基于显色体系和破壁条件的优化,构建了酶解破壁与显色反应相结合的氨基酸脱氢酶突变体高通量筛选方法。以颜色和吸光度表征内消旋-2,6-二氨基庚二酸脱氢酶(StDAPDH)酶活,以碘硝基四唑紫(INT)-吩嗪硫酸甲酯(PMS)为显色剂,通过其与StDAPD...基于显色体系和破壁条件的优化,构建了酶解破壁与显色反应相结合的氨基酸脱氢酶突变体高通量筛选方法。以颜色和吸光度表征内消旋-2,6-二氨基庚二酸脱氢酶(StDAPDH)酶活,以碘硝基四唑紫(INT)-吩嗪硫酸甲酯(PMS)为显色剂,通过其与StDAPDH的偶联反应优化了显色体系;以StDAPDH酶活为评价指标,在单因素实验的基础上,采用响应面法探究了菌体量、破壁时间、溶菌酶加量对菌体破壁效果的影响。确定最优显色体系为:INT浓度0.1 mg·mL^(-1)、PMS浓度0.5μg·mL^(-1)、检测波长510 nm;确定最优菌体破壁条件为:30 mL OD 600值为1.5的菌悬液,离心后用10 mL PBS缓冲液重悬菌体沉淀,加入0.2 mg·mL^(-1)的溶菌酶,在37℃下破壁50 min。该方法简单高效,为氨基酸脱氢酶突变体的高通量筛选提供了一定参考。展开更多
目的应用Ion Torrent半导体靶向测序技术检测1例枫糖尿症(MSUD)患儿的致病基因突变,明确其致病突变,并探讨该技术用于复杂单基因病检测的可行性。方法采集患儿外周血,提取基因组DNA,经多重PCR扩增富集目的基因片段,构建平均片段大小为30...目的应用Ion Torrent半导体靶向测序技术检测1例枫糖尿症(MSUD)患儿的致病基因突变,明确其致病突变,并探讨该技术用于复杂单基因病检测的可行性。方法采集患儿外周血,提取基因组DNA,经多重PCR扩增富集目的基因片段,构建平均片段大小为300bp左右的文库,经乳液PCR及磁珠颗粒富集,最后采用318半导体测序芯片进行高通量测序,应用Ion Torrent Suite v3.0软件进行数据提取、序列比对及SNPs和Indels提取,再用dbSNP 137数据库过滤得到SNPs和Indels,可疑突变经Sanger法测序验证。结果检出患儿1个新发错义点突变并通过Sanger测序验证,突变是BCKDHB基因第6外显子的NM183050:c.586C>T(p.His196Tyr)。结论 Ion Torrent半导体靶向测序技术可对复杂单基因遗传病进行快速、准确地基因诊断。展开更多
A full_length cDNA has been cloned encoding nicotinamide adenine dinucleotide phosphate_specific glutamate dehydrogenase (NADP_GDH) from Chlorella sorokiniana with the RT_PCR method. The complete nucleotide sequence o...A full_length cDNA has been cloned encoding nicotinamide adenine dinucleotide phosphate_specific glutamate dehydrogenase (NADP_GDH) from Chlorella sorokiniana with the RT_PCR method. The complete nucleotide sequence of NADP_GDH gene had 94% homology to the previously reported one . The NADP_GDH gene was constructed into a vector highly expressed in plants. The specific activity of NADP_GDH in transformants was detected, but not in the control plants. All transformed shoots on MS medium containing lower concentration of nitrogen and the transformed seedlings grown in lower concentration of nitrogen vermiculite had higher growth rate and more leaves than the control plants. Transformed leaf discs cultured on MS medium containing different nitrogen concentrations had more chlorophyll contents compared to the controls. These results suggested that exogenous NADP_GDH may enhance the absorption and utilization to ammonium in plants. The increased weight of transformed leaf discs cultured on medium supplemented with different concentrations of phosphinothricin (PPT) was more than that of control discs. 0.5 μg/mL PPT could be used as a selecting drug instead of kanamycin to develop the transformants. These results suggested that the NADP_GDH gene might be used as a new selecting gene in the future research of plant gene engineering.展开更多
Gastrointestinal stromal tumors(GISTs) are the most common mesenchymal tumors of the gastrointestinal tract and have gained considerable research and treatment interest,especially in the last two decades. GISTs are dr...Gastrointestinal stromal tumors(GISTs) are the most common mesenchymal tumors of the gastrointestinal tract and have gained considerable research and treatment interest,especially in the last two decades. GISTs are driven by mutations commonly found in the KIT gene and less commonly in the platelet-derived growth factor receptor alpha gene,BRAF gene and succinate dehydrogenase gene. GISTs behave in a spectrum of malignant potential,and both the tumor size and mitotic index are the most commonly used prognostic criteria. Whilst surgical resection can offer the best cure,targeted therapy in the form of tyrosine kinase inhibitors(TKIs) has revolutionized the management options. As the first-line TKI,imatinib offers treatment for advanced and metastatic GISTs,adjuvant therapy in high-risk GISTs and as a neoadjuvant agent to downsize large tumors prior to resection. The emergence of drug resistance has altered some treatment options,including prolonging the first-line TKI from 1 to 3 years,increasing the dose of TKI or switching to second-line TKI. Other newer TKIs,such as sunitinib and regorafenib,may offer some treatment options for imatinib-resistant GISTs. New molecular targeted therapies are being evaluated,such as inhibitors of BRAF,heat shock protein 90,glutamine and mitogenactivated protein kinase signaling,as well as inhibitors of apoptosis proteins antagonist and even immunotherapy. This editorial review summarizes the recent research trials and potential treatment targets that may influence our future patient-specific management of GISTs. The current guidelines in GIST management from Europe,North America and Asia are highlighted.展开更多
