OBJECTIVETo screen the hepatocyte proteins that interact with hepatitis B virus X protein (HBx).

METHODSThe recombinant plasmid pSos-HBx was constructed by inserting Sos-HBx fragment into the bait vector, and after sequence verification the plasmid was transformed into competent yeast cells. The expression and self-activation of Sos-HBx protein was detected in the yeast cells. The hepatocyte proteins interacting with the bait protein was screened with CytoTrap yeast two-hybrid technique.

RESULTSThe reconstructed plasmid harboring HBx gene expressed Sos-HBx protein in the yeast cells without self-activation of the protein. CytoTrap yeast two-hybrid system identified 6 hepatocyte proteins that interacted with HBx, including fibronectin 1, translationally controlled tumor protein, IQ motif and WD repeats 1, follistatin, orosomucoid 1, and disulfide isomerase family A member 3.

CONCLUSIONSix HBx-binding hepatocyte proteins have been identified using the CytoTrap yeast two-hybrid system, which provides clues for further investigation of the role of HBx protein in hepatitis and liver cancer.

Genetic Vectors ; Hepatocytes ; metabolism ; Humans ; Plasmids ; Protein Interaction Domains and Motifs ; Proteins ; metabolism ; Trans-Activators ; metabolism ; Two-Hybrid System Techniques

Objective: To study the interaction between hepatitis B virus X protein(HBx) and mitochondrial elongation factor G1 (EFG1) in yeast cells or hepatoma cells. Methods: After verification the interaction between HBx and EFG1 by CytoTrap yeast two-hybrid system, EFG1 genome was amplified by means of polymerase chain reaction(PCR) and cloned into the pcDNA3.1/myc-His(-)A vector, following verification by sequencing. Expression of HBx and EFG1 protein was verified in Huh7 cells. Then the recombinant vector pcDNA3.1/myc-His(-)A-EFG1 and pFLAG-CMV-2-HBx were transfected into Huh7 cells; 48 h later, the cells were lysed. The direct interaction between HBx and EFG1 was further confirmed by the Co-immunoprecipitation (Co-IP) assay. Results: The interaction between HBx and EFG1 was successfully verified by CytoTrap yeast two-hybrid system. The recombined plasmid pcDNA3.1/myc-His(-)A-EFG1 was obtained. Furthermore, Co-IP assay was used to confirm the interaction between HBx and EFG1 in Huh7 cells. Conclusions The direct interaction between HBx and EFG1 was confirmed. Therefore our findings provide experimental basis for the influence of HBx protein on the expression of mitochondrial protein and provide new insights into the pathogenesis of HBx in the development of hepatocellular carcinoma.

OBJECTIVE: To assess the association of KIR/HLA alleles with hepatocellular carcinoma (HCC) and hepatitis B virus (HBV) infection among ethnic Han Chinese patients from southern China. METHODS: For 95 patients with HCC and 171 healthy controls, the genotype of HLA-C alleles was determined with a PCR sequence-specific oligonucleotides typing method on an Illumina GenDx NGSgo platform. Genotypes comprised of HLA-C and KIR gene alleles were also subjected to statistical analysis. RESULTS: In total 16 KIR genes (2DL2, 2DS2, 2DS3, 2DS5, 3DS1, 2DS1, 2DL5, 2DS4, 3DL1, 3DP1, 2DL3, 2DP1, 3DL3, 2DL1, 3DL2 and 2DL4) were discovered in the two groups. The frequencies of KIR2DL3 alleles and combinational genotypes of KIR2DL3/HLA-C1C2 were significantly lower in the patient group compared with the controls (0.9368 vs. 0.9883, χ²>3.84; P<0.05, OR = 0.1; 0.0112 vs. 0.2663, χ²>3.84; P<0.05, RR = 0.03). The frequency of HLA-C2C2 genotype of the patient group was significantly lower than that of the controls (0.0316 vs. 0.2690, P<0.05, RR = 0.09), while the frequency of HLA-C1C2 genotype was significantly higher than that of the controls (0.2316 vs. 0.0058, P<0.05, RR = 51.23). CONCLUSION Above results suggested that the KIR2DL3 allele is associated with lower risk for HCC. There may be individual difference in patients with HCC and HBV infection but various combinations of KIR/HLA alleles.
Alleles ; Carcinoma, Hepatocellular ; genetics ; China ; Gene Frequency ; Genotype ; Humans ; Liver Neoplasms ; genetics ; Polymorphism, Genetic ; Receptors, KIR