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M. P. Chumakov Institute of Poliomyelitis and Viral Encephalitides A NEW SEROLOGICAL SYSTEM FOR PREDICTING HCV TREATMENT RESPONSE, PRELIMINARY RESULTS 1 Kuznetsova , 1 Tallo , 2 Brjalin , 1 Reshetnjak , Tatiana Tatjana Vadim Irina 3 3 1 Maria Smirnova , Alexei Shevelev , Valentina Tefanova 1Department of Virology, NIHD, Tallinn, Estonia 2West-Tallinn Central Hospital, Tallinn, Estonia 3Federal State Budgetary Institution «M. P. Chumakov Institute of Poliomyelitis and Viral Encephalitides» of RAMS, Moscow, Russia Background Previously we analyzed complete NS5A sequences from Estonian patients with chronic HCV-1b infection who had received combination therapy with PegIFNα-2a plus ribavirin. Phylogenetic analysis revealed four subclades tending to confer a different treatment outcome. These subclades were characterized by several steady inherited amino acid substitutions. However, detection of amino acid substitutions in HCV is precluded by irreproducible PCR of relevant NS5A region. Serological methods, e. g. solid-phase immune-assay (ELISA), provides an efficient and inexpensive alternative to PCR for routine assay. However, there are no commercial kits available for assay of antibody response towards NS5A regions. Additionally, NS5A protein is not easily available as an antigen for serological tests. Objectives Our work was focused on designing short derivatives of NS5A gene adapted for efficient production in E. coli and allowing NS5A putative subclades discrimination within HCV1b genotype by serological testing. The first stage required establishing an innovative screening method for designing truncated antigens. This approach included in vitro constructing libraries of NS5A fragments and their screening for expression efficiency in bacteria. The second stage included selection of an appropriate clones by serological methods (Western-blot). The third stage involved engineering high-efficient producers of designed NS5A mini-derivatives fused with green fluorescent protein (GFP). Results Methods 1. Nicking PCR product with DNAse. NS5A PCR products were treated with DNAse in several dilutions to produce nicks in DNA. Probes with mild extent of degradation were used for the next stage. 1. Selection of DNA probes with mild extent of degradation 2. Generation of deletion derivatives. DNA probes purified by phenol-chloroform extraction were treated with DNA polymerase I for extending the gaps in DNA. Then DNA probes were mixed with random primers bearing constant adapter sequence at 5’-end and ligated. The library of deletion derivatives was eventually produced by PCR with a single adapter primer. 3. Cloning library of gene fragments into specially designed LacZ-based vector was performed. Phenotypical selection for brightness of colonies was carried out. Level of protein expression was evaluated by beta-galactosidase activity test. 2. Results of PCR with a single adapter primer 4. PCR with standard primers for determination of insertion size in the selected clones was performed. Obtained PCR products were sequenced. 4. Insertion size range: from 50 bp to 700 bp 5. LacZ-based protein production and Western-blot with HCVpositive sera from patients were performed. 5. One out of 40 clones was immune-positive 6. Engineering GFP-based producers of selected NS5A derivatives was carried out. 6. Primary purification of a fused protein with GFP Delution of DNAse 104 105 106 conc. 1 kb Ladder Probes: a s n r 100 bp Ladder 3. Quantification of beta-galactosidase activity in soluble cell fractions beta-galactosidase activity Precipitate Supernatant PAGE electrophoresis under denaturing and quasi-native conditions kDa Conclusions and plans Extraction by: Tris SDS Ac.acid Tris SDS Ac.acid The scheme of producing short derivatives library was designed. One water-soluble immune-positive protein was obtained. Its purification and further serological testing in ELISA with panels of serum samples is in a progress. Testing alternative enzymes for extending the gaps in DNA, e.g. exonuclease III and lambda phage nuclease, is in course. Obtaining and testing new NS5A-derived proteins is considered.