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Enhanced plasma factor Ⅷ activity in mice via cysteine mutation using dual vectors

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Science China(Life Sciences) 2012年第6期55卷 521-526页

作者： zhu fuxiang LIU ZeLong MIAO Jing QU HuiGe CHI XiaoYan Life Science College of Ludong University Yantai 264025China

Hemophilia A is caused by a genetic mutation in coagulation factor VIII （FVIII） gene and gene therapy is considered to be a promising strategy for its treatment. We recently demonstrated that co-delivery of two vectors expressing M662C mutated heavy and D1828C mutated light chain genes of B-domain-deleted coagulation factor VIII （BDD-FVIII） leads to inter-chain disulfide cross-linking and improved heavy chain secretion in vitro. In this study, co-injection of both M662C and D1828C mutated BDD-FVIII gene expression vectors into mice resulted in increased heavy chain secretion and coagulation activity in plasma in vivo. Approximately （239＋_56） ng mL-1 above endogenous levels of transgenic FVIII heavy chain was found in mouse plasma using a chain-specific ELISA. For FVIII coagulation activity, approximately （1.09＋_0.25） IU mL-1 above en- dogenous levels were detected in co-injected transgenic mouse plasma using a chromogenic assay. These data demonstrate that inter-chain disulfide bonds likely increase heavy chain secretion and coagulation activity in the plasma of transgenic mice with an improved efficacy of a dual-vector delivery of BDD-FVIII gene. These findings support our ongoing efforts to develop a gene therapy for hemophilia A treatment using dual-AAV vectors.

关键词： coagulation factor VIII dual-vector gene delivery inter-chain disulfide bonding heavy chain secretion coagulation ac-tivity

The effect of a secretion-enhanced heavy chain on improving intein-based dual-vector co-delivery of a full-length factor Ⅷ gene

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Chinese Science Bulletin 2011年第2期56卷 158-163页

作者： zhu fuxiang YANG ShuDe LIU ZeLong MIAO Jing QU HuiGe CHI XiaoYan College of Life Sciences Ludong University Yantai 264025 China

Treatment of hemophilia A by gene therapy is adversely affected by inefficient FVIII secretion and the large FVIII gene,which is difficult to package in the promising adeno-associated virus (AAV) *** secretion of FVIII is caused mainly by inefficient secretion of its heavy ***,we have employed a protein splicing-based dual-vector to co-transfer a B-domain-deleted FVIII (BDD-FVIII) gene,suggesting that the light chain,covalently ligated to a co-expressed heavy chain can improve the secretion of spliced ***,its level of secretion was affected by inefficient secretion the heavy ***,we studied the effect of a mutant heavy chain with L303E/F309S substitutions,which enhance FVIII secretion on the heavy chain itself and spliced FVIII when using a protein splicing-based split-delivery of a full-length FVIII *** vectors expressing Ssp DnaB intein-fused mutant heavy and light chains were transiently co-transfected into cultured COS-7 cells.A spliced FVIII protein was seen in co-transfected cells by Western blot *** heavy chain was secreted by cells transfected with the mutant heavy chain gene alone at (39±11) ng/mL and this secretion increased to (123±13) ng/mL when cells were co-transfected with the light chain gene,which was greater than the secretion of wild-type heavy *** amount of spliced FVIII in the culture supernatant of co-transfected cells was (86±14) ng/mL,with an activity of (0.61±0.08) IU/mL,which was greater than that of wild-type FVIII co-transfected *** FVIII and bioactivity were also detected in the combined culture supernatant of cells individually transfected with mutant heavy and light chain gene at a higher level than that of combined wild-type heavy and light chain *** suggested that the heavy chain with improved secretion markedly increased the efficacy of protein splicing-based split delivery of the full-length FVIII gene using a *** result

关键词：分泌作用基因传递因子和内含肽轻链载体基础蛋白质剪接

Protein trans-splicing based dual-vector delivery of the coagulation factor Ⅷ gene

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Science China(Life Sciences) 2010年第6期53卷 683-689页

作者： zhu fuxiang,LIU ZeLong,CHI XiaoYan & QU HuiGe Life Science College of Ludong University,Yantai 264025,China Life Science College of Ludong University Yantai China

A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eukaryotic expression vectors,expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII),was *** transient co-transfection of the two vectors into 293 and COS-7 cells,the culture supernatants contained (137±23) and (109±22) ng mL–1 spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL–1 for 293 and COS-7 cells,*** spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain *** spliced BDD-FVIII protein bands from cell lysates were visualized by Western *** data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.

