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微生物源果胶酶在猪PK15细胞中异源表达及其酶学性质分析 被引量:5
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作者 莫健新 王豪强 +3 位作者 黄广燕 蔡更元 吴珍芳 张献伟 《遗传》 CAS CSCD 北大核心 2019年第8期736-745,共10页
果胶是植物细胞壁组分之一,是畜禽饲料中主要的抗营养因子,影响畜禽对日粮中能量和氮的利用效率。果胶酶在自然界中广泛存在于细菌、酵母和丝状真菌等微生物中,对解除果胶的抗营养作用、提高饲料利用率具有良好的效果。为了探索在猪细... 果胶是植物细胞壁组分之一,是畜禽饲料中主要的抗营养因子,影响畜禽对日粮中能量和氮的利用效率。果胶酶在自然界中广泛存在于细菌、酵母和丝状真菌等微生物中,对解除果胶的抗营养作用、提高饲料利用率具有良好的效果。为了探索在猪细胞中表达微生物源果胶酶基因的可行性,本研究通过脂质体转染法将微生物源果胶酶基因pg5a、pgI、pga3A和pgaA导入猪PK15细胞中进行异源表达,利用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid,DNS)法测定这4种基因编码果胶酶的酶活力。结果显示,4种果胶酶基因均能在猪PK15细胞中转录出mRNA,但只有pg5a和pgI适应猪细胞表达系统。其中,果胶酶PG5A的最高酶活为0.95 U/mL,最适作用pH为pH4.0,在pH4.6~6.0范围内维持46%以上的酶活;PGI在pH5.0处获得最高酶活,为0.30 U/mL,在pH4.0~6.0范围内维持35%以上的酶活。消化蛋白酶耐受实验结果显示,PG5A和PGI对胃蛋白酶和胰蛋白酶均有较强的耐受能力,用1 mg/mL的猪胃蛋白酶处理2 h后,PG5A和PGI的剩余酶活分别为76%和71%;用1 mg/mL的猪胰蛋白酶处理2 h后,PG5A和PGI的剩余酶活分别为44%和93%。综上所述,果胶酶基因pg5a和pgI能在猪细胞中正常表达,其编码的果胶酶对猪消化道pH条件和消化蛋白酶具有较强的耐受能力,可作为制备转果胶酶基因猪的候选基因。 展开更多
关键词 果胶酶 果胶 抗营养因子 转基因
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MEK抑制剂PD0325901显著提高猪胎儿成纤维细胞ssODN介导的HDR效率
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作者 欧浩 李国玲 +5 位作者 王豪强 黄广燕 蔡更元 李紫聪 吴珍芳 张献伟 《遗传》 CAS CSCD 北大核心 2019年第4期327-336,共10页
基因组DNA发生双链断裂(double strand break, DSB)后主要通过同源定向修复(homologous-directed repair, HDR)和非同源末端连接(nonhomologous end joining, NHEJ)两种途径进行修复,其中单链寡聚核苷酸(single-stranded oligodeoxyribo... 基因组DNA发生双链断裂(double strand break, DSB)后主要通过同源定向修复(homologous-directed repair, HDR)和非同源末端连接(nonhomologous end joining, NHEJ)两种途径进行修复,其中单链寡聚核苷酸(single-stranded oligodeoxyribonucleotide, ssODN)介导的同源定向修复是动物基因组常用的基因组定点修饰技术,具有较大的科研和应用价值。为提高猪基因组ssODN介导HDR效率,本研究以猪胎儿成纤维细胞(porcine fetal fibroblasts, PFFs)为研究对象,利用丝裂原活化的细胞外信号调节激酶(mitogen-activated extracellular signal-regulated kinase, MEK)抑制剂PD0325901培养细胞,研究其对HDR效率的影响及作用分子机理。结果显示,PD0325901能显著提高PFFs的G_2期和S期细胞群百分比,减少G_1期细胞群比率,促进HDR修复因子的表达。在最适浓度250 nmol/L时,PD0325901使ssODN介导的GFP报告载体的修复效率提高了58.8%;同时使PFFs基因组位点DMD和ROSA26定点修饰效率分别提高了48.16%和17.64%。本研究表明,MEK抑制剂PD0325901能显著提高猪基因组ssODN介导的同源定向修复效率,为高效制备定点基因修饰动物模型提供了新思路。 展开更多
关键词 MEK抑制剂 同源定向修复(HDR) PD0325901 基因编辑
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New measurement of the neutron-induced total cross-sections of ^(nat)Cr in a wide energy range on the Back-n at CSNS
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作者 Baoqian Li Min Xiao +17 位作者 Yonghao Chen Jieming Xue Xinxiang Li Han Yi Pinjing Cheng Rong Liu Yiwei Yang Zijie Han Dajun Zhao haoqiang wang Jirong Zhao Peng Luan Jing Liu Zijun Liu Chaomin Chen Wen Luo Bo Zheng Song Feng 《Chinese Physics C》 SCIE CAS CSCD 2024年第10期168-175,共8页
