The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different(from Ⅰ to Ⅵ) concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase(BAD...The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different(from Ⅰ to Ⅵ) concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase(BADH: EC 1.2.1.8) activities have a direct relation with increased stressing time in the same treatment; both exhibit a single peak with increasing the concentration of saline-alkali solution, and number V shows the highest value. The BADH gene of Leyrnus chinensis was cloned by RT-PCR and RACE technology and was designated as LcBADH. The cDNA sequence of LcBADH was 1774bp including the open reading frame(ORF) of 1521bp(coding 506 amino acids). The vector of prokaryotic expression was constructed by inserting the LcBADH gene fragmcnt into pET30a(+) and transformed into E. coli BL21(DE3). The result of SDS-PAGE shows that the idio-protein with a molecular mass of 56.78 kDa was effectively expressed in the recombinant bacteria induced by isopropyl fl-D-thiogalactoside(IPTG).展开更多
The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genom...The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains: a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/(Arg+Lys) ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 (DE3) by pColdlII was also successfully observed in this study.展开更多
The zinc finger proteins belong to the largest family of transcription factors.But there is little research of Cys2/His2 type zinc finger proteins in cotton,and there is no submission of correlating
To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites ...To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.展开更多
BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have bee...BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten. METHODS: The arresten gene was obtained from a healthy puerpera's placenta tissue by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5α, BL21 and BL21 (DE3) by the CaCl_2 transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purlfled, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane (CAM). RESULTS: The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 (DE3). SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities. CONCLUSIONS: The successful construction of recombinant plasmid pBV220/arr and the effective expression in E. coil have laid a foundation for further study of its anti-angiogenic function and may pave the way for future antitumor application.展开更多
Objective To obtain the sequence of Chinese obese (OB) cDNA and establish a method of leptin production in China Methods Han Chinese OB cDNA fragment was obtained by reverse transcriptase polymerase chain reactio...Objective To obtain the sequence of Chinese obese (OB) cDNA and establish a method of leptin production in China Methods Han Chinese OB cDNA fragment was obtained by reverse transcriptase polymerase chain reaction (RT PCR) with total RNA extracted from human adipocytes and was inserted into the expressing vector pBV220 Then the constructed recombinant plasmid pBV220 OB was transformed to E coli DH5α for leptin expression The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression E coli cells were lysed by high pressure homogenization After cell membrane was extracted, the inclusion bodies were mainly renatured and purified primarily by precipitation with ammonium sulfate and gel chromatography through a Sephadex G75 column The activity of recombinant leptin was determined by its influence on the satiety and weight gain of mice Results Analysis of DNA sequence showed that Han Chinese OB cDNA included the glutamine codon at 49 The amount of recombinant leptin expressed in E coli accounted for 31%-47% of total cellular proteins From 1?L of fermentative bacteria about 40?mg of pure recombinant human leptin was isolated with a purity of being above 95% The recombinant human leptin could reduce food intake and inhibit weight gains in mice Conclusion The glutamine codon at 49 is not missing in Chinese OB gene The biologically active human leptin can be obtained by a relatively simple method of recombinant DNA technology展开更多
PLA2 enzyme hydrolyzes arachidonic acid, and other polyunsaturated fatty acids, from the sn-2 position to release free arachidonic acid and a lysophospholipid. Previous studies reported that the PLA2 in invertebrate o...PLA2 enzyme hydrolyzes arachidonic acid, and other polyunsaturated fatty acids, from the sn-2 position to release free arachidonic acid and a lysophospholipid. Previous studies reported that the PLA2 in invertebrate organisms participates in lipid signaling molecules like arachidonic acid release in immune-associated tissues like hemo- cytes and fat bodies. In the present study, we cloned the BmPLA2 gene from fat body tissue of silkworm Bombyx mori, which has a total sequence of 1.031 kb with a 31.90 kDa pro- tein. In silico results of BmPLA2 indicated that the protein has a putative WD40 conserved domain and its phylogeny tree clustered with Danaus plexippus species. We investigated the transcriptional expression in development stages and tissues. The highest expression of BmPLA2 was screened in fat body among the studied tissues of third day fifth instar larva, with a high expression on third day fifth instar larva followed by a depression of expression in the wandering stage of the fifth instar larva. The expression of BmPLA2 in female pupa was higher than that of male pupa. Our RNAi-mediated gene silencing results showed highest reduction of BmPLA2 expression in post-24 h followed by post-48 and post-72 h. The BmPLA2-RNAi larvae and pupa could be characterized by pharate adult lethality and underdevelopment. The phenotypic characters of fat body cells in RNAi-induced larva im- plied that BmPLA2 affects the metabolic functions of fat body tissue in silkworm Bombyx mori.展开更多
