Objective To construct the eukaryotic expression vector of HBEBP2 gene,and screen the exonic genes of proteins in hepatocytes interacting with HBEBP2.Methods The DNA fragment of HBEBP2 was amplified by PCR.The eukaryotic expression vector pGBKT7-HBEBP2 was constructed successfully by yeast-two hybrid system 3 and then transformed into yeast cells AH109.The transformed AH109 were then mated with yeast cells Y187 containing hepatic cDNA library plasmid.The diploid yeast cells were plated on synthetic dropout nutrient medium(SD/-Trp/-Leu/-His/-Ade) containing X-?-gal for the first selection.Library plasmids pACT2-DNA were extracted and co-transformed into yeast cell AH109 together with pGBKT7-HBEBP2.Then the yeast cells were plated on synthetic dropout nutrient medium(SD/-Trp/-Leu /-His/-Ade) containing X-?-gal for the second screening to eliminate false positive clones.The real positive clones were sequenced and analyzed by bioinformatics.Results Six proteins binding to HBEBP2 were screened,including human sapiens lactate dehydrogenase D,human sapiens mitochondrion,human sapiens mannose,human sapiens aldehyde oxidase,human sapiens serpin peptidase inhibitor and other two unknown proteins.Conclusions A novel class of proteins in hepatocytes interacting with HBEBP2 has been obtained.It is presumed that HBEBP2 protein is correlated with glycosylation,lipid metabolism and cell proliferation,etc.

OBJECTIVETo explore the molecular basis of 4 cases with weak D variant of Rh blood type.

METHODSRoutine serological testing was applied to determine the D, C, c, E and e antigens of the Rh blood group. The D antigen was further detected with an indirect antiglobulin test. RHD zygosity was detected by sequence-specific primer PCR method. All exons and flanking intron regions of the RHD gene were sequenced.

RESULTSThe samples were determined as weak D phenotype by serological testing. DNA sequencing showed that the 4 cases were heterozygous for 17C>T mutation in exon 1, 29G>C mutation in exon 1, 1212C>A mutation in exon 9, and IVS4+5G>A mutation in intron 4 of the RHD gene, respectively. According to the rule of Rhesus Base Nomenclature, the 4 samples were respectively named as weak D type 31, weak D type 71, weak D type 72, and weak D type 82.

CONCLUSIONSerological and molecular testing for the weak D can facilitate in-depth understanding of its immunology and genetics, and provide guidance for clinical blood transfusion and prevention of hemolytic disease in newborns.

Exons ; genetics ; Female ; Humans ; Middle Aged ; Mutation ; genetics ; Rh-Hr Blood-Group System ; genetics

OBJECTIVETo explore the molecular mechanism of CisAB01 subtype in the ABO blood group system, and to investigate the expression of A and B antigens in red blood cells (RBCs).

METHODSFor 5 unrelated individuals with the CisAB phenotype, the molecular basis for the blood type was studied with serological assay, DNA sequencing and haplotype analysis. Bioinformatics analysis was carried out to investigate the changes in structure and function of relevant enzymes. Expression of A and B antigens in RBCs of CisAB01 was detected by flow cytometry.

RESULTSAll of the 5 samples were found to have a CisAB01 subtype. The underlying mutations, 467C>T and 803G>C in exon 7, have resulted in replacement of amino acid P156L and G268A. The mean fluorescence intensity (MFI) of A antigen in CisAB01 cases was 135, while the control group was 172. The B antigens in CisAB01 cases (MFI=38) showed significant decrease in MFI compared with the control group (MFI=164).

CONCLUSION803G>C mutation of the ABO gene probably underlies the CisAB01 subtype. Fluorescence intensity of A antigens in CisAB01 subtype cases is slightly lower than the normal type, while the B antigen was significantly lower.

ABO Blood-Group System ; genetics ; Adult ; Base Sequence ; China ; Exons ; Female ; Humans ; Molecular Sequence Data ; Mutation ; Young Adult

OBJECTIVETo identify a novel Bx13 allele.

METHODSSerological characteristics was determined with standard serological methods. All of the seven exons and flanking regions of the ABO gene were analyzed with PCR and direct sequencing. The amplicon of exon 7 was also cloned and sequenced.

RESULTSThe individual was determined as with a rare Bx phenotype by serological tests. Direct DNA sequencing showed that the individual was heterozygous for the B/O01 allele, while there was a novel 893C>T mutation in the B101 allele, which has led to an amino acid substitution Ala298Val in the α,3-D-galactosyl-transferase. The mutation was not found among 100 randomly selected blood donors.

CONCLUSIONA novel Bx13 allele has been identified. Substitution of amino acid in the conserved region of the enzyme may reduce the activity of α,3-D-galactosyl-transferase.

ABO Blood-Group System ; genetics ; Alleles ; Exons ; Female ; Humans ; Middle Aged ; Mutation ; Sequence Analysis, DNA

OBJECTIVE To explore the molecular mechanism for a blood sample with mixed-field hemagglutination upon determination of ABO blood group. METHODS Serological techniques were employed to identify the erythrocyte phenotype. The A and B antigens were detected by flow cytometry. The preliminary genotype of ABO gene was assayed with sequence-specific primer-polymerase chain reaction (PCR-SSP). Exons 6 and 7 of the ABO gene were amplified with PCR and analyzed by direct sequencing. Haplotypes of the ABO gene were analyzed by cloning sequencing as well. RESULTS The serological reaction pattern has supported an O phenotype when all the tubes were centrifuged for the first time. However, a mixed-field hemagglutination of red blood cells (RBCs) with anti-A antibodies was present after the tube was centrifuged five times later. A antigens were detected on the surface of partial red blood cells of the sample by flow cytometry. PCR- SSP results have shown that the preliminary ABO genotype was A/O. Analysis of the fragments of exons 6 and 7 of the ABO gene has indicated that heterozygosis lied as follows: 261G/A, 425T/T, 467C/T, 646A/T, 681A/G, 745C/T, 771C/T, 829A/G, conjecturing the genotype to be A307/O02, which was confirmed by haplotype sequence analysis. Compared with A101 allele, A307 allele has two missense mutations, 467C> T and 745C> T, which have resulted in substitutions Pro156Leu and Arg249Trp in the A glycosyltransferase polypeptide chain. CONCLUSION A variant allele (A307) has been identified for the first time in mainland China, which is responsible for the formation of A3 phenotype.
ABO Blood-Group System ; genetics ; Adult ; Genotype ; Humans ; Male ; Phenotype