الفهرس 
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Abstract
There were some predictions from bioinformatics analysis, that are lacked practical confirmation, like the presence of some uniqueresidues to JFH-1 sequence that may be helpful in increasing theRdRp or improve replication of genotype 4 in cell culture system.Second, the presence of some unique residues to genotypes 2&3 thatmay by a way or another help in shortening the length of interferon/ribavirin treatment period especially in genotype 4. Many resistance mutations found in the naïve sequences of different genotype sequences including our sequence more genotype specific inhibitors are required to be discovered that stands beside that the very rare available molecular characterization studies
conducted on genotype 4. Cloning of NS5B570 coding sequence (eluted band confirmed by PCR) in a mammalian expression vector, pTARGET, was carried
out and colonies contained the cloned insert were selected by amplicillin and blue/white screening. Then white colonies were tested by PCR amplification of the three overlapping fragments. For successful expression, right oriented ligation of NS5B570 insert needed to be detected that was carried out by PCR that was followed by 2 successive semi-nested PCR reactions for more confirmation. Before DNA immunization of mice with the cloned right oriented NS5B570 coding sequence to pTARGET (pTARGETNS5B570 construct), 2 important processes were performed; the first, colony (contained the right oriented insert) was subjected for large-scale preparation of endofree plasmid DNA and the extracted construct was subjected for another PCR for right orientation confirmation that was followed by a nested PCR on the eluted band. The second, was the transfection of HepG2 with the with right oriented pTARGET-NS5B570 construct to predict a successful expression in transformed E. coli and give strong indication of expression reliability in immunized mice tissue. Such expression was detected at the transcription level by RT-PCR after RNA extraction and RT-PCR for detection of extracted RNA integrity and also normal transcription of a house keeping gene in the transfected cells. Mice were DNA immunized by injection of pTARGETNS5B570 construct three successive, 10 days apart, intramuscularly. Pooled collected mice sera were used for expression detection of the cloned NS5B570 at the protein level in E. coli using direct ELISA. The specificity of such interaction was confirmed by sandwich ELISA through capturing the expressed NS5B570 E. coli by commercial specific polyclonal rabbit anti-NS5B antibodies giving higher signal with the IMS than CMS. The specific interaction between the expressed NS5B570 in E. coli and the raised antibody in IMS was strongly confirmed by western blot that gave specific band at the expected molecular weight. All of these results confirm reliability of NS5B570 expression cloned under CMV promotor in a prokaryotic system (E. coli). In Sandwich ELISA, expressed NS5B570 protein and the raised antibodies in IMS were used for differentiation between CHS and IHS and such system was compared with another using commercial specific polyclonal rabbit anti-NS5B antibodies, instead of the present in IMS, to capture NS5B570 expressed in E. coli and differentiation between CHS and IMS. The result indicated that our
system was more reliable in differentiation CHS and IHS than using the commercial antibodies.