinant protein could be recognized by the S. japonicum infected rabbit serum. CONCLUSION: The full-length cDNA sequences encoding S. japonicum casein kinase II beta subunit were firstly sequenced, cloned, and expressed in E. coli.
Assuntos
Caseína Quinase II/genética , Schistosoma japonicum/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Caseína Quinase II/química , Clonagem Molecular , DNA Complementar/química , DNA Complementar/isolamento & purificação , Escherichia coli/genética , Dados de Sequência Molecular , Coelhos , Schistosoma japonicum/genéticaRESUMO
<p><b>BACKGROUND</b>Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase II beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E. coli).</p><p><b>METHODS</b>The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3'RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E. coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot.</p><p><b>RESULTS</b>A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase II beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E. coli JM109. The recombinant protein could be recognized by the S. japonicum infected rabbit serum.</p><p><b>CONCLUSION</b>The full-length cDNA sequences encoding S. japonicum casein kinase II beta subunit were firstly sequenced, cloned, and expressed in E. coli.</p>
Assuntos
Animais , Coelhos , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Caseína Quinase II , Química , Genética , Clonagem Molecular , DNA Complementar , Química , Escherichia coli , Genética , Dados de Sequência Molecular , Schistosoma japonicum , GenéticaRESUMO
Objective To observe the effects of low frequency transcranial magnetic stimulation (LF-TMS) on the electroencephalogram (EEG),expression of NPY in hippocampus in pilocarpine (PLO)-induced epileptic rats. Methods Forty male Sprague-Dawley rats (240-260 g) were used to establish a model of epilepsy by in- tradominal injection of pilocarpine,and then randomized into 2 groups:a control group and an intervention group. The control group was treated by sham LF-TMS,while the intervention group was treated by LF-TMS once daily for 7 days.Ⅰgroup simply celiac inject pilocarpine.Ⅱgroup celiac inject PLO after LF-TMS.The EEG was recorded in both groups and the checked pathology.Pathological item include HE staining,NPY immunohisto chemical staining. Results The latency for seizure attack was significantly lengthened,while the frequency of seizure attack and times of major seizure attack were significantly decreased in the intervention group.The HE staining revealed significant de- generation and necrosis of neurons in the hippocampus,especially in the CA3 region,in rats in the control group. The pathologic changes were significantly less severe in the intervention,Immunohistochemical staining showed a sig- nificantly higher expression of NPY in the hippocampus as compared with the intervention group. Conclusion U- sing the PLO-induced epilepsy model,LF-TMS could not only postpone the generation of kindling but also inhibit the progress of epilepsy.The increased NPY expression in the hippocampusin the intervention group implied a close rela- tionship between NPY and epilepsy attack.
