ABSTRACT
Objective To explore humam cytomegalovirus(HCMV) encoded microRNAs during latent infection in order to help study HCMV virology and latent infection mechanisms.Methods A model of HCMV latent infection via THP-1 cells infected with HCMV was constructed.Deep-sequencing was performed using high-resolution Solexa sequencing platform.The secondary structure of the newly sequenced miRNA was predicted by RNAFold bioinformatics software. Results HCMV encoded various miRNAs during latent infection, including miR-US25-2-3p, miR-US25-2-5p, miR-UL112, miR-US25-1, miR-UL22A and PC-5p-148467 with a predicted length of 25 bp, named hcmv-miR-US33as-5p.Conclusion HCMV can express many types of miRNAs during latent infection.
ABSTRACT
Objective To study the effect of HCV receptors′sequence on virus entry based on the two-dimensional structure and via tandem expression of HCV receptors on mouse hepatocytes.Methods The construced recombinant expression vectors pCDH-hLDLR-hSR-BⅠ-hCD81-GFP, pCDH-hLDLR-hCD81-hSR-BⅠ and pCDH-hCLDN-1-hOCLN-DsRed were cotransfected into 293FT cells with package vectors.The collected recombinant lentivirus expressing hCLDN-1-hOCLN was concentrated and attacked mouse hepatocytes.The transgenic mouse hepatocytes with tandem overexpression of CLDN-1 and OCLN were established after G418-selection.The transduced cells LSCCO/Hepa1-6 and LCSCO/Hepa1-6 were sorted via flow cytometry and puro-G418-selection after recombinant lentivirus expressing hLDLR-hSR-BⅠ-hCD81 and hLDLR-hCD81-hSR-BⅠattacked Hepa1-6 respectively.The infectivity of transduced mouse hepatocytes LSCCO/Hepa1-6 and LCSCO/Hepa1-6 to HCV was analyzed via direct-infection of serum-derived virus.Furthermore, the effect of HCV receptors′sequence on virus entry was studied.Results Both LSCCO/Hepa1-6 and LCSCO/Hepa1-6 enhanced HCV-cell binding.The transduced mouse hepatocytes LSCCO/Hepa1-6 had more HCV endocytosis.Conclusion SR-BⅠhas priority over CD81 in HCV entry in the early stage.
ABSTRACT
Purpose To test the virus inactivation effect of water bath method at 60 ℃ for 10 hours and alcohol treatment for 3 hours which was used in the production of urinary trypsin inhibitor(UTI).Methods Sindbis virus,Pseudorabies virus(PRV) and poliovirus1(PV1) were used as indicated viruses in this test.After being added separately into the UTI raw material in 10% proportion,the viruses were treated with water bath at 60 ℃ for 10 hours and alcohol for 3 hours and then the samples of UTI were taken to inoculate the cell line for assay of cytopathic effect.Results The water bath at 60 ℃ for 10 hours could inactive Sindbis,PRV and PV1 in more than(6.503±0.102)LgTCID_(50),(6.42±0.158) LgTCID_(50) and(6.587±0.061)LgTCID_(50) respectively,and alcohol treatment for 3 hours could inactive Sindbis,PRV and PV1 in more than(5.88±0.204)LgTCID_(50),(6.378±0.268)LgTCID_(50) and(5.963±0.118) LgTCID_(50) respectively.No cytopathic effect was found in the cell line which was inoculated with treated samples after blind passage for three generations.Conclusion The water bath method at 60 ℃ for 10 hours and alcohol treatment for 3 hours which were used in the production of UTI had good effects on virus inactivation and the inactivation efficiency on Sindbis,PRV and PV1 was more than 6 LgTCID_(50)/mL.
ABSTRACT
Objective The gene encoding part of HTLV(Human Tcell leukemia viruses) envelope protein gp21 was cloned and expressed in order to prepare diagnostic agent for detecting HTLV.Methods The gene encoding part of HTLV envelope protein gp21 was obtained by combining four oligonucleotides into a long DNA fragment,then cloned and subcloned into Tvector and pGEX2T respectively.Expression was performed under induction in a fusion way that target protein gene was inserted downstream the carrying protein gene of pGEX2T which makes purification of target protein convenient.At last,Target protein was purified by affinity chromatography in glutathione sepharose 4B column and its antigenicity was confirmed by immunoblotting.Results and conclusion Sequence analysis showed it was feasible to obtain a target gene by ligating short oligonucleotides;As expected,target protein was expressed which exhibited strong antigenicity after being subcloned into fusion expression vector pGEX2T.