关键词： intein protein trans-splicing coagulation factor VIII dual-vector gene delivery

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors

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Science China(Life Sciences) 2013年第4期56卷 262-267页

作者： zhu fuxiang LIU ZeLong WANG XiaoLei MIAO Jing QU HuiGe CHI XiaoYan College of Life Science Ludong University Department of Hematology Yuhuangding Hospital

Protein trans-splicing based dual-vector factor VIII （FVIII） gene delivery is adversely affected by less efficiency of protein splicing. We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins, protein splicing elements, thereby facilitating protein trans-splicing. Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing, increasing the levels of spliced FVIII protein. In this study, C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver. Forty-eight hours post-injection, plasma was collected and analyzed for FVIII antigen concentration and coagulation activity. These mice showed increased circulating FVIII heavy chain polypeptide （442± 151 ng mL i vs. 305±103 ng mL-1） and coagulation activi- ty （1.46±0.37 IU mL i vs. 0.85±0.23 IU mL-1） compared with control mice co-administered dual vectors expressing the heavy and light chains of wild-type FVIII. Moreover, coagulation activity was similar to that of mice receiving a single vector ex- pressing FVIII （1.79_＋0.59 IU mL-l）. These findings indicate that improving protein trans-splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer.

关键词： coagulation factor VIII protein trans-splicing dual-vector gene delivery plasma coagulation activity

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Anti-metastatic Effect of Repeated Cyclic-GRGDSPA Peptide Produced by Intein Protein Trans-splicing on Murine B16 Melanoma Cells

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中国肺癌杂志 2009年第6期12卷 665-667页

作者： fuxiang zhu Zelong LIU Xiaoyan CHI Xianqing LIU Department of Biochemistry and Molecular Biology Life Science College Ludong University Yantai 26402.5 China Department of Biochemistry and Molecular Biology Dalhousie University Halifax B3H 4H7 Canada

Background and objective Metastasis is one of the most important causes of mortality in tumor. The pathological process of metastasis includes several sequential steps as

关键词：肺癌治疗临床药物

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面内端部激励下斜拉索振动方程推导与求解

地震工程与工程振动 2023年第3期43卷 150-160页

作者：朱付祥易江广州大学土木工程学院广东广州510006

为精确描述面内端部激励下拉索非线性振动机制,构建抽象端部激励下的拉索力学描述模型,分别推导了拉索拟静态振动和模态振动控制方程,基于有限差分法给出了拉索模态振动的数值求解方法。由于垂度效应的影响,在轴向、竖向以及轴向和竖向... 详细信息

为精确描述面内端部激励下拉索非线性振动机制,构建抽象端部激励下的拉索力学描述模型,分别推导了拉索拟静态振动和模态振动控制方程,基于有限差分法给出了拉索模态振动的数值求解方法。由于垂度效应的影响,在轴向、竖向以及轴向和竖向共同激励下拉索的拟静态索力与端部位移采用一个非线性方程予以表征。拉索拟静态振动可视作外部激励施加在拉索上,引起拉索的非线性模态振动,通过理论推导分别求得模态振动的控制方程和变形协调方程。在模态振动过程中,拉索的动力特性(如刚度和周期)不断变化,索力和拉索变形相互耦合,使得其求解非常复杂。采用有限差分法进行数值求解,并基于有限元方法验证了数值求解方法的准确性。重点讨论了拉索模态振动共振机制,结果表明,在轴向、竖向以及轴向和竖向激励下,拉索模态振动存在多个共振区域,包括小周期、0.5 T 1、T 1和2T 1等(T 1为成桥状态下拉索基本振动周期);在共振区域内,拉索振动索力幅度较大,模态振动对总索力的贡献系数较高,超过0.4,在实际工程中不容忽视;拉索阻尼对模态振动的影响较大,增加拉索阻尼比将改变拉索模态振动的幅频特征,模态振动索力及其贡献系数均有所降低,但降低程度受激励幅度的影响较大。

