贻贝(Mytilus)中富含各种黏附蛋白,其中贻贝足丝蛋白5(Mytilus galloprovincialis foot protein type 5,Mgfp-5)是贻贝与外界基质进行黏附的主要蛋白分子之一。贻贝黏蛋白天然提取时产量低、易凝固且纯化困难,通过化学合成和基因工程等...贻贝(Mytilus)中富含各种黏附蛋白,其中贻贝足丝蛋白5(Mytilus galloprovincialis foot protein type 5,Mgfp-5)是贻贝与外界基质进行黏附的主要蛋白分子之一。贻贝黏蛋白天然提取时产量低、易凝固且纯化困难,通过化学合成和基因工程等方法重组贻贝黏蛋白解决该问题。本研究在E.coli BL21(DE3)中表达了贻贝蛋白Mgfp-5,亲和层析纯化到Mgfp-5蛋白的含量为12.25%,纯度为96.92%;酪氨酸酶修饰Mgfp-5后,其DOPA(多巴,3,4-二羟基苯丙氨酸)含量为9.60 pmol/g,是修饰前的9.32倍,生物黏性增强到1 116 n N,是Cell-Tak TM黏性的1.60倍;3.0μg/cm2的Mgfp-5蛋白的毒性级别为1级;Mgfp-5浓度≤20μg/m L时对红细胞无溶血现象,具有生物安全性。本研究可为重组Mgfp-5蛋白研究奠定基础,同时为来源于Mgfp-5黏蛋白的临床生物黏合剂的研制提供参考。展开更多
Here,a dopa decarboxylase(DDC)from Harmonia axyridis was heterogeneously expressed in Escherichia coli for the efficient biosynthesis of dopamine.For the production of recombinant DDC,the cultivation conditions includ...Here,a dopa decarboxylase(DDC)from Harmonia axyridis was heterogeneously expressed in Escherichia coli for the efficient biosynthesis of dopamine.For the production of recombinant DDC,the cultivation conditions including IPTG concentration,temperature and induction time were optimized and obtained an optimal specific enzyme activity of 51.72 U·mg^(-1) crude extracts.After the purification of DDC with a recovery yield of 68.79%,its activity was further characterized.The Vmax,Km,Kcat,and Kcat/Km of DDC for d ihyd roxy pheny la la nine(dopa)were 0.02 mmol·ml^(-1)·s^(-1),2.328 mmol·ml^(-1),10435.90 s^(-1) and4482.77 ml,mmol respectively.The highest DDC activity was observed at the condition of pH 7.5 and 45℃.With the purified DDC,the feasibility to produce dopamine from L-dopa was evaluated.The optimal yield was determined at the following bioconversion conditions:pH of 7,0,the reaction temperature of 40℃,0.4 mmol·L^(-1) of PLP and 4 g·L^(-1) of L-dopa,Subsequently,a fed-batch process for the production of dopamine was developed and the effect of oxygen was evaluated.The titer,yield and productivity of dopamine reached up to 21.99 g·L^(-1)80.88%and 14.66 g·L^(-1)·h^(-1) at 90 min under anaerobic condition.展开更多
Traditional Chinese medicine Buyi Ganshen therapy combined with dopa preparation in the therapy of Parkinson’s disease could enhance the clinical safety and efficacy compared with dopa preparation along.The exact mec...Traditional Chinese medicine Buyi Ganshen therapy combined with dopa preparation in the therapy of Parkinson’s disease could enhance the clinical safety and efficacy compared with dopa preparation along.The exact mechanism of how Buyi Ganshen works combined with dopa preparation still unknown and the enrolled articles quality was relatively inadequate,yet it could provide the reference in the clinical application and further studies.Background:To research the influence of“Buyi Ganshen”,a traditional Chinese medicine that commonly used in“Tonifying the Liver and the Kidney”,added dopa preparation for the therapy of Parkinson’s disease.Methods:This meta-analysis examined existing literature by searching 7 electronic databases(CNKI,WanFang,EMBASE,PubMed,Medline,the Cochrane Library,and the VIP database)from inception to December 2019.Randomized controlled trials evaluating the influence of“Buyi Ganshen”combined with dopa preparation for Parkinson’s disease were identified.The main results were clinical efficacy,UPDRS I(mental score),total unified Parkinson’s disease ratingscale score,UPDRS III(motor score),UPDRS II(activities of daily life),and UPDRS IV(complications of treatment).The subordinate results were the SPOCA-AUT scores,traditional Chinese medicine syndromes and other adverse reactions.The Review Manager 5.3 software was used to calculate the pooled estimate effect.The outcomes were showed as relative ratio with 95%confidence interval.The fixed or random model was elected based on the level of homogeneity among researches.The I2 was used to detected heterogeneity.The quality of the researches was also assessed.Results:The meta-analysis include 2,241 individuals from a total of 30 studies.These data suggested that the therapy of Parkinson’s disease with the combination of“Buyi Ganshen”and dopa preparation were more effective compared to dopa preparation alone.Conclusion:“Buyi Ganshen”combined with dopa preparation exists,in some degree,to enhance clinical safety and effectiveness in the cure of Parkinson’s disease,compared with dopa preparation along.As most of the studies included were with low quality,this conclusion must be considered with caution.Thus,more multi-center,high-quality,prospective randomized controlled trials with large enough sample sizes are required to further illuminate the influence of“Buyi Ganshen”combined with dopa preparation for Parkinson’s disease.展开更多
