[Objective] The research aimed to study the self-activation test of inverting T1083 substitution mutation BD fusion vector pGBKT7-TS into yeast,and discuss whether its expression product can be used as bait for furthe...[Objective] The research aimed to study the self-activation test of inverting T1083 substitution mutation BD fusion vector pGBKT7-TS into yeast,and discuss whether its expression product can be used as bait for further two-hybrid screening.[Method] T1083 substitution mutation BD fusion vector pGBKT7-TS was inverted into yeast to make the self-activation and protein expression toxic detection test.[Result] This expression product of the fragment was inactive and had no toxic to yeast cell.It could be used as bait for further two-hybrid screening.[Conclusion] The research laid the experimental foundation for further study of the effect of T1083 substitution mutation on the interaction of NtKrp and its target partner.展开更多
[ Objective ] The paper was to construct bait vector pGBKTT-Lescpth5, and detect its self-activated transcription activity and the toxicity on yeast cells. [ Method] Lescpth5 was amplified by PCR technique, and connec...[ Objective ] The paper was to construct bait vector pGBKTT-Lescpth5, and detect its self-activated transcription activity and the toxicity on yeast cells. [ Method] Lescpth5 was amplified by PCR technique, and connected into bait vector pGBKTT. The recombined vector was transformed into yeast competent ceils AH109 and carried out self-activated detection and toxicity detection in the au.xotrephic medium. [ Result] Digestion and sequencing results showed that bait vector pGBKT7-Lescpth5 was suecessfully constructed with correct reading frame. Self-activated activity and toxicity detection results showed that bait vector had no self- activated activity on yeast strain AH109, which also had no toxicity on yeast. [Condusion] Bait vector successfully constructed could be used in yeast two-hybrid system, which laid the foundation for screening of cDNA library in the next step.展开更多
Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells...Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 μm plasmid. The human interferon αA (IFN αA) gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium. Tbe data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN αA biological activity was 2.6×10^(10) units per liter, demonstrating that high-level expression and secretion of IFN αA were achieved in yeast by using the stable vector pHC11.展开更多
文摘[Objective] The research aimed to study the self-activation test of inverting T1083 substitution mutation BD fusion vector pGBKT7-TS into yeast,and discuss whether its expression product can be used as bait for further two-hybrid screening.[Method] T1083 substitution mutation BD fusion vector pGBKT7-TS was inverted into yeast to make the self-activation and protein expression toxic detection test.[Result] This expression product of the fragment was inactive and had no toxic to yeast cell.It could be used as bait for further two-hybrid screening.[Conclusion] The research laid the experimental foundation for further study of the effect of T1083 substitution mutation on the interaction of NtKrp and its target partner.
基金Supported by Natural Science Foundation of Beijing (6012017)Dr. Funding of Xinjiang Production and Construction Corps~~
文摘[ Objective ] The paper was to construct bait vector pGBKTT-Lescpth5, and detect its self-activated transcription activity and the toxicity on yeast cells. [ Method] Lescpth5 was amplified by PCR technique, and connected into bait vector pGBKTT. The recombined vector was transformed into yeast competent ceils AH109 and carried out self-activated detection and toxicity detection in the au.xotrephic medium. [ Result] Digestion and sequencing results showed that bait vector pGBKT7-Lescpth5 was suecessfully constructed with correct reading frame. Self-activated activity and toxicity detection results showed that bait vector had no self- activated activity on yeast strain AH109, which also had no toxicity on yeast. [Condusion] Bait vector successfully constructed could be used in yeast two-hybrid system, which laid the foundation for screening of cDNA library in the next step.
文摘Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 μm plasmid. The human interferon αA (IFN αA) gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium. Tbe data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN αA biological activity was 2.6×10^(10) units per liter, demonstrating that high-level expression and secretion of IFN αA were achieved in yeast by using the stable vector pHC11.
