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OBJECTIVE@#To explore the molecular mechanism for an individual with Bweak subtype.@*METHODS@#Serological methods were used to identify the proband's phenotype. In vitro enzyme activity test was used to determine the activity of B-glycosyltransferase (GTB) in her serum. The genotype was determined by PCR amplification and direct sequencing of exons 5 to 7 and flanking sequences of the ABO gene. T-A cloning technology was used to isolate the haploids. The primary physical and chemical properties and secondary structure of the protein were analyzed with the ProtParam and PSIPRED software. Three software, including PolyPhen-2, SIFT, and PROVEAN, was used to analyze the effect of missense variant on the protein.@*RESULTS@#Serological results showed that the proband's phenotype was Bweak subtype with anti-B antibodies presented in her serum. In vitro enzyme activity assay showed that the GTB activity of the subject was significantly reduced. Analysis of the haploid sequence revealed a c.398T>C missense variant on the B allele, which resulted in a novel B allele. The 398T>C variant has caused a p.Phe133S substitution at position 133 of the GTB protein. Based on bioinformatic analysis, the amino acid substitution had no obvious effect on the primary and secondary structure of the protein, but the thermodynamic energy of the variant protein has increased to 6.07 kcal/mol, which can severely reduce the protein stability. Meanwhile, bioinformatic analysis also predicted that the missense variant was harmful to the protein function.@*CONCLUSION@#The weak expression of the Bweak subtype may be attributed to the novel allele of ABO*B.01-398C. Bioinformatic analysis is helpful for predicting the changes in protein structure and function.
Assuntos
Feminino , Animais , Sistema ABO de Grupos Sanguíneos/genética , Fenótipo , Genótipo , Éxons , AlelosRESUMO
OBJECTIVE@#To analyze the molecular characteristics of a recombinant allele of the ABO blood group.@*METHODS@#The ABO phenotype was determined with the tube method. The coding regions of the ABO and FUT1 genes were analyzed by PCR-sequence based typing. The ABO alleles of the proband were determined by allele-specific primer sequencing. The full sequences of the ABO gene of the proband and her mother were determined through next generation sequencing.@*RESULTS@#The red blood cells of the proband did not agglutinate with anti-H, and the sequence of the FUT1 gene was homozygous for c.551_552delAG.The proband was thereby assigned as para-Bombay. Bi-directional sequencing also found that she was heterozygous for c.261G/del,467C>T,c.526C>G,c.657C>T,c.703G>A,c.796C>A,c.803G>C and c.930G>A of the coding regions of the ABO gene. Allele-specific primer sequencing also found her to carry a ABO*A1.02 allele and a recombinant allele from ABO*O.01.01 and ABO*B.01. The recombination site was located between nucleotide c.375-269 and c.526, and the allele was maternally derived.@*CONCLUSION@#An recombinant allele of the ABO gene has been identified, which has originated from recombination of ABO*O.01.01 with the ABO*B.01 allele.
Assuntos
Feminino , Humanos , Sistema ABO de Grupos Sanguíneos/genética , Alelos , Tipagem e Reações Cruzadas Sanguíneas , Fucosiltransferases/genética , Genótipo , Fenótipo , Recombinação GenéticaRESUMO
OBJECTIVE@#To explore the molecular basis for an individual with Bw subtype.@*METHODS@#Routine serological reactions were used to determine the surface antigens of erythrocytes and antibodies in serum. PCR-sequence-based typing (PCR-SBT) was used to analyze the coding regions of the ABO gene and erythroid-specific regulatory element in its intron 1. Amplicons for exons 5 to 7 containing the variant site were subjected to TA cloning for the isolation of the haploid and verification of the sequence. The 3D structure of mutant protein was predicted with Pymol software. Changes of amino acid residues and structural stability were also analyzed.@*RESULTS@#Serological assay showed that the individual had weakened B antigen and anti-B antibody in his serum. His genotype was determined as ABO*B.01/ABO*O.01.01. Sequencing of the entire coding region of the ABO gene identified an additional heterozygous c.734C/T variant. No variant was found in the erythroid-specific regulatory element of intron 1. Haploid cloning and isolation has obtained an ABO*O.01.01 allele and a ABO*B.01 allele containing a c.734T variant, which has led to substitution of Thr by Ile at position 245 in the functional center of glycosyltransferase. Based on the 3D structure of the protein, the residues binding with the mutation were unchanged, but the bonding distance between the hydrogens was changed with the amino acid substitution. Meanwhile, the connections with water molecules were increased.@*CONCLUSION@#The c.734C>T variant of the GTB gene can lead to an amino acid substitution in the functional center of the enzyme, which in turn may affect the stability of glycosyltransferase B protein and reduceits enzymatic activity.