基金Supported by Research and Agricultural Project of Science and Technology Department of Zhejiang Province(No.2008C22026)Key Project of Education Department of Zhejiang Province(No.20070430)~~
基金Research and Agricultural Project of Zhejiang Technology Department(2008C22026,2009C32016)Key Project of Zhejiang Education Department(20070430)Xin-Miao Project of Zhejiang Technology Department(2008R40G2110002)
文摘贻贝(Mytilus)中富含各种黏附蛋白,其中贻贝足丝蛋白5(Mytilus galloprovincialis foot protein type 5,Mgfp-5)是贻贝与外界基质进行黏附的主要蛋白分子之一。贻贝黏蛋白天然提取时产量低、易凝固且纯化困难,通过化学合成和基因工程等方法重组贻贝黏蛋白解决该问题。本研究在E.coli BL21(DE3)中表达了贻贝蛋白Mgfp-5,亲和层析纯化到Mgfp-5蛋白的含量为12.25%,纯度为96.92%;酪氨酸酶修饰Mgfp-5后,其DOPA(多巴,3,4-二羟基苯丙氨酸)含量为9.60 pmol/g,是修饰前的9.32倍,生物黏性增强到1 116 n N,是Cell-Tak TM黏性的1.60倍;3.0μg/cm2的Mgfp-5蛋白的毒性级别为1级;Mgfp-5浓度≤20μg/m L时对红细胞无溶血现象,具有生物安全性。本研究可为重组Mgfp-5蛋白研究奠定基础,同时为来源于Mgfp-5黏蛋白的临床生物黏合剂的研制提供参考。
基金funded by the National Natural Science Foundation of China(21576134 and 21706126)the National Key Research and Development Program(2016YFA0204300)。
文摘Here,a dopa decarboxylase(DDC)from Harmonia axyridis was heterogeneously expressed in Escherichia coli for the efficient biosynthesis of dopamine.For the production of recombinant DDC,the cultivation conditions including IPTG concentration,temperature and induction time were optimized and obtained an optimal specific enzyme activity of 51.72 U·mg^(-1) crude extracts.After the purification of DDC with a recovery yield of 68.79%,its activity was further characterized.The Vmax,Km,Kcat,and Kcat/Km of DDC for d ihyd roxy pheny la la nine(dopa)were 0.02 mmol·ml^(-1)·s^(-1),2.328 mmol·ml^(-1),10435.90 s^(-1) and4482.77 ml,mmol respectively.The highest DDC activity was observed at the condition of pH 7.5 and 45℃.With the purified DDC,the feasibility to produce dopamine from L-dopa was evaluated.The optimal yield was determined at the following bioconversion conditions:pH of 7,0,the reaction temperature of 40℃,0.4 mmol·L^(-1) of PLP and 4 g·L^(-1) of L-dopa,Subsequently,a fed-batch process for the production of dopamine was developed and the effect of oxygen was evaluated.The titer,yield and productivity of dopamine reached up to 21.99 g·L^(-1)80.88%and 14.66 g·L^(-1)·h^(-1) at 90 min under anaerobic condition.
基金This study was financially supported by TCM Administration Project of Guangdong Province,China(No.20203010)Science and Technology Program of Guangdong,China(No.2020A151501325).
文摘Traditional Chinese medicine Buyi Ganshen therapy combined with dopa preparation in the therapy of Parkinson’s disease could enhance the clinical safety and efficacy compared with dopa preparation along.The exact mechanism of how Buyi Ganshen works combined with dopa preparation still unknown and the enrolled articles quality was relatively inadequate,yet it could provide the reference in the clinical application and further studies.Background:To research the influence of“Buyi Ganshen”,a traditional Chinese medicine that commonly used in“Tonifying the Liver and the Kidney”,added dopa preparation for the therapy of Parkinson’s disease.Methods:This meta-analysis examined existing literature by searching 7 electronic databases(CNKI,WanFang,EMBASE,PubMed,Medline,the Cochrane Library,and the VIP database)from inception to December 2019.Randomized controlled trials evaluating the influence of“Buyi Ganshen”combined with dopa preparation for Parkinson’s disease were identified.The main results were clinical efficacy,UPDRS I(mental score),total unified Parkinson’s disease ratingscale score,UPDRS III(motor score),UPDRS II(activities of daily life),and UPDRS IV(complications of treatment).The subordinate results were the SPOCA-AUT scores,traditional Chinese medicine syndromes and other adverse reactions.The Review Manager 5.3 software was used to calculate the pooled estimate effect.The outcomes were showed as relative ratio with 95%confidence interval.The fixed or random model was elected based on the level of homogeneity among researches.The I2 was used to detected heterogeneity.The quality of the researches was also assessed.Results:The meta-analysis include 2,241 individuals from a total of 30 studies.These data suggested that the therapy of Parkinson’s disease with the combination of“Buyi Ganshen”and dopa preparation were more effective compared to dopa preparation alone.Conclusion:“Buyi Ganshen”combined with dopa preparation exists,in some degree,to enhance clinical safety and effectiveness in the cure of Parkinson’s disease,compared with dopa preparation along.As most of the studies included were with low quality,this conclusion must be considered with caution.Thus,more multi-center,high-quality,prospective randomized controlled trials with large enough sample sizes are required to further illuminate the influence of“Buyi Ganshen”combined with dopa preparation for Parkinson’s disease.
