| Phage displayed random peptide library of target gene can be constructed through random DNA fragments, which come from a specific gene digested by DNase I. It is the latest application of phage display technique. On the one hand, the foreign peptides displayed in this type of peptide library are limited, on the other hand the fragment size and regions are random. It is easy to analyze and display the complex antigen epitopes. Therefore, target gene phage displayed random peptide library has an advantage in studying new type of virus antigens. In the present study, we carried out the following work of two parts: 1. The construction and identification of phage displayed random peptide library of full length HGV eDNA genome; 2. Phage display of human interferon alpha A-2b and construction of ptiage displayed random peptide library of human interferon alpha A-2b cDNA. 1. Construction and identification of phage displayed random peptide library of full-length HGV cDNA genome Hepatitis 0 virus(HGV) was determined as a member of flaviviridae in 1995. It is a single-stranded RNA virus with a genome of 9400bp in length. HGV is mainly transmitted through transfusion and responsible for chronic liver infection and coinfection with other hepatitis virus. HGV distributed worldwide can induce persistent viremia. Its RNA has been detected in 1-3% of blood donors in China. Because of HGV RNA high prevalence, it is necessary and insistent to study the transmission, pathogenesis, variation and detection of this new type of virus. At present, the known antigen epitopes of HGV are very limited, which block the further research on pathogenesis and immune mechanism of HGV. For attaining the epitopes related to HGV pathogenesis or protection, providing the base of diagnosis and defend of HGV, we constructed a phage displayed random peptide library of full-length HGV genome for HGV epitope screening. Random fragments full-length HGV genome eDNA partially digested with DNase I were cloned into phagemid vector pCANTAB5X. HGV peptides were displayed on the phage surface as fusion proteins at random. The phage displayed library was carefully checked with in-situ hybridization, enzyme digestion and DNA sequencing for the randomization and integrity. The capacity and titer of the library were calculated as 5.34 X iO phage clones and 1.41 X 1O?TU/ml, respectively. Hybridization in situ with different probes of HGV was shown positive. Enzyme digestion and DNA sequencing of inserted fragments showed the randomness of the constructed library. The integrity and randomness of the phage displayed HGV peptide library have been proved good enough for the application in screening of HGV epitopes. Three monoclonal antibodies of HGV E2 were used to select this phage library. After three rounds of affinity selection, 10 clones were isolated and sequenced. The core amino acid sequence "SRGSPRI" was identified and found to be of similar p1 with the 443-449 amino acids of HGV E2 protein. This "SRGSPRI" peptide can be most likely considered an epitope of HOV E2 proteins. 2. Phage display of human interferon alpha A-2b and construction of a phage display library of human interferon alpha A-2b gene fragments Human interferon alpha(hIFN a) family consists of at least 15 subtypes with 78 to 98% amino acid homology. All of these subtypes can regulate immune responses, cell proliferation, and inhibit virus infection. However, hIFN a subtypes differ in the magnitude of their biologic activities through different int... |
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