Objective:To identify white spot syndrome virus(WSSV)entry into the host-cells of the cultured shrimp Penaeus monodon,we have attempted to localize PmRab7(Ras-related in brain)which is playing a vital role in the WSSV...Objective:To identify white spot syndrome virus(WSSV)entry into the host-cells of the cultured shrimp Penaeus monodon,we have attempted to localize PmRab7(Ras-related in brain)which is playing a vital role in the WSSV internalization.Methods:In this study,we have cloned PmRab7 and expressed in Escherichia coli,further purified rPmRab7 was used for antibody production,isolation of lysosomal sub-cellular fractions and western blot against lysosomal protein.Moreover,high fold-change in PmRab7 regulation with increasing copy number of WSSV has been studied by using real-time PCR.Results:651 bp amplicon size gene was successfully amplified,ligated amplicon with pTZ T-tail vector confirmed by colony PCR and retriction enzyme digestion on agarose gel.Subcloned(pRSET-B)651 bp gene transformed successfully in Rosetta and after 6 h of induction expressed rPmRab7 was on SDS page,furthermore soluble fraction of rPmRab7(26 kDa)was purified by ni-NTA column.AntiPmRab7 antibody was received by Merk Pvt.Ltd.,and western blot analysis revealed that PmRab7 is present in the lysosomal sub-cellular fraction.Copy number of WSSV was increased 5 fold in 24 h and 20 fold in 72 h of infection and subsequently transcrtipt of PmRab7 was Ct=1.0 to Ct=8.5.Conclusions:Presence of PmRab7 on lysosome clearly indicating PmRab7 participating in lysosomal maturation,other hand WSSV may follow the same route of entry.WSSV internalization has directly linked with regulation of PmRab7.展开更多
基金Supported by INCOIS(G4/1515/2013),Ministry of Earth Sciences,Govt.of India.
文摘Objective:To identify white spot syndrome virus(WSSV)entry into the host-cells of the cultured shrimp Penaeus monodon,we have attempted to localize PmRab7(Ras-related in brain)which is playing a vital role in the WSSV internalization.Methods:In this study,we have cloned PmRab7 and expressed in Escherichia coli,further purified rPmRab7 was used for antibody production,isolation of lysosomal sub-cellular fractions and western blot against lysosomal protein.Moreover,high fold-change in PmRab7 regulation with increasing copy number of WSSV has been studied by using real-time PCR.Results:651 bp amplicon size gene was successfully amplified,ligated amplicon with pTZ T-tail vector confirmed by colony PCR and retriction enzyme digestion on agarose gel.Subcloned(pRSET-B)651 bp gene transformed successfully in Rosetta and after 6 h of induction expressed rPmRab7 was on SDS page,furthermore soluble fraction of rPmRab7(26 kDa)was purified by ni-NTA column.AntiPmRab7 antibody was received by Merk Pvt.Ltd.,and western blot analysis revealed that PmRab7 is present in the lysosomal sub-cellular fraction.Copy number of WSSV was increased 5 fold in 24 h and 20 fold in 72 h of infection and subsequently transcrtipt of PmRab7 was Ct=1.0 to Ct=8.5.Conclusions:Presence of PmRab7 on lysosome clearly indicating PmRab7 participating in lysosomal maturation,other hand WSSV may follow the same route of entry.WSSV internalization has directly linked with regulation of PmRab7.
