大鼠ZFP580基因的克隆与原核表达
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摘要
目的:克隆和分析ZFP580基因cDNA开读框架全编码区(编码172个氨基酸)、N端编码区(编码1-88位氨基酸)、C端C2H2型锌指主构域编码区(编码89-172位氨基酸)。构建ZFP580基因原核表达载体并鉴定,诱导表达GST-ZFP580融合蛋白。
方法:1从正常大鼠小脑组织中提取总RNA。
2根据GenBank公布的大鼠ZFP580基因cDNA开读框架序列设计引物,用RT-PCR的方法扩增ZFP580基因cDNA开读框架以及ZFP580基因的N端编码区和C端编码区。
3用T-A克隆方法将目的基因克隆至pMD18-T-Easy载体中,挑取琼脂板上的白色菌落,接种于5ml含有Amp的LB液体培养基中,37℃摇菌培养过夜。用碱裂解法小剂量制备重组质粒。采用BglII和XhoI双酶切法、PCR法及测序法鉴定重组质粒。构建成功的阳性重组质粒记作pMD18-T-ZFP580。同法成功克隆ZFP580基因N端编码区和C端编码区,阳性重组质粒记作pMD18-T-N、pMD18-T-C。
4用BglII和XhoI对克隆载体pMD18-T-ZFP580和原核表达载体pET-42a(+)分别进行双酶切,琼脂糖凝胶电泳酶切产物,纯化回收带有粘端的ZFP580基因和pET-42a(+)载体。将ZFP580基因和pET-42a(+)表达载体用T4DNA连接酶连接过夜。连接产物转化DH5α感受态细胞,加LB液体培养基,37℃培养60min。将菌液均匀涂布于含Kan的LB琼脂平板上,于37℃培养箱中培养12-16h。挑取琼脂板上的单菌落,接种于5ml含有Kan的LB液体培养基中,37℃摇菌培养过夜。用碱裂解法小剂量制备质粒进行鉴定。阳性重组质粒记作pET-42a-ZFP580。同法成功构建ZFP580基因的C端编码区,阳性重组质粒记作pET-42a-C。阳性重组质粒用双酶切的方法鉴定并双向测序,确认有无读码框错误。
5阳性重组载体pET-42a-ZFP580转化E.coli BL21(DE3),IPTG诱导表达,优化表达条件,确定最佳表达参数,SDS-PAGE分析鉴定GST-ZFP580蛋白的表达。
6生物信息学分析ZFP580基因结构和功能。
结果:1甲醛变性琼脂糖凝胶电泳呈现清晰的28S和18S两条带,并且28S宽度以及亮度是18S的2倍,5S量较少。证实所提取总RNA完整性高。测OD260/OD280比值1.9左右,证明总RNA纯度高,无蛋白质和酶等抑制物残留。
2 RT-PCR扩增后,在519bp附近出现了一条特异性条带,此DNA条带与大鼠ZFP580基因cDNA开读框架全编码区的大小相符合。测序证明已获得ZFP580基因开读框架,其序列与GenBank中预测序列完全一致。同法获得264bp和252bp大鼠ZFP580基因的N端编码区和C端编码区。序列比对结果证实与GenBank中预测的一致。
3 T-A克隆构建获得的阳性克隆载体pMD18-T-N、pMD18-T-C、pMD18-T-ZFP580,用Bgl II和Xho I双酶切和PCR法鉴定。结果表明,上述三个克隆载体已成功构建。
4原核表达载体pET-42a-ZFP580、pET-42a-C经鉴定证实构建成功。测序证实pET-42a-ZFP580载体无读码框的错误,转化入表达菌株E.coli BL21(DE3), IPTG诱导,SDS-PAGE鉴定分析GST-ZFP580融合蛋白表达情况。
5小剂量多次进行蛋白表达实验,未能诱导表达目的蛋白。深入探讨了未能表达目的蛋白的原因。
6大鼠ZFP580基因cDNA全长1100bp,完整开读框架为519bp,编码一个含有172个氨基酸的蛋白质,所编码的蛋白与锌指蛋白相似有2个功能结构域:N端的富含脯氨酸结构域和C端的3个C2H2型锌指结构。
结论:1大鼠ZFP580基因cDNA开读框架全编码区、大鼠ZFP580基因的N端编码区和C端编码区与GenBank中预测序列完全一致。
2成功构建克隆载体pMD18-T-N、pMD18-T-C、pMD18-T- ZFP580和原核表达载体pET-42a-ZFP580、pET-42a-C。
3原核表达载体pET-42a-ZFP580读码框正确无误,IPTG诱导表达,经SDS-PAGE分析表达情况。小剂量多次进行蛋白表达实验,未能诱导表达目的蛋白。
4详细分析大鼠ZFP580基因结构和功能,大鼠ZFP580基因开读框架编码一个含有172个氨基酸的蛋白质,所编码的蛋白与锌指蛋白相似有2个功能结构域。
Objective: To clone the full-length open reading frame cDNA sequence of Zinc Finger Protein (ZFP580, encoding 172 amino acids) gene from rat cerebellum. To clone and analyze the cDNA sequence of amino terminal encoding region (encoding 1-88 amino acids) and carboxyl terminal encoding region (encoding 89-172 amino acids) of ZFP580 gene. To construct prokaryotic fusion expression vectors and induces the expression of fusion protein GST-ZFP580.