Assuntos
Humanos , Masculino , Sistema ABO de Grupos Sanguíneos/genética , Alelos , Éxons/genética , Genótipo , Glicosiltransferases/genética , FenótipoRESUMO
<p><b>OBJECTIVE</b>To explore the molecular basis of an individual with Bel variant of the ABO blood group.</p><p><b>METHODS</b>The ABO antigen and serum antibody of the individual were detected by serological method. All coding regions and flanking introns of the ABO gene were amplified with PCR and sequenced bidirectionally. The haplotypes of the individual were analyzed by cloning and sequencing. A three dimensional model of the mutant protein was constructed and analyzed.</p><p><b>RESULTS</b>The individual has expressed a very weak B antigen on its red blood cells by absorption and elution testing, which was identified as a Bel variant phenotype. The heterozygous sites in exon 6 (261del/G) and exon 7 (297A/G, 484del/G, 526C/G, 657C/T, 703G/A, 796C/A, 803G/C, 930G/A) of the coding region of the ABO gene were identified by direct sequencing. Haplotype analysis showed that the individual has carried an O01 allele and a novel B allele. The sequence of the novel B allele was identical to B101 except for a del G at nucleotide position 484 (484delG), which was nominated as B120 by the Blood Group Antigen Gene Mutation Database (dbRBC NCBI). The 484delG mutation of the B allele has led to a reading frame shift and created a premature terminal codon for the glycosyltransferase (GT) enzyme. Prediction of the 3D structure suggested that the GT enzyme has become an incomplete protein only with its N-terminal region.</p><p><b>CONCLUSION</b>The 484delG mutation of the glycosyltransferase B gene has probably abolished or reduced the enzymatic activity and resulted in the Bel variant phenotype.</p>
Assuntos
Feminino , Humanos , Sistema ABO de Grupos Sanguíneos , Genética , Alelos , Sequência de Bases , Éxons , Genótipo , Glicosiltransferases , Genética , Dados de Sequência Molecular , Mutação , Deleção de SequênciaRESUMO
<p><b>OBJECTIVE</b>To generate recombinant GPⅢa as an alternative source for HPA-1a antigen and combine it with Luminex xMAP beads for the detection of HPA-1a-specific alloantibody.</p><p><b>METHODS</b>The full coding region of ITGB3 gene was amplified and ligated with pcDNA3.1. The recombinant plasmid was transfected into CHO cells, and those with stable expression were screened with G418. Expressed protein was identified and coupled with Luminex xMAP beads, which were then reacted with sera samples. Subsequently, phycoerythrin-labeled anti-species IgG antibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer.</p><p><b>RESULTS</b>DNA sequencing confirmed that the cloned ITGB3 gene was HPA-1aa. The recombinant GPⅢa was coupled with Luminex xMAP beads. The sensitivity of Luminex beads assay to detect HPA-1a antibody was dilution 1/32 (3.125 U/mL). The Luminex beads assay could specifically identify the HPA-1a antibody from the test sera, and the results were consistent with that of monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technology. Cross-reactivity was not observed with the samples containing HLA, ABO and other HPA antibodies (HPA-3a and HPA-5b). The results illustrated that to detect HPA antibody with Luminex xMAP beads technology is feasible.</p><p><b>CONCLUSION</b>Recombinant GPⅢa was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.</p>
Assuntos
Animais , Cricetinae , Humanos , Antígenos de Plaquetas Humanas , Alergia e Imunologia , Autoanticorpos , Alergia e Imunologia , Sequência de Bases , Western Blotting , Células CHO , Cricetulus , Imunoensaio , Métodos , Integrina beta3 , Genética , Alergia e Imunologia , Metabolismo , Microesferas , Proteínas Recombinantes , Alergia e Imunologia , Metabolismo , Reprodutibilidade dos TestesRESUMO
Objective To construct a prokaryotic expression system for major histocompatibility complex class Ⅰ chain-related gene B ( MICB) and to establish an ELISA method for the detection of anti-MICB antibodies in patients with kidney transplantation. Methods The MICB cDNA fragments were ob-tained by RT-PCR with a pair of specific primers. The MICB cDNA and the prokaryotic expression vector pET-28a were digested by two restriction enzymes and ligated to construct the recombinant expression plas-mid pET-28a-MICB. The transformed E. coli BL21 DE3 strains carrying recombinant expression plasmid were induced by IPTG to express MICB protein. The expressed recombinant proteins were identified by Western blot assay and purified by Ni-NTA Spin column. The purified proteins were coupled to ELISA for the detection of anti-MICB antibodies in patients with kidney transplantation. Results Three common MICB fragments contained the exons 2 and 3 were obtained. The recombinant proteins were expressed in E. coli BL21 DE3 strains carrying pET-28a-MICB and successfully purified by the Ni-NTA Spin column. Results of the Western blot assay confirmed that the obtained proteins were the target proteins. The ELISA method was successfully established and used for the detection of anti-MICB antibodies in 24 patients with kidney trans-plantation. The absorbance values indicated that the sensitivities of three recombinant MICB proteins were different. Conclusion The expression system for MICB gene was successfully constructed. The established ELISA for the detection of anti-MICB antibodies would pave the way for further investigation on the correla-tions between MICB protein and transplantation immunity.
