Screening Of Proteins Binding To HCV Core Protein From Human Pancreas CDNA Library By Yeast Two-hybrid

【Objective】The paper is based on National Natural Science Foundation project"Chronic hepatitis B and C influence factors associated with MS and mechanism of preliminary study".To screen proteins in pancreas cDNA library interacting with hepatitis core protein by yeast two-hybrid,which suppy some new clues for HCV core action of developing the abnormality of the lipid metabolism and the glycometabolism in the patients with chronic hepatitis virus infection.【Methods】After amplified and purified,the human pancreas cDNA library was evaluated.The HCVcore gene was amplified by polymerase chain reaction(PCR) and inserted into eukaryotic expressive vector pGBKT7.First of all,pGBKT7-core was contransformated into AH109 with human pancreas cDNA library plasimid.The yeast cells were plated on nutrient deficiency medium(SD/-Trp/-Leu/-His/-Ade) and nutrient deficiency(SD/-Trp/-Leu/-His/-Ade) medium containing X-α-gal for selection twice.The plasmids in diploid yeast cells were extracted and electrotransformed into E.coli DH5α.The plasmids in DH5αwere extracted,sequenced and analyzed by bioinformatic methods.Then applied yeast marion system,the puried library plasmids were transformed into yeast strain Y187 and screened on the nutrient deficiency medium SD/-Leu.The reconstructed plasmid pGBKT7-core was transformed into yeast strain AH109 and screened on the nutrient deficiency medium SD/-Trp.The transformed AH109 mated with Y187 containing the library plasmid.The diploid yeast cells were plated on nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade and the same medium containing X-α-gal for selecting.Yeast plasmids were extracted and transformed to E.coli DH5α.After sequencing,genes were submitted to GenBank database.【Result】The human pancreas cDNA library was about 4×10~7,the purified DNA was about 0.34mg/ml,and the built in fragments were different in size after digested with EcoRⅠ,X hoⅠ.Six proteins binding to HCV core were screened by yeast contransformation system,including homo sapiens protease,serine 1(PRSS1),Homo sapiens carboxypeptidase A1(CPA1),Homo sapiens chymotrypsin C(CTRC),Homo sapiens lysine-rich coiled-coil 1,Homo sapiens zinc finger,matrin type 1(ZMAT1), Homo sapiens uncharacterized bone marrow protein BM044.Eleven proteins binding to HCVcore were screened by yeast mation system,including Homo sapiens colipase, Homo sapiens kinesin family member 3B(KIF3B),Homo sapiens carboxypeptidase B1(CPB1),Homo sapiens mitochondrion,Homo sapiens protease,serine,1(PRSS1), Homo sapiens protease,serine,2(PRSS2) et al.【Conclusions】This study used the yeast two-hybrid were transfected with technical and human pancreas cDNA library screening,respectively 6 and 11 and HCVcore protein binding protein gene.To further investigate the results of HCV infection caused MS provides a new idea,but also confirmed the superior technology with a total of transfection technology,the yeast two-hybrid system's progress and development.