Methods: 1 Total RNA was extracted from rat cerebellum with Trizol solution.
2 The specific primers were designed according to the cDNA encoding sequence of ZFP580 full-length open reading frame published by GenBank. The cDNA encoding sequences of ZFP580 full-length open reading frame, amino terminal encoding region and carboxyl terminal encoding region were obtained by RT-PCR. Sequence analysis of these fragments and its deduced amino acids were accomplished online at the National Center for Biotechnology Information servers and edited using the Primer5,Clustalx and DNAMAN programs.
3 These cDNA fragments of ZFP580 gene were cloned into pMD18-T-Easy vector according to the instructions of the T-A clone kit and sequenced. Plasmids were extracted according to the instructions of plasmid extraction kit and digested respectively by restriction enzyme Xho I and Bgl II to select positive recombinants. The recombinant plasmids were named as pMD18-T-ZFP580, pMD18-T-C and pMD18-T-N respectively. The recombinant plasmids were sent to Takara Company to be sequenced by using the dideoxy chain-termination method.
4 The pMD18-T-ZFP580 recombinant vector and pET-42a(+) fusion expression vector was digested by the Bgl II and Xho I respectively. The digested products were purified, recoverd and then linked by T4DNA ligase at 16°C for 1h. The recombinants were then transformed into E.coli DH5αcompetent cells. Several clones were randomly selected, then inoculated in 5mL LB liquid culture medium(containing Kan, 10μg/ml) respectively, and cultured 37°C at 220 rpm overnight. Plasmids were extracted according to the instructions of plasmid extraction kit and digested respectively by restriction enzyme Bgl II and Xho I to select positive recombinants. The cDNA encoding sequence of ZFP580 full-length open reading frame was sub-cloned into prokaryotic expressing plasmid pET-42a (+), generating pET-42a-ZFP580. Prokaryotic expression vector pET-42a-ZFP580 was sequenced by using the dideoxy chain-termination method and confirmed by reading both strands.
5 Prokaryotic expression recombinant plasmids were transformed into E.coli BL21(DE3) to express the proteins. After the recombinant bacterium was induced with IPTG, the expressed recombinant protein was analyzed with SDS-PAGE.
Results: 1 The total RNA product was examined by the electrophoresis of agarose gel containing formaldehyde. Results showed that there were two clear bands of 18S and 28S.The ratio A260/A280 and A260/A230 of RNA sample was all about 1.9, which demonstrated that RNA quantity and purity were well.It can be used in the following RT-PCR.
2 The cDNA open reading frame sequence of ZFP580, amino terminal encoding region and carboxyl terminal encoding region were obtained by RT-PCR successfully. DNA sequencing results showed that the ZFP580 open reading frame sequence, the sequences of ZFP580 amino terminal encoding region and ZFP580 carboxyl terminal encoding region were exactly consistent with the sequence reported in GenBank.
3 The results of double enzyme digestion with Bgl II and Xho I showed the recombinant plasmids construction of pMD18-T- ZFP580, pMD18-T-C and pMD18-T-N of ZFP580 gene is successful.
4 The recombinant plasmids of pET-42a-ZFP580 and pET-42a- C were confirmed by restriction endonuclease digestion and DNA sequencing respectively. The result indicates that the recombinant plasmid of pET-42a-ZFP580 contains the correct open reading frame.
