Cloning And Expression Of Hepatitis C Virus Nonstructural Protein 5A, And Screening Of Genes Transgulated By Its Deletion Mutant Protein Of Hepatitis C Virus With CDNA Microarray

Background and Objective:Hepatitis C virus (HCV) is the transfusion-associated hepatitis viruses, within the family Flaviviridae. And it is the most common infectious cause of chronic liver disease in the United States and many other developed countries . It is estimated that 170 million individuals are infected with this virus (Anonymous, 1999), and in 80% of cases the virus establishes a chronic infection, resulting in fibrosis, cirrhosis and, increasingly, hepatocellular carcinoma (HCC).It harms human sapiens greatly. The predominant site of replication and probably infection is the liver, yet several studies have suggested that HCV may infect other cell types, such as B cells, monocytes/macrophages, and dendritic cells where it could induce immune dysfunctions. Understanding the molecular basis by which HCV targets liver cells and extra-hepatic sites is critical to unravel the mechanisms of its pathogenesis and disease chronicity. The HCV genome is a positive, single strand RNA molecule which includes two untranslated regions at the 5' and 3' ends,and a large open reading frame encoding for a 3010±3030 amino acid polyprotein. This polyprotein is post-translationally processed into structural and non-structural proteins, the cleavage being dependent on host and viral encoded enzymes (Figure 1). The structural proteins, encoded in the N-terminal region, include the core protein followed by two envelope glycosylated proteins: E1 and E2. The nonstructural domain encodes for six proteins: NS2, 3, 4A, 4B and 5A, 5B. NS5A protein(447 amino acid residues for HCV-1b) is phosphorylated at its serine residues for basal phosphorylation in the C-terminal region. In an NS4A protein-dependent manner,NS5A protein is hyperphosphorylated in serine residues in the central region.NS4A protein also associates with and directs hyperphosphorylation of the NS5A protein. Although the role of NS5A in the HCV life cycle is not yet known,namely,a region(amino acid 2209 to 2248) of NS5A is associated with sensitivity to IFN. And it has been demonstrated that this region has been designated as the IFN-sensitivity determining region(ISDR),and it is useful for predicting the response to IFN. On the other hand ,it has been observed that NS5A protein repressed PKR through a direct interaction with the protein catalytic domain ,and that NS5A protein disrupted PKR dimerization in vivo ,which resulted in the inhibition of PKR-mediated eIF-2 alpha phosphorylation.However,it remains unclear whether or not NS5A protein is involved in the resistance of infected cells to the antiviral effects of IFN. It has been demonstrated that NS5A protein functions as a potent transcriptional activator when 129-146 amino residues at the N-terminus of the ns5a protein are deleted.The transcriptional activation domain of NS5A includes two acidic amino acid regions(2 143-2 184 2 220-2 273 amino residues ) and a praline-enriched region. (2 282-2 327 amino residues)These regions are known as transcriptional activation regions. NS5A protein could influence the gene expression level in the hepatocytes, which related possibly with HCV pathogenesis mechanism. The investigation on the NS5A protein interaction with the celluar genes in the hepatocytes can further explain the HCV infection mechanism and the achievements will conduce to seek valid means for curing the disease.Methods:1. The specific primers were designed based on the reference and the HCV-1b sequence published by NCBI,and the PCR products were prospected to contain NS5A sequence. A 1632bp DNA fragment was extracted and amplified by reverse transcription polymerase chain reaction (RT-PCR), HCV-1b RNA extracted from the patient's serum as the template, and ligated into pGEM-T cloning vector. The recombinant pGEM-T vector was sent to be sequenced . The other pair of primers of NS5A were designed according to the sequence we got. A 1341 bp DNA fragment was extracted and amplified by polymerase chain reaction (PCR), recombinant pGEM-T vector as the template, and ligated into pGEM-T cloning vector again.2. After being sequenced, the correct DNA fragment of NS5A was inserted into inducible proeukaryotic expressive vector pET-32a(+). The competent BL21 (DE3) E. coli was transformed, and then cultured and induced by 0.5mM/ml IPTG at 30℃for 5hrs.3.According to the NS5A sequence we got,the NS5Adml primers were designed. The NSSAdm1 fragment was extracted and amplified by polymerase chain reaction (PCR), , recombinant pET-32a(+)-NS5A as the template, and ligated into pCDNA-3.1(-) cloning vector. Sequencing it showed we got the correct one.4. The DNA fragment of NS5Adml with KpnI and HindIII cut sites was constructed into eukaryotic expressive vector pcDNA3.1/myc-His(-) A, and transfected into HepG2 cells . The RNA was isolated from HepG2 cells transfected with pcDNA3.1/myc-His(-)A-NS5Adm1,andpcDNA3. 1/myc-His(-) A empty vector respectively.The cDNA microarray was employed respectively to analyze the differentially expressed DNA sequence between the two groups.Results:1. After being sequenced ,the NS5A sequence we got was 91.4 % similar to the one on NCBI. Thus NS5A was cloned successfully. 2. The recombinant NS5A- pET-32a(+) was expressed optimistically and confirmed by Western Blotting. 3. Sequencing NS5Adm1 showed we got the correct one. 4. There were 3 kinds of hepatocytes gene up regulated by NS5Adm1 and 10 kinds of hepatocyte gene down regulated by NS5Adm1.Conclusions1.NS5A is cloned successfully. The successful expression of NS5A formed the baisis of preparing its monoclonal antibody and polyclonal antibody. 2. The up regulation genes: (1) Homo sapiens ATPase, Ca++ transporting, plasma membrane 2 (ATP2B2); (2) Homo sapiens primase, polypeptide 1, 49kDa (PRIM1); (3)ring finger 185 The down regulation genes: (1) Human sapiens hbrm mRNA; (2) Homo sapiens SCL/TAL1 interrupting locus (STIL3).; (3) Homo sapiens cDNA FLJ16813 fis, clone THYMU3030231, highly similar to Ribonucleoside-diphosphate reductase M2 chain; (4)Homo sapiens zinc finger protein 253; mRNA(5) interleukin 2 enhanced factor;(6) Homo sapiens U2-associated SR140 protein (SR140); (7) Homo sapiens neuroblastoma-amplified protein (NAG);(8) Homo sapiens calponin 3, acidic (CNN3); (9) Homo sapiens large conductance calcium-activated potassium channel beta2 subunit (KCNMB2);(10) Homo sapiens ralA binding protein 1 (RALBP1). These results by screening and analyzing indicated that NSSAdm1 was involved into signal transduction, ionic transporting tumor and apoptosis. 3.The above results established a material foundation for the further study on the biologic activities of NS5A, and provided a clue for the regulatory mechanism in vivo.