| ObjectiveTo prepare HBV esiRNA library and to study its inhibition effects on HBV replication in HepG2.215 cells.Method1,The purification of GST-tagged Escherichia coli endoribonucleaseⅢ(GST-RNaseⅢ) fusion protein The plasmid pGEX-KG-RNaseⅢLac-operator-controlled expressing GST-RNaseⅢfusion protein was constructed and transformed to Escherichia coli BL21(DE3).Monoclonal was selected and in mass culture,then was induced by adding lactose analogs IPTG.The induced bacteria were collected and lysed.Then GST-RNaseⅢfusion protein was extracted by GST-agarose beads affinity chromatography and purified by dialysis.2,The two-orientation-transcription template of 1-540nt of HBV genome(T7-HBV-1 DNA) and its products identification Design primers that each has T7 promoter at 5'end for T7-HBV-1 DNA PCR amplification.T7 RNA polymerase was involved into in vitro two-orientation-transcription of T7-HBV-1 DNA and the products were annealed and dsRNA confirmation,i.e.,T7-HBV-1 dsRNA.3, The test and optimization of activity and reaction condition about GST-RNaseⅢenzyme,and the preparation of T7-HBV-1 esiRNA library Using identical amount of GST-RNaseⅢand same substrate of T7-HBV-1 dsRNA involving in reaction to explore most the appropriate reaction time for the purpose of obtaining most products. Upon optimized reaction protocol, more T7-HBV-1 dsRNA was cleaved into T7-HBV-1 esiRNA library which was finally separated and purified.4,Analysis of T7- HBV-1 esiRNA library efficiency T7-HBV-1 esiRNA library generated from above digestion was then used 0,50,100 and 150nmol/L to transfect into HepG2.215 cells carrying HBV after which HepG2.215 cells were cultured for 3 days.Apply semiquantitative PCR and QRT-PCR to test the HBsAg mRNA level of HepG2.215 cells treated above.Employ the HBsAg ELISA to test HBsAg protein changes in supernatants of HepG2.215 cells treated above.Results1,GST-RNaseⅢenzyme was successfully induced and isolated The length of RNaseⅢDNA products by PCR was consistent with the theoretical length of it. The recombinant plasmid was confirmed to be right constructed by double restriction enzyme digestion identification and DNA sequencing. The SDS-PAGE of induced and non-induced Escherichia coli BL-21(DE3) transformed by pGEX-KG-RNaseⅢplasmid showed:the induced bacteria lane emerged a significantly deep protein band at relative molecule weight between 5.5-7.2×104 which was consistent with GST-RNaseⅢ,indicating that induced Escherichia coli BL-21(DE3) successfully expressed GST-RNaseⅢ.The purity of GST-RNaseⅢafter dialysed was more than 95%,and the concentration 1.665g/L.2,In vitro two-orientation-transcription products of T7-HBV-1 DNA was confirmed to be dsRNA The length of T7-HBV-1 DNA by PCR was the same as its theoretical length(586bp),and so as the transcription products.While the transcription products couldn't be degraded by DNasel and RNaseA before denaturation, and on the contrary by RNaseA when keeping single strand,which, summarized that the transcription products were dsRNA.3, GST-RNaseⅢenzyme could perform the capacity of cleaving T7-HBV-1 dsRNA into esiRNA library With the reaction time extending,products length were shrinking,and the quantity was decreasing.One hour was the reaction time when the products quantity was more and length mostly at 20bp.Purified esiRNA library length approach to that of siRNA.4, The HBsAg mRNA and protein level were significantly reduced in HepG2.215 cells after transfection of the esiRNA library Semiquantitative PCR and QRT-PCR showed:HBsAg mRNA level was downregulated more significantly with T7- HBV-1 esiRNA concentration increased to be transfected to HepG2.215 cells.And HBsAg ELISA revealed that the HBsAg protein in supernatants was decreased as well.ConclusionBiological prepared esiRNA library could successfully inhibit the replication of HBV in HepG2 cells opening up a novel idea for the treatment of HBV infection.In further saying,gene interference by esiRNA library provide a suggestion for clinical therapy against virus infection. |
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