5 The expression of the GST-ZFP580 fusion protein in E.coli BL21 (DE3) was analyzed by SDS-PAGE. No expected protein band was observed with induction of IPTG
6 The sequence of the whole length cDNA of ZFP580 gene is 1100bp. It contains an open reading frame (519bp), encoding a protein consisted of 172 amino acids. The result of amino acids sequence analysis of zinc finger protein online demonstrated that the encoding protein of ZFP580 has two domains. The amino terminal region between amino acids 5 and 88 is also remarkable rich in Proline residues. The carboxyl terminal region between amino acids 94-172 includes three high conserved C2H2 Zinc finger motifs.
Conclusion: 1 The cDNA open reading frame sequence of ZFP580, amino terminal encoding region and carboxyl terminal encoding region were successfully cloned. Those cDNA sequences are identical to the predicted ZFP580 gene of GenBank.
2 The clone vectors of pMD18-T-N、pMD18-T-C、pMD18-T- ZFP580 and pET-42a-ZFP580 fusion expression vector were constructed successfully.
3 No expected protein band was observed with induction of IPTG.
4 The open reading frame of ZFP580 gene encodes a 172 amino acid protein containing two domains.
方法:1从正常大鼠小脑组织中提取总RNA。
2根据GenBank公布的大鼠ZFP580基因cDNA开读框架序列设计引物,用RT-PCR的方法扩增ZFP580基因cDNA开读框架以及ZFP580基因的N端编码区和C端编码区。
3用T-A克隆方法将目的基因克隆至pMD18-T-Easy载体中,挑取琼脂板上的白色菌落,接种于5ml含有Amp的LB液体培养基中,37℃摇菌培养过夜。用碱裂解法小剂量制备重组质粒。采用BglII和XhoI双酶切法、PCR法及测序法鉴定重组质粒。构建成功的阳性重组质粒记作pMD18-T-ZFP580。同法成功克隆ZFP580基因N端编码区和C端编码区,阳性重组质粒记作pMD18-T-N、pMD18-T-C。
4用BglII和XhoI对克隆载体pMD18-T-ZFP580和原核表达载体pET-42a(+)分别进行双酶切,琼脂糖凝胶电泳酶切产物,纯化回收带有粘端的ZFP580基因和pET-42a(+)载体。将ZFP580基因和pET-42a(+)表达载体用T4DNA连接酶连接过夜。连接产物转化DH5α感受态细胞,加LB液体培养基,37℃培养60min。将菌液均匀涂布于含Kan的LB琼脂平板上,于37℃培养箱中培养12-16h。挑取琼脂板上的单菌落,接种于5ml含有Kan的LB液体培养基中,37℃摇菌培养过夜。用碱裂解法小剂量制备质粒进行鉴定。阳性重组质粒记作pET-42a-ZFP580。同法成功构建ZFP580基因的C端编码区,阳性重组质粒记作pET-42a-C。阳性重组质粒用双酶切的方法鉴定并双向测序,确认有无读码框错误。
5阳性重组载体pET-42a-ZFP580转化E.coli BL21(DE3),IPTG诱导表达,优化表达条件,确定最佳表达参数,SDS-PAGE分析鉴定GST-ZFP580蛋白的表达。
6生物信息学分析ZFP580基因结构和功能。
结果:1甲醛变性琼脂糖凝胶电泳呈现清晰的28S和18S两条带,并且28S宽度以及亮度是18S的2倍,5S量较少。证实所提取总RNA完整性高。测OD260/OD280比值1.9左右,证明总RNA纯度高,无蛋白质和酶等抑制物残留。
2 RT-PCR扩增后,在519bp附近出现了一条特异性条带,此DNA条带与大鼠ZFP580基因cDNA开读框架全编码区的大小相符合。测序证明已获得ZFP580基因开读框架,其序列与GenBank中预测序列完全一致。同法获得264bp和252bp大鼠ZFP580基因的N端编码区和C端编码区。序列比对结果证实与GenBank中预测的一致。
3 T-A克隆构建获得的阳性克隆载体pMD18-T-N、pMD18-T-C、pMD18-T-ZFP580,用Bgl II和Xho I双酶切和PCR法鉴定。结果表明,上述三个克隆载体已成功构建。
4原核表达载体pET-42a-ZFP580、pET-42a-C经鉴定证实构建成功。测序证实pET-42a-ZFP580载体无读码框的错误,转化入表达菌株E.coli BL21(DE3), IPTG诱导,SDS-PAGE鉴定分析GST-ZFP580融合蛋白表达情况。
5小剂量多次进行蛋白表达实验,未能诱导表达目的蛋白。深入探讨了未能表达目的蛋白的原因。
6大鼠ZFP580基因cDNA全长1100bp,完整开读框架为519bp,编码一个含有172个氨基酸的蛋白质,所编码的蛋白与锌指蛋白相似有2个功能结构域:N端的富含脯氨酸结构域和C端的3个C2H2型锌指结构。
结论:1大鼠ZFP580基因cDNA开读框架全编码区、大鼠ZFP580基因的N端编码区和C端编码区与GenBank中预测序列完全一致。
2成功构建克隆载体pMD18-T-N、pMD18-T-C、pMD18-T- ZFP580和原核表达载体pET-42a-ZFP580、pET-42a-C。
3原核表达载体pET-42a-ZFP580读码框正确无误,IPTG诱导表达,经SDS-PAGE分析表达情况。小剂量多次进行蛋白表达实验,未能诱导表达目的蛋白。
4详细分析大鼠ZFP580基因结构和功能,大鼠ZFP580基因开读框架编码一个含有172个氨基酸的蛋白质,所编码的蛋白与锌指蛋白相似有2个功能结构域。
Objective: To clone the full-length open reading frame cDNA sequence of Zinc Finger Protein (ZFP580, encoding 172 amino acids) gene from rat cerebellum. To clone and analyze the cDNA sequence of amino terminal encoding region (encoding 1-88 amino acids) and carboxyl terminal encoding region (encoding 89-172 amino acids) of ZFP580 gene. To construct prokaryotic fusion expression vectors and induces the expression of fusion protein GST-ZFP580.
