Human serum albumin(HSA) is widely utilized for medical purposes and biochemical research.Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA(rHSA).However,transgene spread...Human serum albumin(HSA) is widely utilized for medical purposes and biochemical research.Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA(rHSA).However,transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value.This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds,and a simple process for recovery and purification of rHSA for economical manufacture.An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference(RNAi) cassette suppressing the CYP81A6 gene.This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) cassette which confers glyphosate tolerance.ANX Sepharose Fast Flow(ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance(Butyl HP) hydrophobic interaction chromatography was used to purify rHSA.A transgenic rice line,HSA-84,was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds.This line also demonstrated high sensitivity to bentazon,and thus could be killed selectively by a spray of bentazon.A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%.Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA.This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.展开更多
A female-specific protein, vitellogenin （Vg）, and its corresponding egg vitellin （Vt） are identified in the ectoparasitic wasp Nasonia vitripennis. Both native Vt and Vg have a molecular mass of about 350 kDa, whi...A female-specific protein, vitellogenin （Vg）, and its corresponding egg vitellin （Vt） are identified in the ectoparasitic wasp Nasonia vitripennis. Both native Vt and Vg have a molecular mass of about 350 kDa, which is composed of two subunits of approximately 190 kDa and 165 kDa under reducing and denaturing conditions （sodium dodecyl sulfate-polyacrylamide gel electrophoresis）. An indirect sandwich enzyme-linked immunosorbent assay developed with both monoclonal and polyclonal antibodies against N. vitripennis Vt. Vg was first detected in the hemolymph on the 10th day after parasitism, and was first observed in oocytes on the 12th day. In adults deprived of food, the highest hemolymph Vg level occurred at the time of adult eclosion and the highest level of Vt in ovaries was found at 30 h after eclosion. In contrast, feeding adults with 20% sucrose resulted in the reduction of Vt uptake by ovaries and the extension of life span, but had little effect on Vg production. Deprived of hosts, starvation of female wasps had no significant effects on ovariole growth and oocyte maturation before the wasps died. However, starvation of female wasps supplied with hosts accelerated the wasps laying progeny into hosts, but resulted in a decrease of total progeny production by comparison with wasps fed with 20% sucrose.展开更多
基金Project supported by the Fundamental Research Funds for the Central Universities(No.2017FZA6011)the National Key Transgenic Research Projects(No.2016ZX08010003)of China
基金Project(No.2011ZX08010-003)supported by the Ministry of Agriculture of China
文摘Human serum albumin(HSA) is widely utilized for medical purposes and biochemical research.Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA(rHSA).However,transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value.This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds,and a simple process for recovery and purification of rHSA for economical manufacture.An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference(RNAi) cassette suppressing the CYP81A6 gene.This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) cassette which confers glyphosate tolerance.ANX Sepharose Fast Flow(ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance(Butyl HP) hydrophobic interaction chromatography was used to purify rHSA.A transgenic rice line,HSA-84,was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds.This line also demonstrated high sensitivity to bentazon,and thus could be killed selectively by a spray of bentazon.A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%.Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA.This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.
基金Acknowledgments This work was supported by the National Basic Research and Development Program of China （2006CB 102005）, the National Natural Science Foundation of China （Grant No. 30270899）, the program for New Century Excellent Talents in the University of the Ministry of Education of China （NCET-05-0513）, and the Innovation Research Team Program of the Ministry of Education of China （Grant No. IRT0535）.
文摘A female-specific protein, vitellogenin （Vg）, and its corresponding egg vitellin （Vt） are identified in the ectoparasitic wasp Nasonia vitripennis. Both native Vt and Vg have a molecular mass of about 350 kDa, which is composed of two subunits of approximately 190 kDa and 165 kDa under reducing and denaturing conditions （sodium dodecyl sulfate-polyacrylamide gel electrophoresis）. An indirect sandwich enzyme-linked immunosorbent assay developed with both monoclonal and polyclonal antibodies against N. vitripennis Vt. Vg was first detected in the hemolymph on the 10th day after parasitism, and was first observed in oocytes on the 12th day. In adults deprived of food, the highest hemolymph Vg level occurred at the time of adult eclosion and the highest level of Vt in ovaries was found at 30 h after eclosion. In contrast, feeding adults with 20% sucrose resulted in the reduction of Vt uptake by ovaries and the extension of life span, but had little effect on Vg production. Deprived of hosts, starvation of female wasps had no significant effects on ovariole growth and oocyte maturation before the wasps died. However, starvation of female wasps supplied with hosts accelerated the wasps laying progeny into hosts, but resulted in a decrease of total progeny production by comparison with wasps fed with 20% sucrose.
