【Objective】 To determine the rare ABO blood subgroups rapidly and ensure the blood transfusion safety of five patients by a series of serological tests and family investigation, as their preliminary serological results of ABO blood grouping was inconsistent. 【Methods】 ABO blood grouping, antibody screening and Coombs′ tests were performed by the routine serological methods, including manual tube and automatic blood group analyzer, which had matched micro-column gel cards from Diagnostic Grifols. Polymerase chain reaction (PCR) was used to amplify the 6 and 7 exons as well as their adjacent intron region of ABO gene. The patients and their relatives′ ABO blood group and subgroup were analyzed and identified through the comparison with serological phenotype database of ABO blood group. The products of PCR were sequenced directly, and the gene mutation was identified through the comparison with the Blood Group Antigen Gene Mutation Database. 【Results】 Whether micro-column gel cards or manual tube test, the forward and reverse tests of serological grouping were not supported by each other on the five patients′ ABO blood grouping. The forward tests of patients No.1~3 showed AwB phenotype and the reverse tests showed B group. No.4 patient was the forward ABw phenotype and the reverse A group, and No.5 patient was the forward normal AB phenotype and the reverse B group, respectively. All of 5 patients′ Rh C/D/E blood grouping showed clearly; the IDT test and antibody screening result of patient No.1 was positive, while the antibody screening result of patient No.4 was negative. 【Conclusion】 The blood group serological characterization of patient No.1~3 met B(A) blood group, and patient No.4~5 met CisAB blood group. These tests can make a preliminary diagnosis of blood group phenotype, which are verified correctly via blood group genotype.

Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 β-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarS_cyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarS_cyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarS_cyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarS_cyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.
Humans ; Histidine Kinase/metabolism* ; Fusobacterium nucleatum/metabolism* ; Automobiles ; Protein Kinases/genetics* ; Escherichia coli/metabolism* ; Colorectal Neoplasms