Methods: 1 Total RNA was extracted from rat cerebellum with Trizol solution.
2 The specific primers were designed according to the cDNA encoding sequence of ZFP580 full-length open reading frame published by GenBank. The cDNA encoding sequences of ZFP580 full-length open reading frame, amino terminal encoding region and carboxyl terminal encoding region were obtained by RT-PCR. Sequence analysis of these fragments and its deduced amino acids were accomplished online at the National Center for Biotechnology Information servers and edited using the Primer5,Clustalx and DNAMAN programs.
3 These cDNA fragments of ZFP580 gene were cloned into pMD18-T-Easy vector according to the instructions of the T-A clone kit and sequenced. Plasmids were extracted according to the instructions of plasmid extraction kit and digested respectively by restriction enzyme Xho I and Bgl II to select positive recombinants. The recombinant plasmids were named as pMD18-T-ZFP580, pMD18-T-C and pMD18-T-N respectively. The recombinant plasmids were sent to Takara Company to be sequenced by using the dideoxy chain-termination method.
4 The pMD18-T-ZFP580 recombinant vector and pET-42a(+) fusion expression vector was digested by the Bgl II and Xho I respectively. The digested products were purified, recoverd and then linked by T4DNA ligase at 16°C for 1h. The recombinants were then transformed into E.coli DH5αcompetent cells. Several clones were randomly selected, then inoculated in 5mL LB liquid culture medium(containing Kan, 10μg/ml) respectively, and cultured 37°C at 220 rpm overnight. Plasmids were extracted according to the instructions of plasmid extraction kit and digested respectively by restriction enzyme Bgl II and Xho I to select positive recombinants. The cDNA encoding sequence of ZFP580 full-length open reading frame was sub-cloned into prokaryotic expressing plasmid pET-42a (+), generating pET-42a-ZFP580. Prokaryotic expression vector pET-42a-ZFP580 was sequenced by using the dideoxy chain-termination method and confirmed by reading both strands.
5 Prokaryotic expression recombinant plasmids were transformed into E.coli BL21(DE3) to express the proteins. After the recombinant bacterium was induced with IPTG, the expressed recombinant protein was analyzed with SDS-PAGE.
Results: 1 The total RNA product was examined by the electrophoresis of agarose gel containing formaldehyde. Results showed that there were two clear bands of 18S and 28S.The ratio A260/A280 and A260/A230 of RNA sample was all about 1.9, which demonstrated that RNA quantity and purity were well.It can be used in the following RT-PCR.
2 The cDNA open reading frame sequence of ZFP580, amino terminal encoding region and carboxyl terminal encoding region were obtained by RT-PCR successfully. DNA sequencing results showed that the ZFP580 open reading frame sequence, the sequences of ZFP580 amino terminal encoding region and ZFP580 carboxyl terminal encoding region were exactly consistent with the sequence reported in GenBank.
