Cloning And Expression Of Gene 5 Transactivated By Pre-S1 Protein Of Hepatitis B Virus, A Human Novel Gene, And Its Partial Functions

The persistence infection of Hepatitis B virus(HBV)is still a global health problem.Acute infection with HBV may result in chronic infection,which is a leading cause of cirrhosis and hepatocellular carcinoma.However,there is no effectively therapic technique and means for HBV infection at present.Insights into the viral hepatitis pathogenesis of HBV should be sure to obtain an exciting advance in its treatment.When HBV enters the hepatocytes,the molecular mechanism responsible for HBV DNA duplication and expression,immune escape of HBV, chronic infection,hepatic fibrosis,malignant transformation caused by HBV infection are strongly associated with the interaction between virus proteins and hapetocellular proteins.In recent years,the HBV pathogenetic mechanism was researched deeply. The complicated trans-regulation mechanism involved in the course of HBV interaction with hepatocytes.Hepatitis virus protein could influence the gene expression level in the hepatocytes,which related possibly with HBV pathogenesis mechanism.The investigation on the protein-protein interaction with each antigen of HBV in the hepatocytes can further explain the HBV infection mechanism and the achievements will conduce to seek valid means for curing the disease.The study on cloning and expression of many function-unknown genes screeined and their functions is an important source of creative intelligence in viral hepatitis pathogenesis research and the important content of human genome and postgenome project.The recombinant expression plasmids pcDNA3.1(-)with functional fragments of Pre-S1 Protein of Hepatitis B Virus were constructed,and were transfected into HepG2 cells.Plasmid pcDNA3.1(-)empty vector was used as control.The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-preS1 and pcDNA3.1(-) empty vector respectively,and suppression subtractive hybridization(SSH)method was employed to analyze the differentially expressed DNA sequences between the two groups.One new gene with unknown functions was screened and enrolled in GenBank after verification by dot blot hybridization.The novel gene was named Human Gene 5 Transactivated by Pre-S1 Protein of Hepatitis B Virus(PS1TP5) (GenBank Accession:AY427953).To clarify the structure and biological function of the protein coded by the new gene,the interrelationships between biology and clinical medicine,and regulatory mechanisms is the most challenging works in the molecular biology research. Accoding to our work,linking molecular biology technology based on gene cloning and expression as the main purpose with bioinformatic technology is the foundation work to study the functions of a new gene.Besides,it is the important way of educing the biological functions of a new gene that protein-protein interaction and gene trans-regulation effects.At the present stage,the study aimed at human novel gene PS1TP5 to explore its regulation effects on hepatocellular gene expression profiles,and the interaction with hepatocellular proteins.The main work is as followings:1.A 438bp DNA fragment of PS1TP5 with EcoRⅠand BglⅡcut sites was extracted and amplified by reverse transcription polymerase chain reaction(RT-PCR), mRNA from HepG2 cells as the template,and ligated into pGEM-T cloning vector. After sequencing,the correct DNA fragment was inserted into inducible proeukaryotic expressive vector pET-32a(+).The competent BL21(DE3)E.coli was transformed,and then cultured and induced by 1mmol/L IPTG at 30℃for 2hrs,3hrs,4hrs.The recombinant PS1TP5 was expressed optimistically with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and confirmed by Western Blotting hybridization.2.ExPASy proteomics server was employed to analyze and forecast the physic-chemical property,structure,and function of PS1TP5.Results showed that it's molecular weight was 15853.4,theoretical pI was 9.41,half-life was 30 hrs (mammalian reticulocytes,in vitro),and extinction coefficient was 17335 M^-1cm^-1or 16960 M^-1cm^-1(280nm)in difference conditions.It also showed that the protein had no coiled coil region,but had four hydrophobic regions.The secondary structure prediction showed that it had helix regions and strand regions.In addition,the motif structure prediction showed that it had three protein kinase C phosphorylation sites. The protein had no transmembrane helix structure and signal peptide.Therefore,the subcellular localization of PS1TP5 was at cytoplasm.3.The DNA fragment of PS1TP5 with EcoRⅠand BglⅡcut sites was constructed into yeast expressive vector pGBKT7,and transformed into AH109 yeast strains.The yeast protein was isolated and detected by Western blotting analysis. Yeast two-hybrid screening was performed by mating AH109 with Y187 containing leukocyte cDNA library plasmid.There were 23 kinds of leukocyte proteins interacting with PS1TP5 leukocyte adhesion protein p150,95,interleukin 2 receptor gamma chain,PALM2-AKAP2 protein(PALM2-AKAP2),eukaryotic translation initiation factor 4A,beta-2-microglobin,solute carrier family 9(sodium/hydrogen exchanger),calreticulin,asialoglycoprotein receptor 1(ASGR1),MHC classⅡlymphocyte antigen,cytochrome c oxidase subunit 1,lymphocyte antigen 86(LY86), lymphocyte cytosolic protein 1,and so on.These results by screening and analyzing indicated that PS1TP5 was involved into cell proliferation,cell differentiation,signal transduction,growth,and metabolism.In addition,One new gene with unknown functions was also screened and enrolled in GenBank,was named human PS1TP5-binding protein 1(PS1TP5BP1)(GenBank accession DQ471327).4.The DNA fragment of PS1TP5 with EcoRⅠand BglⅡcut sites was constructed into eukaryotic expressive vector pcDNA3.1/myc-His(-)A,and transfected into HepG2 cells.Both were mutually the parallel control.The mRNA was isolated from HepG2 cells transfected pcDNA3.1/myc-His(-)A -PS1TP5 and pcDNA3.1/myc-His(-)A empty vector,respectively,and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups.The subtractive library of genes transactivated by PS1TP5 was constructed successfully. We got the sequences of the 23 genes with known functions,including transmembrane 4 superfamily(TM4SF),solute carrier family 7 member 5(SLC7A5),inositol monophosphatase 2(IMPA2),signal sequence receptor-β,epoxide hydrolase 1, protein kinase BRPK,etc.These results indicated that PS1TP5 are closely correlated with immunoregulation,carbohydrate metabolism,signal transduction,the formation mechanism of hepatic fibrosis and occurrence and development of tumor.In addition, One new gene with unknown functions was also screened and enrolled in GenBank, was named human PS1TP5-transactivated protein 1(PS1TP5TP1),(GenBank accession DQ487761).The above results established a material foundation for the further study on the biologic activities of PS1TP5,and provided a clue for the regulatory mechanism in vivo.