RESUMEN
Objective: To design and confirm the cleavage activity of ribozyme Rz217 to oncogene ki-rasG12V messenger RNA and search for a new method for gene therapy targeting oncogene ki-ras. Methods: According to Symon' s principle,design an ribozyme specific for ki-rasc12v mRNA, both the constructs for transcription in vitro of ribozyme Rz217 and ki-ras exonl and the mammalian expression constructs of ribozyme Rz217 were constructed by DNA recombinant technique,ribozyme Rz217 and ki-ras exonl mRNA was obtained by transcription in vitro with T7 and SP6 RNA polymerase. Pancre atic carcinoma cell line PaTu8988 and human hepatocellular carcinomacell line BEL7404 were transfected with Rz217 mammalian expression constructs and the level of endogenous ki-rasG12V mRNA or ki-ras mRNA was determined by semiquantitative RT-PCR. Results: Not only in vitro but also in vivo, ribozyme Rz217 can cleave the mRNA of oncogene ki-ras (G12V) in site-specific manner and can not cleave the mRNA of wild-type ki-ras. Conclusion: Ribozyme Rz217 can cleave oncogene ki-rasc12v mRNA and the cleveage is specific for ki-rasG12V mRNA.
RESUMEN
190-kilodalton glycoprotein (P190) of Plasmodium falciparum. precursor of the major surface protein of merozoites, is considered a promising candidate for blood stage malarial vaccine. We designed six primers according to the sequence of MAD20 strain, with a GC clamp and BamHI site at the 5'- end of each one, and a GC clamp and Xbal site at the 3'- end of each one. The primers were synthesized by phosphoramidite approach (User's Manual of ABI Company) and purified using HPLC. Three fragments in the second, third and fourth conserved regions of P190 gene of Plasmodium falciparum FCC1/HN strain isolated from the blood of patients in Hainan Province of China were amplified by the polymerase chain reaction (PCR) technique. The amplified fragments were subcloned into pUC18 vectors and sequenced using the dideoxy chain termination method. All three regions of P190 gene of FCCl/HN strain also were highly conservative as compared with P190 gene of MAD20 (Papua New Guinea isolate), K1 (Thailand isolate), Wellcome (West Africa isolate) and CAMP (Malaysia) strains of Plasmodium falciparum. The C at position 81 in the second conserved block of P190 gene of FCC1/HN isolate was substituted by T, which did not change the amino acid determined by the coden corresponding to the substitution.
RESUMEN
Two DNA fragments, designated as P190CRI (AA1-55) and P190CRV (AA1597-1667) respectively, which encoded amino acid residues of conserved region I and V on the P190 antigen, were amplified by polymerase chain reaction from genomic DNA in FCC1/HN strain of Plasmodium falcipamm isolated from Hainan Province, China. It was found that there were five bases substitution in the P190CRV, in comparison with the nucleotide sequences of MAD20 strain. These two sequenced fragments were inserted into pGEX-2T plasmid. E.coli JM109 (DE3) were transformed with the recombinant plasmids and the parental plasmid. The results show that the two fragments were expressed at high level as C-terminal fusions with glutathione s-transferase (GST). The fusion proteins were easily purified from bacterial lysates by affinity chromatography using glutathione sepharose 4B.
RESUMEN
An recombinant vector pCSV-EPO for expression of EPO cDNA in mammalian cells was constructed by the techniques of gene recombinant, PCR amplification and region-specific mutagenesis. The pCSV-EPO was introduced into COS7 cells by DEAE-dextran-mediated transfection. The expression of the EPO was demonstrated by EPO-ELISA assay. At 48h post transfection, the EPO level was 25ng/ml and 72 h was 17ng/ml.