3 The results of double enzyme digestion with Bgl II and Xho I showed the recombinant plasmids construction of pMD18-T- ZFP580, pMD18-T-C and pMD18-T-N of ZFP580 gene is successful.
4 The recombinant plasmids of pET-42a-ZFP580 and pET-42a- C were confirmed by restriction endonuclease digestion and DNA sequencing respectively. The result indicates that the recombinant plasmid of pET-42a-ZFP580 contains the correct open reading frame.
5 The expression of the GST-ZFP580 fusion protein in E.coli BL21 (DE3) was analyzed by SDS-PAGE. No expected protein band was observed with induction of IPTG
6 The sequence of the whole length cDNA of ZFP580 gene is 1100bp. It contains an open reading frame (519bp), encoding a protein consisted of 172 amino acids. The result of amino acids sequence analysis of zinc finger protein online demonstrated that the encoding protein of ZFP580 has two domains. The amino terminal region between amino acids 5 and 88 is also remarkable rich in Proline residues. The carboxyl terminal region between amino acids 94-172 includes three high conserved C2H2 Zinc finger motifs.
Conclusion: 1 The cDNA open reading frame sequence of ZFP580, amino terminal encoding region and carboxyl terminal encoding region were successfully cloned. Those cDNA sequences are identical to the predicted ZFP580 gene of GenBank.
2 The clone vectors of pMD18-T-N、pMD18-T-C、pMD18-T- ZFP580 and pET-42a-ZFP580 fusion expression vector were constructed successfully.
3 No expected protein band was observed with induction of IPTG.
4 The open reading frame of ZFP580 gene encodes a 172 amino acid protein containing two domains.
引文
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35 Miller JF, Dower WJ, Tompkin LS. High-voltage electroporation of bacteria: genetic transformation of Campylobater jejuni with plasmid DNA. Proe Natl Acad SciUSA, 1988, 85(3): 856-860
36 Chung T, R.Miller. A rapid and convenient method for the preparation and storage of competent bacterial cells. Nuclei Acids Res, 1988, 16: 3580
37 Li FY, Zhu XH, Huang YH, et al. expression of superantigen SED in Escherichia coli. Immunological journal, 2002, 18(2): 92-94
38 Xie ZHH, Wang AM, Ma CH, et al. Cloning of Thioredoxin (TRX) cDNA from PC12 cells and expression in E.coli. Basic Medical Sciences and Clinics, 2002, 22(1): 59-62
39 张健, 刘新平, 张伟, 等. 人NDRG1的融合表达、纯化及抗体制备.中国生物化学与分子生物学报, 2003, 19(6): 698-703
40 郑文婕, 刘敬忠, 吕星, 等. 重组人FtJT3配体的克隆、表达及功能鉴定.生物工程学报, 2000, 16(6): 708-712
41 黄培堂等译. 分子克隆实验指南, 第三版.2002, 1217
42 Novagen. pET系统操作手册(第十版). 2003, 5-12
43 马建岗, 基因工程学原理, 西安交通大学出版社。
44 张惠展. 基因工程概论. 上海: 华东理工大学出版社, 1999: 250-255
45 吴冠芸等主编. 生物化学与分子生物学实验常用数据手册.北京:科学出版社. 1999: 63-70
46 Smith DB. purification of glutathione-S-transferase fusion Proteins, Methods MOI.Cell Biol, 1993, 4: 220一229
47 Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions withglutathione-S-transferase. Gene, 1988, 67: 31-40
48 The Rat Genome Project Sequencing Consortiu. Genome sequence of the brown Norway rat yields insight in mammalian evolution. Nature, 2004, 428: 493–521
1 赵莲, 薛爽, 陈芳. 炎症、动脉粥样硬化和心脑血管疾病.心血管病学进展, 2005, 6(2): 193-196
2 Antonio C, Peter T. PPARs in atnerosclerosis: the clot thickens. J Clin Invest, 2004, 114(11): 1538-1540