关键词：桥梁工程参数振动振动方程斜拉索面内端部激励共振周期

Understanding and therapeutically modulating the stability of regulatory T cells to treat inflammatory diseases

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Science Foundation in China 2015年第1期23卷 78-88页

作者： LI YangYang LI Dan WANG AiTing zhu fuxiang Tsun Andy LI Bin Key Laboratory of Molecular Virology and Immunology Unit of Molecular ImmunologyInstitut Pasteur of ShanghaiShanghai Institutes for Biological SciencesChinese Academy of Sciences

Forkhead box P3(FOXP3)^+regulatory T(Treg)cells are crucial for the homeostasis of the immune system,and harbor suppressive capacities of which are maintained by or inherited with distinct epigenetic *** functional features of Treg cells are facilitated by multiple T cell differentiation-related transcription factors that therefore endow them with adaption capability to dynamic *** studies also recalled the importance of immune cell metabolism,which is intimately linked with Treg cell differentiation,proliferation and *** the molecular mechanism underlying the functional stability of Treg cells could provide therapeutic opportunities to treat major human inflammatory diseases including infection,autoimmunity,allergy and cancer.

关键词： Inflamation RegulatoryTcell Epigenetics Proteomics Cellmetabolism doi:10.16262/j.cnki.1005—0841.2015.01.002

Control of Foxp3+ Regulatory T Cell Lineage Stability by Usp21

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Control of Foxp3+ Regulatory T Cell Lineage Stability by Usp...

尿激酶氨基末端基因抗乳腺癌细胞转移的实验研究

第十届全国免疫学学术大会

作者： Yangyang Li Yue Lv Shuaiwei Wang fuxiang zhu Bin Li Institut Pasteur of Shanghai CAS

Foxp3+Regulatory T(Treg)cells harbor immune suppressive capacities and are crucial for the maintenance of immune homeostasis and control of dominant immune *** cells are considered to be a heterogenic cell lineage,where compromised Foxp3 expression results in generation of ex Treg cells and acquisition of alternative effector or hybrid *** we report that E3 deubiquitinase Usp21 prevents the loss of Foxp3 protein and ex Treg ***

关键词： ex Treg cells Usp21 Foxp3

来源： cnki会议评论

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cnki会议

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中华肿瘤杂志 2001年第2期23卷 115-117页

作者：朱甫祥邢桂春贺福初军事医学科学院放射医学研究所北京100850

目的　探讨尿激酶氨基末端 (ATF)基因转移对肿瘤转移的抑制作用。方法　构建重组ATF基因真核表达载体pcDNA3 ATF ,用脂质体Lipofectin介导 ,将其导入其证明尿激酶 (uPA)和尿激酶受体 (uPAR)均有表达的人乳腺癌细胞系MCF 7。用Westernblo... 详细信息

目的　探讨尿激酶氨基末端 (ATF)基因转移对肿瘤转移的抑制作用。方法　构建重组ATF基因真核表达载体pcDNA3 ATF ,用脂质体Lipofectin介导 ,将其导入其证明尿激酶 (uPA)和尿激酶受体 (uPAR)均有表达的人乳腺癌细胞系MCF 7。用Westernblot检测转染细胞ATF基因的表达 ;体外观察并比较了野生型和转基因MCF 7细胞侵袭人工基底膜能力 ;裸鼠皮下接种癌细胞 ,复制自发性肿瘤转移模型 ,观察成瘤性和转移性。结果　uPA/uPAR在MCF 7中有较高水平的表达 ;转ATF基因后 ,可检测到ATF的明显表达 ,体外侵袭穿透人工基底膜的能力明显降低 ,体内原位成瘤性不受影响 ,但自主性肺转移能力有一定程度的下降。结论　ATF基因转移后 ,ATF的表达可能竞争性抑制内源性uPA与uPAR的结合 ,在一定程度上抑制癌细胞的侵袭和转移。

关键词：乳腺肿瘤肿瘤转移尿激酶基因转移乳腺癌基因治疗