The neutron total cross-section of ^(nat)Cr plays a crucial role in new nuclear engineering design and fundamental science.A new measurement of the neutron-induced total cross-sections of ^(nat)Cr was performed using ... The neutron total cross-section of ^(nat)Cr plays a crucial role in new nuclear engineering design and fundamental science.A new measurement of the neutron-induced total cross-sections of ^(nat)Cr was performed using the transmission method on the back-streaming white neutron beamline(Back-n)at the China Spallation Neutron Source(CSNS).The neutron energy was determined using the time-of-flight technique.The neutron total cross-sections of ^(nat)Cr were obtained across a broad energy range(0.3 eV−20 MeV)in one experiment for the first time.The resulting effective total cross-sections were compared with the existing experimental data in different energy ranges,which revealed good agreement with the evaluated libraries.Theoretical calculation of the total cross-section in the energy range of 1.5 to 20 MeV was then conducted using TALYS-1.96 and compared with the present results.The measurement provides a high-quality total cross-section of ^(nat)Cr,including detailed uncertainty data across a wide energy range,offering a valuable reference for nuclear data re-evaluation and nuclear engineering design. 展开更多
关键词 neutron total cross-section natural chromium Back-n white neutron beamline NTOX
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SDT联用安罗替尼对非小细胞肺癌的协同杀伤作用
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作者 雷月 朱丽全 +2 位作者 刘鹏 王浩强 谢波 《中国肿瘤临床》 CAS 2024年第20期1027-1033,共7页
目的:探究声动力疗法(sonodynamic therapy,SDT)联合安罗替尼对非小细胞肺癌(non-small cell lung cancer,NSCLC)的协同杀伤效果及作用机制。方法:本研究选取A549和H1299肺癌细胞作为研究对象,设置空白对照组、单药安罗替尼组、单纯SDT... 目的:探究声动力疗法(sonodynamic therapy,SDT)联合安罗替尼对非小细胞肺癌(non-small cell lung cancer,NSCLC)的协同杀伤效果及作用机制。方法:本研究选取A549和H1299肺癌细胞作为研究对象,设置空白对照组、单药安罗替尼组、单纯SDT组及SDT联合安罗替尼治疗组。通过CCK-8和细胞划痕实验检测细胞活性与细胞迁移能力;流式细胞术检测活性氧(reactive oxygen species,ROS)水平、凋亡状态和细胞周期;蛋白质印迹法(Western blot)检测凋亡蛋白Caspase-3和周期蛋白Cyclin D1的表达;ROS清除实验等探讨SDT联合安罗替尼协同作用机制。结果:与单纯使用安罗替尼相比,SDT联合安罗替尼可显著抑制肺癌细胞的增殖[细胞存活率:A549:(49.96±4.82)%vs.(86.79±2.64)%,P<0.01;H1299:(31.91±4.87)%vs.(88.04±2.16)%,P<0.001]与迁移[愈合率:A549:(4.23±0.17)%vs.(14.28±0.05)%,P<0.05;H1299:(13.68±2.16)%vs.(42.81±8.11)%,P<0.001]。联合治疗组可明显诱导A549细胞凋亡[凋亡率:(12.58±0.815)%vs.(8.43±0.56)%,P<0.05]。机制研究发现,安罗替尼耐药与ROS水平相关,经活性氧清除剂N-乙酰半胱氨酸(NAC)作用后,细胞内ROS含量减少,安罗替尼IC50增大,敏感性下降。SDT联合安罗替尼后,肿瘤细胞内的ROS水平显著高于单药安罗替尼组(934.14±2.01 vs.166.75±1.45,P<0.001),并激活Caspase-3,下调Cyclin D1的表达。结论:SDT联合安罗替尼具有明显的协同抗肿瘤作用,其机制与ROS通路激活下游凋亡蛋白Caspase-3及下调Cyclin D1,抑制肺癌细胞增殖,诱导细胞凋亡相关。 展开更多
关键词 非小细胞肺癌 声动力疗法 安罗替尼 ROS
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Measurement and analysis of the neutron-induced total cross-sections of 209Bi from 0.3 eV to 20 MeV on the Back-n at CSNS 被引量:2
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作者 薛洁明 冯松 +15 位作者 陈永浩 易晗 李鑫祥 肖敏 程品晶 刘荣 羊奕伟 韩子杰 赵大俊 王豪强 李宝钱 赵继荣 刘梓隽 陈朝敏 罗文 郑波 《Chinese Physics C》 SCIE CAS CSCD 2023年第12期56-63,共8页