3 Jaber J, Murin J, Kinova S, et al. The role of infection and inflammation in the pathogenesis of atherosclerosis. Vnitr Lek, 2002, 48(7): 657-666
4 LaRose JC, Grundy SM, Waters PD, et al. Intensive lipid lowering with atorvastatin patients with stable coronary disease. N Engl J Med, 2005, 352(14): 1425-1435
5 李颖莉.低密度脂蛋白受体相关蛋白与动脉粥样硬化的关系. 中国分子心脏病学杂志, 2003, 3(3): 167-173
6 李素敏. 弱氧化型低密度脂蛋白的致动脉粥样硬化作用. 中国动脉硬化杂志, 2002, 10(3): 271-274
7 Yong IS, Mc Eneny J. Lipoprotein oxidation and atheroscle- rosis. Biochemical Society Transactions, 2001, 29(2): 358-362
8 Berliner JA, Watson AD. A Role for Oxidized Phospholipids in Atherosclerosis. N Engl J Med, 2005, 353(1): 9-11
9 Zalewski A, Macphee C. Role of lipoprotein-associated phospholipase A2 in atherosclerosis: biology, epidemiology,and possible therapeutic target. Arterioscler Thromb Vase Biol, 2005, 25(5): 923-931
10 陈良, 张梅, 李长江, 等. 复方丹参滴丸对动脉粥样硬化粘附因子的作用. 中国动脉硬化杂志, 2007, l5(2): 101-104
11 Navab M. The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids. J Lipid Res, 2004, 45(6): 993-1007
12 官秀梅, 钱民章. 单核细胞趋化蛋白-1对人脐静脉平滑肌细胞增殖的影响. 中国动脉硬化杂志, 2005, 13(3): 309-312
13 李静, 孙雷, 庄永杰, 等. 单核细胞趋化蛋白-1(MCP-1)和核因子-κB与巨噬细胞浸润及动脉粥样硬化斑块形成的关系. 大连医科大学学报, 2007, 29 (1):14-17
14 Mozes G, Mohacsi F, Glovjozjk P, et al. Adenovirus- mediated gene transfer of macrophage colony stimulating factor to the arterial wall in vivo. Arterioscler Thromb Vasc Biol, 1998, 18(7): 1157-1163
15 Rallidis L S, Zolindaki M G, Pentzeridis P C, et a1. Raised concentrations of macrophage colony stimulating factor in severe unstable angina beyond the acute phase are strongly predictive of long term outcome. Heart, 2004, 90 (1): 25-29
16 Kim H H, Ha H j, Kim S O, et al. KR31372, a benzopyran derivative, inhibits oxidized LDL-stimulated proliferation and migration of vascular smooth muscle cells. Fundam Clin Pha-rmacol, 2000, 14 (5): 469-476
17 Cucina A, Pagliei S, Borrelli V, et al. Oxidized LDL induces production of platelet derived growth factor AA ( PDGF AA) from aortic smooth muscle cells. Eur J Vasc Endovasc Surg, 1998, 16(3): 197-202
18 卢维晟,王一尘. MMP-2表达与大鼠动脉粥样硬化和血管钙化的关系及辛伐他汀对其影响. 心脏杂志(Chin Heart J) 2006, 18(4): 376-378
19 Landmesser U, Horning B, Drexler H. Endothelial function: a critical determinant in atherosclerosis. Circulation, 2004, 109 (21 Suppl 1): Ⅱ27-33
20 Davignon J, Ganz P. Role of endothelial dysfunction in atherosclerosis. Circulation, 2004, 109 (23 Suppl 1): Ⅲ27-32
21 石翠格, 胡刚, 汪海. 天然药物槟榔碱对氧化低密度脂蛋白致血管内皮细胞损伤的保护作用研究. 科学技术与工程, 2007, 7(12): 2780-2783
22 De Ciuceis C, Amiri F, Brassard P, et al. Reduced vascular remodeling, endothelial dysfunction, and oxidative stress in resistance arteries of angiotensin II-Infused macrophage colony stimulating factor-deficient mice. Evidence for a role in inflammation inangiotensin-induced vascular injury. Arterioscler Thromb Vasc Biol, 2005, 25(10): 2106-2113
23 雷新军,马爱群,任冰稳,等. Ox-LDL直接诱导人外周血单核细胞表达致炎细胞因子mRNA.中国免疫学杂志, 2004, 20 (2): 93-96
24 Ianaro A, Maffia P, Grassia G, et al. Cloricromene in endoto-xemia: role of NF-κappaB. Naunyn Schmiedebergs Arch Pharmacol, 2004, 370(2): 140-145
25 Monaco C, Andreakos E, Kiriakidis S, et al. Canonical pathway of nuclear factor kappa B activation selectively regulates proinflammatory and prothrombotic responses in human atherosclerosis. Proc Natl Acad Sci USA, 2004, 101(15): 5634–5639
