Study On The Screening Of Proteins Binding To HCV E1 And HBsAg From Human Pancreas CDNA Library By Yeast Two-hybrid System

Objective To construct the recombinant expression plasmids pGBKT7 with HCV E1,and then transform AH109 yeast strain.After screening proteins by yeast two-hybrid system in pancreas cDNA library interacting with HCV E1 protein and HBsAg proteins,several binding proteins were cloned in order to study the biological functions.Methods Fistly,the bait plasmid pGBKT7-E1 was constructed and transformed into AH109 yeast strains,then screened on the nutrient deficiency medium SD/-Trp and the yeast protein was isolated and detected by Western blotting analysis. Simultaneously,the library was amplifed,purified,evaluated,and transformed into yeast strain Y187 subsequently.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 SD/-Trp/-Leu/-His/-Ade containing X-α-gal for selecting. 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.A few of selected positive proteins were crossed back with bait plasmid in order to verify whether they were able to interact with PAN-1 cells cDNA library.Furthermore,Total RNA were extracted from PAN-1 cells,which as template to amplifiy some selected genes by everse transcription polymerase chain reaction (RT-PCR).Recombinant expression plasmids of these genes were then constructed.Results The recombinant bait plasmids pGBKT7-E1 was constructed successfully.Western blotting results showed that it's molecular weight was 38.9kD. By yeast two-hybrid system Sixteen proteins interacting with HCV E1 and eleven proteins binding HBsAg were founded.Most of them were listed as follows: colipase(CLPS),carboxypeptidase B1(CPB1),mitochondrion,protease serine 1 (PRSS1),chymotrypsinogen B1(CTRB1),carboxypeptidase A1(CPA1),elastase 3A (ELA3A),pancreatic stone protein(REG1A),HSPC142 protein(HSPC142),zinc finger protein 593(ZNF593),aconitase 1(ACO1) and CDK5 regulatory subunit associated protein 3(CDK5RAP3).etc.Recombinant plasmids pcDNA3.1/myc-CPLS and pcDNA3.1/myc-ELA3A were constructed according to project ultimately. Conclusions Screening proteins by yeast two-hybrid system in pancreas cDNA library interacting with HCV E1 and HBsAg in domestic for the first time.These results show that HBV and HCV may be related with metabolism of glucose and lipid. Most of these proteins binding to HCV E1 and HBsAg pay significant roles in the evolution of 2 type diabetes mellitus,which may supply some new evidences and clues for the pathogensis of developing the abnormality of the lipid metabolism and the glycometabolism in the patients with chronic hepatitis virus infection.