Cloning And Expression Of XTP11 Spliced Variant Transregulated By Hepatits B Virus X Protein, And The Study Of Its Function

Background and Objective: The high incidence of hepatitis B and liver cancer is found in China. Hepatitis B virus (HBV) X gene and multifun- ctional X protein (HBx) translated by X gene are necessary for gene transcription of hepatitis B virus, and they play an important role in the occurrence of hepatitis B and hepatocellular carcinoma.Oligonucleotide DNA chip technology and bioinformatics analysis were employed to identify target gene transactivated by hepatitis B virus X protein (HBxAg), and the target gene is called XTP11. In the process of revealing the biological function of XTP11, its spliced variant was found.The purpose of the study is to clarify biological function of XTP11 spliced variant in the course of HBV incidence through the following aspects, such as bioinf- ormatics analysis, subcellular orientation, prokaryotic expression.Methods: (a), In the process of designing primers to amplify XTP11 according to its full length coding gene, we got XTP11 spliced variant, and ,then made bioinformatics analysis of the XTP11 spliced variant. (b) Constructed recombined green fluorescent protein expressive vector pEGFP -C1-XTP11 spliced variant were transfected to HepG2 cells and observed 24hr after transfection by inverted fluorescence microscope. (c)The recombined prokaryotic expression vector pET-32a(+)-XTP11 spliced variant was constructed and transformed into the competent BL21 E. coli. The recombined protein was induced by IPTG and optimization expression of recombinant fusion protein with histidine(His-) tag was obtained. SDS-PAGE and Western blot were used to analyze and validate the specif- icity of recombinant fusion protein.The expressed product was purified by Ni+ affinity column chromatography.Results: (a) XTP11 spliced variant was analyzed by using BLASTn in NCBI database according to the Kozak rule of initiation codons and conservative polyA signal sequence in downstream of terminal codons. The open reading frame of XTP11 spliced variant was spliced and its full length of coding sequence was obtained. The XTP11 spliced variant accession number FJ21106 was obtained in banked in NCBI database.. XTP11 has the same sequence with its spliced variant except GGGDFGGGDF 10 amino acids.(b) XTP11 spliced variant can be subcellularly located in cytoplasm by its visible green fluorescent signal. (c) The XTP11 spliced variant fusion protein was highly expressed in prokaryotic expression system. SDS-PAGE analysis showed that the protein mainly existed in the form of inclusion bodies, its high specificity was testified by Western blot and its purified protein was obtained successfully.Conclusions: ProtParam software was used to analyze the instability coefficient,hydrophobicity index and overall average hydrophilicity of XTP11 spliced variant.The protein mainly consists of turning and folding, and almost has no helix and random coil. Its hydrophobicity diversity ranges from -0.322 to 2.267; SignalP-signal peptide predicted that the probability of anchoring protein was 0.195, suggesting the protein a nonsecreting protein.Subcellular localization of XTP11 spliced variant confirmed that it located in cytoplasm. The XTP11 spliced variant fusion protein was induced and expressed successfully using E. coli prokaryotic expression system and purified by affinity column chromatography. These results provide an valuable information for clarifying the role of XTP11 spliced variant in the occurrence of HBV, and for polyclonal antibody by immunizing animal, establish a method for detecting immunohistochemistry and enzyme-linked immunosorbent assay.