胰岛素原(Proinsulin,Pins)是胰岛素的合成前体。在大肠杆菌表达系统中,其一般以包涵体的形式存在,需要经过变性复性等后续加工过程才能得到有活性的胰岛素。而无细胞蛋白合成体系(Cell-free protein synthesis,CFPS)作为一种新型体外...胰岛素原(Proinsulin,Pins)是胰岛素的合成前体。在大肠杆菌表达系统中,其一般以包涵体的形式存在,需要经过变性复性等后续加工过程才能得到有活性的胰岛素。而无细胞蛋白合成体系(Cell-free protein synthesis,CFPS)作为一种新型体外蛋白合成手段,突破了细胞的生理限制,已成功应用于多种重组蛋白药物的生产。为了探索胰岛素合成的新方法以满足其在新型给药途径研发中的需求,本研究运用CFPS体系进行胰岛素原的可溶性表达。通过将胰岛素原与荧光蛋白进行融合来增加其可溶性,成功在CFPS体系中表达了胰岛素原融合蛋白。最后使用Western blotting对融合红色荧光蛋白的胰岛素原(Pins-mCherry)进行鉴定,利用酶标仪对融合绿色荧光蛋白的胰岛素原(Pins-eGFP)在上清中的表达进行定量分析,结果表明Pins-eGFP部分可溶,其表达量为(12.28±3.45)μg/m L。本研究首次实现了融合胰岛素原在CFPS系统中的可溶性表达,其融合荧光蛋白的策略显著提升了胰岛素原的可溶性,该结果为探究胰岛素合成新方法及开发基于CFPS系统的新型胰岛素给药途径奠定了基础。展开更多
[Objective]The aim was to study the sensitive response characteristics of the main agronomic traits of somaclonal and the variation of sensitive response of the lines of somaclonal to sowing date. [Method]The RITSD ch...[Objective]The aim was to study the sensitive response characteristics of the main agronomic traits of somaclonal and the variation of sensitive response of the lines of somaclonal to sowing date. [Method]The RITSD changing of agronomic traits was studied by using 24 rice somaclonal and its donor parent under two sowing dates. [Result]The average values of RITSD were higher （over one） for the traits of pant height （PH）,density of spikelets （DS）,filled spikelets per panicle （FSP）,spikelets per panicle （SP） and grain weight per plant （GWP）,but lower for days of sowing to heading （DSH）,effective panicles （EP）,panicle length （PL）,seed setting rate （SS） and 1 000-grain weight （1 000-GW）. These showed that PH,DS,FSP,SP and GWP were easily affected by sowing date and tended to higher with the later sowing; the RITSD of somaclonal changed with the agronomic traits. The frequency of RITSD＇ variation of DSH and PH was higher than that of PL. The RITSD＇ value of EP,PL,DS,FSP,SP and SS became higher,and higher or lower for DSH and GWP in different somaclonal; compared with the donor,20 of 24 was significantly different,and the frequency of RITSD variation was over 83. 3%,however,RITSD of the most lines （75%） changed only for 1-2 characters; cluster analysis showed that the RITSD of agronomic traits belonged to different combinations of somaclonal in rice. [Conclusion]the study had provided a theoretical basis for the screening and using of the somaclonal.展开更多
作为一种快速高效的体外蛋白合成手段,无细胞蛋白表达体系(Cell-free Protein Synthesis,CFPS)一直以来就被广泛应用于基础生物学领域的研究。与传统的基于细胞的体内表达体系相比,CFPS突破了细胞的生理限制,其可调控性强、对毒性蛋白...作为一种快速高效的体外蛋白合成手段,无细胞蛋白表达体系(Cell-free Protein Synthesis,CFPS)一直以来就被广泛应用于基础生物学领域的研究。与传统的基于细胞的体内表达体系相比,CFPS突破了细胞的生理限制,其可调控性强、对毒性蛋白的耐受力高,使得许多很难在体内合成的复杂蛋白在体外顺利表达。近年来随着研究人员不断对CFPS进行优化,通过简化制备工艺、开发价格低廉的能量再生系统、稳定底物供应、促进蛋白正确折叠等方式,成功研发出生产效率高、成本低廉、反应体积大的表达体系。凭借其高通量和大规模的蛋白表达优势,CFPS为解决生物制药领域中面临的难题提供了新的解决思路,并成功地应用于高通量药物筛选、大规模生产重组蛋白药物、个体化定制肿瘤疫苗等领域,显示出其在生物制药领域的重要应用潜力。展开更多