26 郑建杰, 马爱群, 王鸿雁, 等. I型胶原及核因子-κB与动脉粥样斑块的关系.临床心血管病杂志, 2006, 22 (7): 410-413
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14 J.撒姆布鲁克, E.F.弗里奇, T.曼尼阿蒂斯著. 金冬雁, 黎孟枫 等译. 分子克隆实验指南, 北京: 科学出版社, 1993
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19 Wu XQ, Oppermann U. High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system. Journal of chromatography B, 2003, 786: 177-185
20 The RIKEN Genome Exploration Research Group Phase II Team and the FANTOM Consortium. Functional annotation of a full-length mouse cDNA collection Nature, 2001, 409: 685-690
21 王琳芳, 杨克恭, 主编. 蛋白质和核酸, 1997, 北京医科大学-协和医科大学联合出版社
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27 Tommerup N, Vissing, H. Isolation and fine mapping of 16 novel human zinc finger-encoding cDNAs identify putativecandidate genes for developmental and malignant disorders. Genomics, 1995, 27(2): 259-264
28 Noce T, Fujiwara Y, Sezaki M, et al. Expression of a mouse zinc finger protein gene in both spermatocytes and oocytes during meiosis. Dev Biol, 1992, 153: 356-367
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33 Jones N. Structure and function of transcription factors. Seminars Cancer Biol, 1990, 1: 5-17
34 Shalloway D, Zelenetz AD, and Cooper GM, Molecular cloning and characterization of the chicken gene homologous to the transforming gene of rous sarcoma virus. Cell. 1981, 24: 531-54
35 Miller JF, Dower WJ, Tompkin LS. High-voltage electroporation of bacteria: genetic transformation of Campylobater jejuni with plasmid DNA. Proe Natl Acad SciUSA, 1988, 85(3): 856-860
36 Chung T, R.Miller. A rapid and convenient method for the preparation and storage of competent bacterial cells. Nuclei Acids Res, 1988, 16: 3580
37 Li FY, Zhu XH, Huang YH, et al. expression of superantigen SED in Escherichia coli. Immunological journal, 2002, 18(2): 92-94
38 Xie ZHH, Wang AM, Ma CH, et al. Cloning of Thioredoxin (TRX) cDNA from PC12 cells and expression in E.coli. Basic Medical Sciences and Clinics, 2002, 22(1): 59-62
39 张健, 刘新平, 张伟, 等. 人NDRG1的融合表达、纯化及抗体制备.中国生物化学与分子生物学报, 2003, 19(6): 698-703
40 郑文婕, 刘敬忠, 吕星, 等. 重组人FtJT3配体的克隆、表达及功能鉴定.生物工程学报, 2000, 16(6): 708-712
41 黄培堂等译. 分子克隆实验指南, 第三版.2002, 1217
42 Novagen. pET系统操作手册(第十版). 2003, 5-12
43 马建岗, 基因工程学原理, 西安交通大学出版社。
44 张惠展. 基因工程概论. 上海: 华东理工大学出版社, 1999: 250-255
45 吴冠芸等主编. 生物化学与分子生物学实验常用数据手册.北京:科学出版社. 1999: 63-70
46 Smith DB. purification of glutathione-S-transferase fusion Proteins, Methods MOI.Cell Biol, 1993, 4: 220一229
47 Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions withglutathione-S-transferase. Gene, 1988, 67: 31-40
48 The Rat Genome Project Sequencing Consortiu. Genome sequence of the brown Norway rat yields insight in mammalian evolution. Nature, 2004, 428: 493–521
1 赵莲, 薛爽, 陈芳. 炎症、动脉粥样硬化和心脑血管疾病.心血管病学进展, 2005, 6(2): 193-196
2 Antonio C, Peter T. PPARs in atnerosclerosis: the clot thickens. J Clin Invest, 2004, 114(11): 1538-1540
3 Jaber J, Murin J, Kinova S, et al. The role of infection and inflammation in the pathogenesis of atherosclerosis. Vnitr Lek, 2002, 48(7): 657-666
