Screening And Identification Of Human Leukocyte CDNA Library Proteins Interacting HBV MHBs～t With Yeast Two-hybrid System

Hepatitis B virus (HBV) infection is the main cause of acute and chronic liver diseases, including chronic active hepatitis, as well as liver cirrhosis (LC) and hepatocellular carcinoma (HCC) in our country. The molecular mechanism responsible for hapetocellμlar damage caused by HBV infection is strongly associated with the interaction between virus proteins and host cellular proteins. Therefore, the investigation on interactions between virus proteins and host cellular proteins can partly explain the mechanism of HBV infection, and may provide some new clues for the prevention and cure of hepatitis B.The genome DNA of HBV is characterized by four overlapping ORFs (S,C,P,X). The S-ORF consists of a single open reading frame which can be divided into three coding regions: pre-S1, pre-S2 and S, each starting with an in-frame ATG codon. Through alternate translational initiation at each of the three AUG codons, a large (LHBs; pre-S1+pre-S2+S), a middle (MHBs; pre-S2+S) and a small (SHBs;S) envelope glycoprotein can be synthesized. The wild-type middle hepatitis B virus surface protein (MHBs) consists of a 55 aa preS2-domain and a 206 aa S-domain. C-terminally truncated middle surface protein of hepatitis B virus (MHBs~t) has been shown to be a transcriptional activator and may be relevant to hepatocarcinogenesis by transactivating gene expression. However, the binding protein of MHBs~t in leukocyte library is unclear.To investigate the biological function of MHBs~t protein, in the experiment, yeast two-hybrid technique was used to screen the proteins in leukocyte library which can interact with MHBs~t. The yeast two-hybrid system was originally developed for studying protein-protein interaction in vivo, it is a powerful, reliable and large scale screening technique which can be used for cDNA library screening. The yeast two-hybrid system-3 used in this experiment is an improved system, and compared with its initial version; it contains three report genes and has a true positive ratio of 95%. Based on the fast that the co-expression portion can interact in diploid yeast cell, produced by mating two yeasts of system-3 type a and typeα, the low efficiency of co-transfection of bait plasmid and library plasmid might be avoided. Using this technique, PCR was performed to amplify the gene of C-terminally truncated at 167 of middle surface protein of HBV and cloned it into a yeast expression plasmid pGBKT7 to construct the bait plasmid pGBKT7-MHBs~t. Then the bait plasmid was transformed into yeast AH109 (type a) and expressed in it. Afterward, diploid yeast cells was prepared by mating transformed AH109 yeast cells with Y187(typeα) yeast cells containing cDNA library plasmid pCAT2 in 2×YPDA, and then, the diploid yeast cells were plated for screening positive colonies using synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal. The results indicated that MHBs~t protein was successfully constructed and expressed in yeast cells as shown by Western blotting, 10 blue clones were obtained. After extracting plasmid from blue colonies, plasmid was transformed into competence Escherichia coli and analyzed by DNA sequencing. Sequences were analyzed in GenBank, 9 genes in positive colonies were obtained using yeast two-hybrid technique, including two ADP-ribosylation factor 1, one RAB6 interacting protein 1 (RAB6IP1), one CCR4-NOT transcription complex subunit 10, one lipopolysaccharide binding protein, one aldolase B, one complement component 3, one pyruvate dehydrogenase and one bacterial artificial chromosome(BAC). These findings may explain the possible mechanisms of MHBs~t in vivo.To further verify the interaction bewteen the binding protein and the bait protein in vitro, we amplified the integrity sequence of LPS-binding protein (LBP) gene and cloned it into pGADT7 vector, the recombination plasmid of pGADT7-LBP was translated by using reticulocyte lysate and analyzed by immunoprecipitation technique in vitro together with MHBs~t.In the experiment, MHBs~t binding proteins from leukocytes were successfully cloned and these results provided some new clues for further studying the biological functions of HBV.