Objective To perform a DNA-based prenatal diagnosis in a family with recessive dys-trophic epidermolysis bullosa, and to develop a strategy to eliminate matemal cell contamination in arnniotic fluid samples. Methods Amniocentesis was carried out at gestation week 16, amniotic fluid culture was used to separate fetal cells from maternal blood cells. Peripheral blood was obtained from the proband, and her parents. Genomic DNA was extracted from peripheral blood and aminotic cells. Subsequently, PCR and direct sequencing were performed to detect pathogenic mutations in the COL7A1 gone. Karyotype analysis was used to confirm paternal information in amniotic fluid. Linkage analysis between micro-satellite markers was performed to confirm the fetal genotype. Resulta Centrifugation showed visible contamination of aminotic cells by blood cells. Direct sequencing revealed that the proband was a carrier of both maternal mutation, R525X in exon 12, and paternal mutation, R2610X in exon 105, while the fetus only carried the maternal mutation, R525X. The second direct sequencing and hapiotype analysis after elimination of mater-nal blood cells by amniotic fluid culture confirmed that the fetus was a carrier of maternal mutation with nor-real phenotype. The pregnancy continued and a clinically unaffected girl was born at gestation week 40.Conclusion The accuracy of DNA-based prenatal diagnosis could be improved by the combination of direct sequencing, amniotic fluid culture, karyotype analysis and linkage analysis, etc.

AIM: To express chimeric toxin Stx2a’-LHRH a nd to investigate the cytotoxic activity of recombinant toxin Stx2a’-LHRH to huma n carcinoma cells.METHODS: Stx2a’-LHRH sequences that added the res tri ction endonucleases NcoⅠ and EcoRⅠ at the 5' and 3 ends were amplified by PCR a nd digested with appropriate restriction enzymes. The digested fragment was subc loned into the vector obtatined by digestion of plasmid pET-28a(+) with NcoⅠ an d EcoRⅠ. E. coli BL21 (DE3) cells were transformed with plasmids of interst and cultured in LB medium containing ampicillin. Expression of the recombinant protein was induced by the addition of isopropylthio-?-D-galactoside (IPTG). T h e cytotoxity of Stx2a’-LHRH to Hep-2 cells was observed under the microscop y. RESULTS: Recombitant plasmid pET-SL was constructed successfu lly and the clones expressing pET-SL stablely were obtained. A special electroph oretic band in SDS-PAGE (a glycoprotein of 28kD) was noted. Stx2a’-LHRH killed He lp-2 cells clearly. CONCLUSION: In this study, construction of c himeric toxin Stx2a’-LHRH and its expression were described. Moreover, it has o bvious cytotoxity to Hep-2 cell. These finding could open up new vistas in the s tudy of targeted durgs.

Objective To prepare human alpha segment of high affinity IgE receptor(FcεRIα)protein by genetic engineering technology and to identify its biological function for laying the foundation for further researching the role of FcεRIα in allergic disea-ses.Methods The human FcεRIα gene was obtained by the PCR based accurate synthesis(PAS)method and the prokaryotic ex-pression vector pET-28a(+)was constructed.The FcεRIα was expressed at low temperature induction and the recombinant protein was purified by His tag.The biological function of recombinant human FcεRIα protein was identified by ELISA.Results The hu-man FcεRIα gene was amplified by PAS with a size of approximately 560 bp.The pET-FcεRIα plasmid was correct through the double enzyme digestion and sequencing identification.The human FcεRIα with a molecular weight of approximately 22 000 was in-duced and purified.The recombinant human FcεRIα could effectively detect human serum anti-FcεRIα autoantibody and could com-bined with serum IgE antibodies with high efficiency.Conclusion Human FcεRIα protein is successfully prepared,which prelimina-rily has the ability for detecting the human serum anti-FcεRIα autoantibodies and IgE antibodies,and provides a favorable practical base for further study.