4 LaRose JC, Grundy SM, Waters PD, et al. Intensive lipid lowering with atorvastatin patients with stable coronary disease. N Engl J Med, 2005, 352(14): 1425-1435
5 李颖莉.低密度脂蛋白受体相关蛋白与动脉粥样硬化的关系. 中国分子心脏病学杂志, 2003, 3(3): 167-173
6 李素敏. 弱氧化型低密度脂蛋白的致动脉粥样硬化作用. 中国动脉硬化杂志, 2002, 10(3): 271-274
7 Yong IS, Mc Eneny J. Lipoprotein oxidation and atheroscle- rosis. Biochemical Society Transactions, 2001, 29(2): 358-362
8 Berliner JA, Watson AD. A Role for Oxidized Phospholipids in Atherosclerosis. N Engl J Med, 2005, 353(1): 9-11
9 Zalewski A, Macphee C. Role of lipoprotein-associated phospholipase A2 in atherosclerosis: biology, epidemiology,and possible therapeutic target. Arterioscler Thromb Vase Biol, 2005, 25(5): 923-931
10 陈良, 张梅, 李长江, 等. 复方丹参滴丸对动脉粥样硬化粘附因子的作用. 中国动脉硬化杂志, 2007, l5(2): 101-104
11 Navab M. The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids. J Lipid Res, 2004, 45(6): 993-1007
12 官秀梅, 钱民章. 单核细胞趋化蛋白-1对人脐静脉平滑肌细胞增殖的影响. 中国动脉硬化杂志, 2005, 13(3): 309-312
13 李静, 孙雷, 庄永杰, 等. 单核细胞趋化蛋白-1(MCP-1)和核因子-κB与巨噬细胞浸润及动脉粥样硬化斑块形成的关系. 大连医科大学学报, 2007, 29 (1):14-17
14 Mozes G, Mohacsi F, Glovjozjk P, et al. Adenovirus- mediated gene transfer of macrophage colony stimulating factor to the arterial wall in vivo. Arterioscler Thromb Vasc Biol, 1998, 18(7): 1157-1163
15 Rallidis L S, Zolindaki M G, Pentzeridis P C, et a1. Raised concentrations of macrophage colony stimulating factor in severe unstable angina beyond the acute phase are strongly predictive of long term outcome. Heart, 2004, 90 (1): 25-29
16 Kim H H, Ha H j, Kim S O, et al. KR31372, a benzopyran derivative, inhibits oxidized LDL-stimulated proliferation and migration of vascular smooth muscle cells. Fundam Clin Pha-rmacol, 2000, 14 (5): 469-476
17 Cucina A, Pagliei S, Borrelli V, et al. Oxidized LDL induces production of platelet derived growth factor AA ( PDGF AA) from aortic smooth muscle cells. Eur J Vasc Endovasc Surg, 1998, 16(3): 197-202
18 卢维晟,王一尘. MMP-2表达与大鼠动脉粥样硬化和血管钙化的关系及辛伐他汀对其影响. 心脏杂志(Chin Heart J) 2006, 18(4): 376-378
19 Landmesser U, Horning B, Drexler H. Endothelial function: a critical determinant in atherosclerosis. Circulation, 2004, 109 (21 Suppl 1): Ⅱ27-33
20 Davignon J, Ganz P. Role of endothelial dysfunction in atherosclerosis. Circulation, 2004, 109 (23 Suppl 1): Ⅲ27-32
21 石翠格, 胡刚, 汪海. 天然药物槟榔碱对氧化低密度脂蛋白致血管内皮细胞损伤的保护作用研究. 科学技术与工程, 2007, 7(12): 2780-2783
22 De Ciuceis C, Amiri F, Brassard P, et al. Reduced vascular remodeling, endothelial dysfunction, and oxidative stress in resistance arteries of angiotensin II-Infused macrophage colony stimulating factor-deficient mice. Evidence for a role in inflammation inangiotensin-induced vascular injury. Arterioscler Thromb Vasc Biol, 2005, 25(10): 2106-2113
23 雷新军,马爱群,任冰稳,等. Ox-LDL直接诱导人外周血单核细胞表达致炎细胞因子mRNA.中国免疫学杂志, 2004, 20 (2): 93-96
24 Ianaro A, Maffia P, Grassia G, et al. Cloricromene in endoto-xemia: role of NF-κappaB. Naunyn Schmiedebergs Arch Pharmacol, 2004, 370(2): 140-145
25 Monaco C, Andreakos E, Kiriakidis S, et al. Canonical pathway of nuclear factor kappa B activation selectively regulates proinflammatory and prothrombotic responses in human atherosclerosis. Proc Natl Acad Sci USA, 2004, 101(15): 5634–5639
26 郑建杰, 马爱群, 王鸿雁, 等. I型胶原及核因子-κB与动脉粥样斑块的关系.临床心血管病杂志, 2006, 22 (7): 410-413