To obtain the protein of duck interferon alpha and study its biological activities, the prokaryotic expression vector of DuIFN-αwas constructed and expressed in BL21 (DE3) plysS. Using PCR technique, the protein gene...To obtain the protein of duck interferon alpha and study its biological activities, the prokaryotic expression vector of DuIFN-αwas constructed and expressed in BL21 (DE3) plysS. Using PCR technique, the protein gene of DuIFN-αwas cloned from pMD-18-duIFN-αrecombinant. The gene was then inserted to pGEM-T vector and identified by restriction endonuclease analysis and sequencing. DuIFN-αwas ligated with the prokaryotic expression vector of pET30 a, then transformed into BL21 (DE3) plysS. The best inducing time and IPTG concentration for the expression of this recombinant protein was tested through the expression of the positive recombinant with different time span and different IPTG concentration. Lots of the protein of DuIFN-αwere expressed in BL21(DE3)plysS with 1 mmol·L-1 IPTG for 4 hours and its molecular weight for 34 000.展开更多
When recombinant E.coli BL21(DE3)is induced to express Bacillus subtilis lipase A(BsLipA)using Isopropylβ-D-1-thiogalactopyranoside(IPTG),the one-time addition of IPTG leads to problems such as limited cell growth,a ...When recombinant E.coli BL21(DE3)is induced to express Bacillus subtilis lipase A(BsLipA)using Isopropylβ-D-1-thiogalactopyranoside(IPTG),the one-time addition of IPTG leads to problems such as limited cell growth,a short enzyme production period,and low yield.To address these issues,this study proposes an innovative IPTG feeding strategy,where the IPTG feeding rate is adjusted based on cell growth rate between 4 and 10 h,followed by a constant IPTG feeding rate after 12 h.Fermentation experiments in a 5 L bioreactor demonstrated that IPTG feeding according to this strategy resulted in continuous enhancement of BsLipA activity,reaching 288.50 U/mL.Compared to a batch induced with a one-time addition of 0.2 mmol/L IPTG,BsLipA activity increased by 6.67 times.This IPTG feeding strategy was applied to a low-nutrient fermentation process with DO-start glucose feeding,leading to a further increase in BsLipA enzyme activity,with the highest activity reaching 580.29 U/mL.The results indicate that this strategy significantly reduces the toxic effects of IPTG on the cells,improves biomass,extends the enzyme production phase,and enhances BsLipA expression levels by balancing the induction strength and cell growth conditions.展开更多
Objective To construct and express a recombinant plasmid pGEX-Sj26GST of Schistosoma japonicum(Sj)in Escherichia coli(E.coli)BL21(DE3).Methods Total RNA was extracted from Sj adult worms by RNeasy Mini kit,26 kilodalt...Objective To construct and express a recombinant plasmid pGEX-Sj26GST of Schistosoma japonicum(Sj)in Escherichia coli(E.coli)BL21(DE3).Methods Total RNA was extracted from Sj adult worms by RNeasy Mini kit,26 kilodalton glutathione-S-transferases of Schistosoma japonicum(Sj26GST)antigen gene was amplified by real-time PCR(RT-PCR)from the total RNA,then cloned into a prokaryotic expression plasmid pGEX-1λT and transformed into E.coli BL21(DE3)to construct pGEX-Sj26GST;BL21(pGEX-展开更多
The Hough mode decomposition (HMD) is used to investigate the global structures of the eastward propagating diurnal tide of zonal wavenumber 3 (DE3). The tide is delineated by using the SABER/ TIMED temperatures colle...The Hough mode decomposition (HMD) is used to investigate the global structures of the eastward propagating diurnal tide of zonal wavenumber 3 (DE3). The tide is delineated by using the SABER/ TIMED temperatures collected during 2002―2006. The HMD analysis results show that the DE3 tide is primarily dominated by two leading propagating Hough modes, i.e., (-3, 3) and (-3, 4) modes; the in-fluences .of the other Hough modes including trapped modes can be neglected. Based upon the HMD analysis results, this paper first reported the maximum of the tidal activity in the MLT region. The re-sults show that the DE3 tide exhibits annual unimodal distribution with the maximal amplitude occur-ring at 110 km in late summer (around July each year). Moreover, characteristic 2-year period variation is observed in the (-3, 3) Hough mode. And this type of inter-annual variation is further reflected in the tidal amplitude at 110 km height. For example, corresponding to the 2-year variation of the (-3, 3) mode, the DE3 tidal amplitude exhibits two substantially enhanced activities with maximal amplitude exceed-ing 12 K in 2002 and 2004, respectively. Moreover, current investigation results indicate that the influ-ence of the second propagating Hough mode, (-3, 4) mode, is important, in particular at the height un-der 100 km, where the DE3 amplitudes exhibit antisymmetric distribution with respect to the equator. The (-3, 4) mode exhibits bimodal distribution over a yearly course, which dominates the DE3 tide in the lower mesosphere. For example, two maximal DE3 activities were observed in late-winter-to-early- spring and late-autumn-to-early-winter, respectively. The first maximum is seen in the south of the equator, and the second maximum is in the north of it.展开更多
基金Studying Abroad and Coming Back Home Fund (LC02C08).
