Objective To clone human cell division cycle gene 2(CDC2)from human liver cancer tissue and express CDC2 protein in E.coli BL-21(DE3).Methods The total RNA was extracted from liver cancer tissue and amplified by reverse transcription polymerase chain reaction(RT-PCR).The PCR products were cloned into the prokaryotic expression vector pET28a(+)followed by DNA sequencing.The recombinant pET28a(+)/CDC2(rCDC2)plasmid was transformed into E.coli.The rCDC2 was induced with IPTG and characterized by SDS-PAGE.Results The cloned CDC2 gene was composed of 894 nucleotides,and is accordance with that reported in GenBank.The prokaryotic expression vector was constructed successfully.The CDC2 protein was successfully expressed in E.coli.Conclusion The CDC2 cDNA is successfully cloned from human liver cancer tissue,and can express in E.coli.

Objective:To construct a eukaryotic expression vector for wild-type human Interleukin-13(whIL-13)and its mutant(mhIL-13) gene fused with enhanced green fluorescent protein(EGFP) and to analyze the expression and subcellular location of the fusion protein in COS-7 cells.Methods:The whole whIL-13 and mhIL-13 coding gene was amplified by RT-PCR. And then the expression vector for EGFP-whIL-13, EGFP-mhIL-13 fusion protein were constructed.The recombinant fusion gene were transfected into COS-7 cells.The expression and subcellular location of the fusion protein were detected by confocal microscopy.Results:The construction of expression vector were all corrected.In the positive cells, green fluorescent was found to be localized in cytoplasm,while empty in nucleus.Conclusion:The expression vectors for EGFP-whIL-13, EGFP-mhIL-13 fusion protein were constructed successfully and expressed in COS-7 cells. Fusion proteins were distributed in cytoplasm and no significant difference was observed between them.

Objective To clone the rat heme oxygenase-1(RHO-1) from rat spleen and express RHO-1 in E.coli BL-21.Methods The total RNA was extracted from rat spleen and amplified by reverse transcription polymerase chain reaction(RT-PCR).PCR products were cloned into pMD18-T(TA)vector followed by DNA sequencing.RHO-1 cDNA fragments in TA vector were subcloned into the prokaryotic expression vector pET28a(+).The recombinant pET28a(+)/RHO-1(rRHO-1) plasmid was transformed into E.coli.The rRHO-1 was induced with IPTG and characterized by SDS-PAGE.Results The cloned RHO-1 gene was composed of 870 nucleotides,and was accordance with the sequence reported in GenBank.The prokaryotic expression vector was constructed successfully.The RHO-1 protein was successfully expressed in E.coli.Conclusion The prokaryotic expression vector of rRHO-1 has been constructed,and the fusion protein has been successfully expressed.

OBJECTIVE: To analyze the clinical presentation and gene of 2 pedigrees with suspected oculocutaneous albinism(OCA), and provide basis for clinical classification, genetic counseling and prenatal diagnosis. METHODS: Variants were identified using next-generation sequencing(NGS) and confirmed by Sanger sequencing in 2 pedigrees with suspected OCA. The pathogenicity of the variants was analyzed according to the American College of Medical Genetics and Genomics (ACMG) standard. RESULTS: Two compound heterozygous mutations of TYR and OCA2 genes were identified respectively in 2 pedigrees with suspected OCA. The mutation of c.819+3insATATGCC in TYR and the mutation of c.1870G>C in OCA2 are first reported in this study. The pathogenicity analysis shows that two novel mutations are likely pathogenic by combination of prediction of SIFT, Polyphen-2 and Human Splicing Finder. CONCLUSION The findings of this study expand the mutational spectrum of OCA. Compound heterozygous mutations in the TYR and OCA2 gene may be responsible for clinical manifestations of 2 pedigrees with suspected OCA.
Albinism, Oculocutaneous ; DNA Mutational Analysis ; Female ; High-Throughput Nucleotide Sequencing ; Humans ; Membrane Transport Proteins ; Monophenol Monooxygenase ; Mutation ; Pedigree ; Pregnancy

Objective To explore the pathogenesis of Russell-Silver syndrome (RSS). Methods Two milliliter peripheral blood samples were collected from 6 male patients aged 6 to 8 years with suspected RSS phenotype, the parents of 2 patients and 5 healthy boys. Mononuclear cells were isolated and genomic DNA was extracted. The methylation level of the H19 imprinting control region(ICR)1 on chromosome 11p15.5 was detected by pyrosequencing.The methylation status and the copy number variation in the corresponding region of one RSS patient with positive results by pyrosequencing were analysed by methylation-specific multiplex-ligation-dependent probe amplification assay (MS-MLPA). Results Pyrosequencing analysis revealed that the methylation rates on the 6 CpG targeting sites in H19 differentially methylated region(DMR)in the 6 RSS patients were about 11%~29%, which were significantly lower than those in their parents and normal controls (44%~59%). The MS-MLPA results of one patient with positive pyrosequencing showed that the methylation rates of 4 sites in H19-DMR were about 10%,which was obviously lower than the normal level.The methylation rates of the 4 sites in KCNQ1OT1 gene were about 50%, which was in the normal range. The copy number variations from all samples detected were in the normal range. Conclusion There is methylation aberration of H19-DMR in ICR1 in children with RSS.