The neutron-induced total cross-section of ^(209)Bi is crucial for the physical design and safety assessment of lead-based fast reactors, and the quality of experimental data should be improved for evaluation and appl... The neutron-induced total cross-section of ^(209)Bi is crucial for the physical design and safety assessment of lead-based fast reactors, and the quality of experimental data should be improved for evaluation and application.A recent experiment was conducted on the back-streaming white neutron beamline(Back-n) at the China Spallation Neutron Source(CSNS) using the neutron total cross-section spectrometer(NTOX). The neutron energy was determined using a fast multi-cell fission chamber and the time-of-flight technique. Two high-purity bismuth samples,6 mm and 20 mm in thickness, were chosen for neutron transmission measurements and comparisons. The neutron total cross-sections of ^(209)Bi, ranging from 0.3 e V to 20 Me V, were derived considering neutron flight time determination, flight path calibration, and background subtraction. A comparison of the experimental results with the data in the ENDF/B-VⅢ.0 library showed fair agreement, and the point-wise cross-sections were found to be consistent with existing experimental data. Special attention was given to the determination of resonance parameters, which were analyzed using the R-matrix code SAMMY and Bayesian method in the 0.5 ke V to 20 ke V energy range. The extracted resonance parameters were compared to previously reported results and evaluated data. This study is recognized as the first one where the neutron total cross-section of bismuth across such a broad energy spectrum is measured in a single measurement or experiment, and it provides valuable data for the assessment of related reaction information for evaluated libraries and the advancement of lead-bismuth-based nuclear systems. 展开更多
关键词 Neutron-induced total cross-section BISMUTH Back-n white neutron beamline NTOX neutron resonance parameter
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An engineered xCas12i with high activity,high specificity,and broad PAM range 被引量:2
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作者 Hainan Zhang Xiangfeng Kong +20 位作者 Mingxing Xue Jing Hu Zikang wang Yinghui Wei haoqiang wang Jingxing Zhou Weihong Zhang Mengqiu Xu Xiaowen Shen Fengcai Yin Zhiyuan Ai Guangyan Huang Junhui Xia Xueqiong Song Hengbin Li Yuan Yuan Jinhui Li Na Zhong Meiling Zhang Yingsi Zhou Hui Yang 《Protein & Cell》 SCIE CSCD 2023年第7期540-545,共6页
Dear Editor,The clustered regularly interspaced short palindromic repeats-Cas(CRISPR-Cas)systems,including type II Cas9 and type V Cas12 systems,which serve in the adaptive immunity of prokaryotes against viruses,have... Dear Editor,The clustered regularly interspaced short palindromic repeats-Cas(CRISPR-Cas)systems,including type II Cas9 and type V Cas12 systems,which serve in the adaptive immunity of prokaryotes against viruses,have been developed into genome-editing tools(Anzalone et al.,2020;Doudna,2020).Compared with type II systems,the type V systems including V-A to V-K showed more functional diversity(Yan et al.,2019).Amongst them,Cas12i has a relatively smaller size(1,033-1,093 aa),compared to SpCas9 and Cas12a,and has a 5'-TTN protospacer adjacent motif(PAM)preference(Yan et al.,2019). 展开更多
关键词 HIGH SPECIFICITY