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<p><b>OBJECTIVE</b>To explore the molecular basis for an individual with rare p phenotype in the P1Pk blood group system.</p><p><b>METHODS</b>Erythrocyte blood group antigens and antibodies in serum were identified in the proband and five family members with a serological method. Coding regions and flanking untranslated regions of the α1,4-galactosyltransferase gene (A4GALT) encoding P1Pk antigens were amplified with polymerase chain reaction and directly sequenced. The haplotypes of A4GALT in the parents of the proband were also analyzed by cloning sequencing.</p><p><b>RESULTS</b>The proband was found with a rare p phenotype with anti-Tja antibody in his serum by serological method. The other family members all had a common P2 phenotype. The results of DNA sequencing showed that a cytosine was inserted at nucleotide position 1026 to 1029 (1026_1029insC) of both alleles of the A4GALT gene in the proband. The mutation has caused a reading frame shift and formed a mutant protein by extending 92 amino acid residues. The other family members were either heterozygous for the insertion or of the wild type at above position.</p><p><b>CONCLUSION</b>The 1026_1029insC mutation of the A4GALT gene is probably responsible for the p phenotype identified for the first time in Chinese population. The individual with the p phenotype possesses anti-Tja antibody.</p>
Assuntos
Adulto , Feminino , Humanos , Masculino , Adulto Jovem , Sistema ABO de Grupos Sanguíneos , Genética , Alelos , Povo Asiático , Genética , Sequência de Bases , Mutação da Fase de Leitura , Galactosiltransferases , Genética , Dados de Sequência Molecular , Mutagênese Insercional , Linhagem , FenótipoRESUMO
<p><b>OBJECTIVE</b>To analyze specific expression of blood group genes using nucleated erythroid cells cultured from un-mobilized peripheral stem cells in vitro.</p><p><b>METHODS</b>Hematopoietic stem cells(HSC) bearing the CD34 antigen were isolated from peripheral blood by centrifugation and magnetic beads sorting, followed by suspension culture in vitro. Cells were collected from medium on various stages and analyzed by immunofluorescence. The RNA transcription of RH and ABO blood group genes was analyzed using culture cells on day 12.</p><p><b>RESULTS</b>A total of(3.19±0.13) ×10 (4) CD34+cells were isolated from about 50 mL peripheral blood with a recovery rate of 67.3%±2.7%. The cells amount in erythroid-lineage culture system on day 9 reached a plateau of a 237.1±15.5-fold amplification of the initial cell input. The stem cell-specific CD34 antigen was dropped off, while the erythroid-specific CD235a and CD240D antigens were increased in culture period. RHD/CE and ABO genes can be amplified using RNA extracted from culture cells on day 12, and genotypes of Rh and ABO systems by DNA sequencing were consistent with their serologic phenotypes.</p><p><b>CONCLUSION</b>A method was established to analyze the gene expression of erythroid blood group derived from un-mobilized peripheral stem cells cultured in vitro. It can be used to study the expression of various erythroid-specific genes.</p>
Assuntos
Humanos , Antígenos CD34 , Genética , Sequência de Bases , Antígenos de Grupos Sanguíneos , Genética , Células Cultivadas , Eritrócitos , Biologia Celular , Citometria de Fluxo , Células-Tronco Hematopoéticas , Biologia Celular , Dados de Sequência MolecularRESUMO
<p><b>OBJECTIVE</b>To delineate serological features and genetic basis for a rare p phenotype of P1Pk blood group system found in a Chinese individual.</p><p><b>METHODS</b>Serological assaying was carried out for a proband with unexpected antibody found in his serum using specific antibodies and panel cells. Coding regions and flanking introns of α 1,4-galactosyltransferase gene (A4GALT) associated with the p phenotype were screened with polymerase chain reaction and DNA sequencing.</p><p><b>RESULTS</b>A rare p phenotype of the P1Pk blood group system has been identified with red blood cells from the proband, whose serum contained anti-Tja antibody which can agglutinate and hemolyze with other common red blood cells. Other members of the proband's family were all normal with P1 or P2 phenotype. DNA sequencing has identified in the proband a homozygous 26 bp deletion at position 972 to 997 of the A4GALT gene. The deletion has caused a shift of the reading frame, resulting in a variant polypeptide chain with additional 83 amino acid residues compared with the wild-type protein. Other family members were either heterozygous for above deletion or non-deleted.</p><p><b>CONCLUSION</b>A 26 bp deletion at position 972 to 997 of the A4GALT gene has been identified in a Chinese individual with p phenotype.</p>