文摘To obtain the protein of duck interferon alpha and study its biological activities, the prokaryotic expression vector of DuIFN-αwas constructed and expressed in BL21 (DE3) plysS. Using PCR technique, the protein gene of DuIFN-αwas cloned from pMD-18-duIFN-αrecombinant. The gene was then inserted to pGEM-T vector and identified by restriction endonuclease analysis and sequencing. DuIFN-αwas ligated with the prokaryotic expression vector of pET30 a, then transformed into BL21 (DE3) plysS. The best inducing time and IPTG concentration for the expression of this recombinant protein was tested through the expression of the positive recombinant with different time span and different IPTG concentration. Lots of the protein of DuIFN-αwere expressed in BL21(DE3)plysS with 1 mmol·L-1 IPTG for 4 hours and its molecular weight for 34 000.
基金funded by the Jiangsu Basic Research Center for Synthetic Biology(Grant No.BK20233003)the Key Project of the Bayannur National Agricultural High-Tech Industry Demonstration Zone(Grant Nos.NMKJXM202210,NMKJXM202309).
文摘When recombinant E.coli BL21(DE3)is induced to express Bacillus subtilis lipase A(BsLipA)using Isopropylβ-D-1-thiogalactopyranoside(IPTG),the one-time addition of IPTG leads to problems such as limited cell growth,a short enzyme production period,and low yield.To address these issues,this study proposes an innovative IPTG feeding strategy,where the IPTG feeding rate is adjusted based on cell growth rate between 4 and 10 h,followed by a constant IPTG feeding rate after 12 h.Fermentation experiments in a 5 L bioreactor demonstrated that IPTG feeding according to this strategy resulted in continuous enhancement of BsLipA activity,reaching 288.50 U/mL.Compared to a batch induced with a one-time addition of 0.2 mmol/L IPTG,BsLipA activity increased by 6.67 times.This IPTG feeding strategy was applied to a low-nutrient fermentation process with DO-start glucose feeding,leading to a further increase in BsLipA enzyme activity,with the highest activity reaching 580.29 U/mL.The results indicate that this strategy significantly reduces the toxic effects of IPTG on the cells,improves biomass,extends the enzyme production phase,and enhances BsLipA expression levels by balancing the induction strength and cell growth conditions.
文摘Objective To construct and express a recombinant plasmid pGEX-Sj26GST of Schistosoma japonicum(Sj)in Escherichia coli(E.coli)BL21(DE3).Methods Total RNA was extracted from Sj adult worms by RNeasy Mini kit,26 kilodalton glutathione-S-transferases of Schistosoma japonicum(Sj26GST)antigen gene was amplified by real-time PCR(RT-PCR)from the total RNA,then cloned into a prokaryotic expression plasmid pGEX-1λT and transformed into E.coli BL21(DE3)to construct pGEX-Sj26GST;BL21(pGEX-
基金Supported by Knowledge Innovation Project of the Chinese Academy of Sciences (CAS) (Grant Nos. KZCX2-YW-123 and KGCX3-SYW-403)National Natural Science Foundation of China (Grant No. 40333034)Institute of Atmospheric Physics of CAS (Grant No. IAP07212)
文摘The Hough mode decomposition (HMD) is used to investigate the global structures of the eastward propagating diurnal tide of zonal wavenumber 3 (DE3). The tide is delineated by using the SABER/ TIMED temperatures collected during 2002―2006. The HMD analysis results show that the DE3 tide is primarily dominated by two leading propagating Hough modes, i.e., (-3, 3) and (-3, 4) modes; the in-fluences .of the other Hough modes including trapped modes can be neglected. Based upon the HMD analysis results, this paper first reported the maximum of the tidal activity in the MLT region. The re-sults show that the DE3 tide exhibits annual unimodal distribution with the maximal amplitude occur-ring at 110 km in late summer (around July each year). Moreover, characteristic 2-year period variation is observed in the (-3, 3) Hough mode. And this type of inter-annual variation is further reflected in the tidal amplitude at 110 km height. For example, corresponding to the 2-year variation of the (-3, 3) mode, the DE3 tidal amplitude exhibits two substantially enhanced activities with maximal amplitude exceed-ing 12 K in 2002 and 2004, respectively. Moreover, current investigation results indicate that the influ-ence of the second propagating Hough mode, (-3, 4) mode, is important, in particular at the height un-der 100 km, where the DE3 amplitudes exhibit antisymmetric distribution with respect to the equator. The (-3, 4) mode exhibits bimodal distribution over a yearly course, which dominates the DE3 tide in the lower mesosphere. For example, two maximal DE3 activities were observed in late-winter-to-early- spring and late-autumn-to-early-winter, respectively. The first maximum is seen in the south of the equator, and the second maximum is in the north of it.