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CRISPR/Cas13系统敲减HEK293T细胞ku70和lig4基因表达 被引量:1
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作者 王豪强 李国玲 +7 位作者 黄广燕 李紫聪 郑恩琴 徐铮 杨化强 吴珍芳 张献伟 刘德武 《生物工程学报》 CAS CSCD 北大核心 2020年第7期1414-1421,共8页
成簇的规律间隔的短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白(CRISPR-associated proteins,Cas)系统是目前基因编辑、基因表达研究的热点,其中靶向RNA的CRISPR/Cas13系统的开发... 成簇的规律间隔的短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白(CRISPR-associated proteins,Cas)系统是目前基因编辑、基因表达研究的热点,其中靶向RNA的CRISPR/Cas13系统的开发为RNA的干扰和编辑提供了新的方向。文中针对HEK293T细胞非同源末端连接(Nonhomologousendjoining,NHEJ)通路修复关键因子Ku70和Lig4的编码序列,设计并合成CRISPR/Cas13a、b系统相应的gRNA,检测其对ku70和lig4基因表达的影响。结果显示,Cas13a对ku70和lig4的RNA敲减效果可以达到50%以上,Cas13b对ku70和lig4的RNA敲减效果分别达到92%和76%;同时Cas13a、b系统能将Ku70和Lig4蛋白水平分别下调至68%和53%。CRISPR/Cas13系统可有效敲减HEK293T细胞RNA和蛋白质表达,为基因功能和调控研究提供一种新的策略。 展开更多
关键词 CRISPR/Cas13 KU70 lig4 基因敲减
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PIK-75 promotes homology-directed DNA repair
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作者 Guoling Li Xianwei Zhang +4 位作者 Hao Ou haoqiang wang Dewu Liu Huaqiang Yang Zhenfang Wu 《Journal of Genetics and Genomics》 SCIE CAS CSCD 2019年第3期141-144,共4页
Homo!ogy-directed repair(HDR)is one of two major DNA repair pathways to mend the double-strand breaks(DSBs)formed in the genome(Liang et al.,1998;Pardo et al.,2009).Although less efficient compared with another DNA re... Homo!ogy-directed repair(HDR)is one of two major DNA repair pathways to mend the double-strand breaks(DSBs)formed in the genome(Liang et al.,1998;Pardo et al.,2009).Although less efficient compared with another DNA repair pathway,nonhomologous end joining(NHEJ),HDR is a type of precise repair to restore DNA damage and sustain genomic stability(Pardo et al.,2009;Ceccaldi et al.,2016).By contrast,NHEJ usually introduces mutations into the repaired site,thus probably harming the genomic integrity(Lieber et al.,2003).The error-free property enables HDR to be harnessed to correct a faulty mutation for therapeutic purpose in cells or in the body(Wu et al.,2013).In add让ion,HDR possesses great potential in the generation of genome-edited animals with precise genetic modifications,such as point mutation,DNA replacement,and DNA insertion in a specific genomic site(Wang et al.,2013).However,the low repair frequency mediated by HDR significantly limits让s application for efficient gene correction or establishment of various genetically modified animal models.Currently,multiple site-specific endonucleases have emerged as highly efficient tools to create targeted DSBs and markedly promote subsequent DNA repair either via HDR or NHEJ(Gaj et al.,2013).Nonetheless,the HDR-mediated modifications following the cleavage of engineering nucleases are still inefficient,usually with an efficiency less than 20%in cultured mammalian cells and embryos(Mali et al..2013;Wang et al.,2013;Yang et al.,2013). 展开更多
关键词 ogy-directed REPAIR MAJOR DNA REPAIR MAMMALIAN cells
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