文摘胰岛素原(Proinsulin,Pins)是胰岛素的合成前体。在大肠杆菌表达系统中,其一般以包涵体的形式存在,需要经过变性复性等后续加工过程才能得到有活性的胰岛素。而无细胞蛋白合成体系(Cell-free protein synthesis,CFPS)作为一种新型体外蛋白合成手段,突破了细胞的生理限制,已成功应用于多种重组蛋白药物的生产。为了探索胰岛素合成的新方法以满足其在新型给药途径研发中的需求,本研究运用CFPS体系进行胰岛素原的可溶性表达。通过将胰岛素原与荧光蛋白进行融合来增加其可溶性,成功在CFPS体系中表达了胰岛素原融合蛋白。最后使用Western blotting对融合红色荧光蛋白的胰岛素原(Pins-mCherry)进行鉴定,利用酶标仪对融合绿色荧光蛋白的胰岛素原(Pins-eGFP)在上清中的表达进行定量分析,结果表明Pins-eGFP部分可溶,其表达量为(12.28±3.45)μg/m L。本研究首次实现了融合胰岛素原在CFPS系统中的可溶性表达,其融合荧光蛋白的策略显著提升了胰岛素原的可溶性,该结果为探究胰岛素合成新方法及开发基于CFPS系统的新型胰岛素给药途径奠定了基础。
基金Supported by Chongqing Major Research Project "Innovation in Hybrid Rice Breeding and New Materials" ( CSTC, 2007, AA1019 )Sichuan Province Breeding Research Project (06yzgg-01)~~
文摘[Objective]The aim was to study the sensitive response characteristics of the main agronomic traits of somaclonal and the variation of sensitive response of the lines of somaclonal to sowing date. [Method]The RITSD changing of agronomic traits was studied by using 24 rice somaclonal and its donor parent under two sowing dates. [Result]The average values of RITSD were higher （over one） for the traits of pant height （PH）,density of spikelets （DS）,filled spikelets per panicle （FSP）,spikelets per panicle （SP） and grain weight per plant （GWP）,but lower for days of sowing to heading （DSH）,effective panicles （EP）,panicle length （PL）,seed setting rate （SS） and 1 000-grain weight （1 000-GW）. These showed that PH,DS,FSP,SP and GWP were easily affected by sowing date and tended to higher with the later sowing; the RITSD of somaclonal changed with the agronomic traits. The frequency of RITSD＇ variation of DSH and PH was higher than that of PL. The RITSD＇ value of EP,PL,DS,FSP,SP and SS became higher,and higher or lower for DSH and GWP in different somaclonal; compared with the donor,20 of 24 was significantly different,and the frequency of RITSD variation was over 83. 3%,however,RITSD of the most lines （75%） changed only for 1-2 characters; cluster analysis showed that the RITSD of agronomic traits belonged to different combinations of somaclonal in rice. [Conclusion]the study had provided a theoretical basis for the screening and using of the somaclonal.
文摘作为一种快速高效的体外蛋白合成手段,无细胞蛋白表达体系(Cell-free Protein Synthesis,CFPS)一直以来就被广泛应用于基础生物学领域的研究。与传统的基于细胞的体内表达体系相比,CFPS突破了细胞的生理限制,其可调控性强、对毒性蛋白的耐受力高,使得许多很难在体内合成的复杂蛋白在体外顺利表达。近年来随着研究人员不断对CFPS进行优化,通过简化制备工艺、开发价格低廉的能量再生系统、稳定底物供应、促进蛋白正确折叠等方式,成功研发出生产效率高、成本低廉、反应体积大的表达体系。凭借其高通量和大规模的蛋白表达优势,CFPS为解决生物制药领域中面临的难题提供了新的解决思路,并成功地应用于高通量药物筛选、大规模生产重组蛋白药物、个体化定制肿瘤疫苗等领域,显示出其在生物制药领域的重要应用潜力。