文摘基于显色体系和破壁条件的优化,构建了酶解破壁与显色反应相结合的氨基酸脱氢酶突变体高通量筛选方法。以颜色和吸光度表征内消旋-2,6-二氨基庚二酸脱氢酶(StDAPDH)酶活,以碘硝基四唑紫(INT)-吩嗪硫酸甲酯(PMS)为显色剂,通过其与StDAPDH的偶联反应优化了显色体系;以StDAPDH酶活为评价指标,在单因素实验的基础上,采用响应面法探究了菌体量、破壁时间、溶菌酶加量对菌体破壁效果的影响。确定最优显色体系为:INT浓度0.1 mg·mL^(-1)、PMS浓度0.5μg·mL^(-1)、检测波长510 nm;确定最优菌体破壁条件为:30 mL OD 600值为1.5的菌悬液,离心后用10 mL PBS缓冲液重悬菌体沉淀,加入0.2 mg·mL^(-1)的溶菌酶,在37℃下破壁50 min。该方法简单高效,为氨基酸脱氢酶突变体的高通量筛选提供了一定参考。
文摘目的应用Ion Torrent半导体靶向测序技术检测1例枫糖尿症(MSUD)患儿的致病基因突变,明确其致病突变,并探讨该技术用于复杂单基因病检测的可行性。方法采集患儿外周血,提取基因组DNA,经多重PCR扩增富集目的基因片段,构建平均片段大小为300bp左右的文库,经乳液PCR及磁珠颗粒富集,最后采用318半导体测序芯片进行高通量测序,应用Ion Torrent Suite v3.0软件进行数据提取、序列比对及SNPs和Indels提取,再用dbSNP 137数据库过滤得到SNPs和Indels,可疑突变经Sanger法测序验证。结果检出患儿1个新发错义点突变并通过Sanger测序验证,突变是BCKDHB基因第6外显子的NM183050:c.586C>T(p.His196Tyr)。结论 Ion Torrent半导体靶向测序技术可对复杂单基因遗传病进行快速、准确地基因诊断。
文摘A full_length cDNA has been cloned encoding nicotinamide adenine dinucleotide phosphate_specific glutamate dehydrogenase (NADP_GDH) from Chlorella sorokiniana with the RT_PCR method. The complete nucleotide sequence of NADP_GDH gene had 94% homology to the previously reported one . The NADP_GDH gene was constructed into a vector highly expressed in plants. The specific activity of NADP_GDH in transformants was detected, but not in the control plants. All transformed shoots on MS medium containing lower concentration of nitrogen and the transformed seedlings grown in lower concentration of nitrogen vermiculite had higher growth rate and more leaves than the control plants. Transformed leaf discs cultured on MS medium containing different nitrogen concentrations had more chlorophyll contents compared to the controls. These results suggested that exogenous NADP_GDH may enhance the absorption and utilization to ammonium in plants. The increased weight of transformed leaf discs cultured on medium supplemented with different concentrations of phosphinothricin (PPT) was more than that of control discs. 0.5 μg/mL PPT could be used as a selecting drug instead of kanamycin to develop the transformants. These results suggested that the NADP_GDH gene might be used as a new selecting gene in the future research of plant gene engineering.
文摘Gastrointestinal stromal tumors(GISTs) are the most common mesenchymal tumors of the gastrointestinal tract and have gained considerable research and treatment interest,especially in the last two decades. GISTs are driven by mutations commonly found in the KIT gene and less commonly in the platelet-derived growth factor receptor alpha gene,BRAF gene and succinate dehydrogenase gene. GISTs behave in a spectrum of malignant potential,and both the tumor size and mitotic index are the most commonly used prognostic criteria. Whilst surgical resection can offer the best cure,targeted therapy in the form of tyrosine kinase inhibitors(TKIs) has revolutionized the management options. As the first-line TKI,imatinib offers treatment for advanced and metastatic GISTs,adjuvant therapy in high-risk GISTs and as a neoadjuvant agent to downsize large tumors prior to resection. The emergence of drug resistance has altered some treatment options,including prolonging the first-line TKI from 1 to 3 years,increasing the dose of TKI or switching to second-line TKI. Other newer TKIs,such as sunitinib and regorafenib,may offer some treatment options for imatinib-resistant GISTs. New molecular targeted therapies are being evaluated,such as inhibitors of BRAF,heat shock protein 90,glutamine and mitogenactivated protein kinase signaling,as well as inhibitors of apoptosis proteins antagonist and even immunotherapy. This editorial review summarizes the recent research trials and potential treatment targets that may influence our future patient-specific management of GISTs. The current guidelines in GIST management from Europe,North America and Asia are highlighted.