基金Supported by the National Natural Science Foundation of China(Nos30590382 and 30570273)
文摘The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different(from Ⅰ to Ⅵ) concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase(BADH: EC 1.2.1.8) activities have a direct relation with increased stressing time in the same treatment; both exhibit a single peak with increasing the concentration of saline-alkali solution, and number V shows the highest value. The BADH gene of Leyrnus chinensis was cloned by RT-PCR and RACE technology and was designated as LcBADH. The cDNA sequence of LcBADH was 1774bp including the open reading frame(ORF) of 1521bp(coding 506 amino acids). The vector of prokaryotic expression was constructed by inserting the LcBADH gene fragmcnt into pET30a(+) and transformed into E. coli BL21(DE3). The result of SDS-PAGE shows that the idio-protein with a molecular mass of 56.78 kDa was effectively expressed in the recombinant bacteria induced by isopropyl fl-D-thiogalactoside(IPTG).
基金supported by the National High Technology Research and Development Program of China (Grant no.2007AA091905)the Basic Scientific Research Operation Fund of China (Grant no. GY02-2007-T11)
文摘The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains: a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/(Arg+Lys) ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 (DE3) by pColdlII was also successfully observed in this study.
文摘The zinc finger proteins belong to the largest family of transcription factors.But there is little research of Cys2/His2 type zinc finger proteins in cotton,and there is no submission of correlating
文摘To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.
基金This work was supported by a grant from Science and Technology Fund of Shanxi Province, China (No. 042082).
文摘BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten. METHODS: The arresten gene was obtained from a healthy puerpera's placenta tissue by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5α, BL21 and BL21 (DE3) by the CaCl_2 transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purlfled, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane (CAM). RESULTS: The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 (DE3). SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities. CONCLUSIONS: The successful construction of recombinant plasmid pBV220/arr and the effective expression in E. coil have laid a foundation for further study of its anti-angiogenic function and may pave the way for future antitumor application.
文摘Objective To obtain the sequence of Chinese obese (OB) cDNA and establish a method of leptin production in China Methods Han Chinese OB cDNA fragment was obtained by reverse transcriptase polymerase chain reaction (RT PCR) with total RNA extracted from human adipocytes and was inserted into the expressing vector pBV220 Then the constructed recombinant plasmid pBV220 OB was transformed to E coli DH5α for leptin expression The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression E coli cells were lysed by high pressure homogenization After cell membrane was extracted, the inclusion bodies were mainly renatured and purified primarily by precipitation with ammonium sulfate and gel chromatography through a Sephadex G75 column The activity of recombinant leptin was determined by its influence on the satiety and weight gain of mice Results Analysis of DNA sequence showed that Han Chinese OB cDNA included the glutamine codon at 49 The amount of recombinant leptin expressed in E coli accounted for 31%-47% of total cellular proteins From 1?L of fermentative bacteria about 40?mg of pure recombinant human leptin was isolated with a purity of being above 95% The recombinant human leptin could reduce food intake and inhibit weight gains in mice Conclusion The glutamine codon at 49 is not missing in Chinese OB gene The biologically active human leptin can be obtained by a relatively simple method of recombinant DNA technology
基金Acknowledgments The work was supported by the Nalional Basic Research Program of China under Grand No. 2012CB 114601 and the National Natural Science Foundation of China (No. NSFC:31372374/C 1703), the Science and Technology Innovation Team of Zhejiang Province (No. 2010R50031 ) and Chinese Universities Scientific Fund to Yun-Gen Miao.
文摘PLA2 enzyme hydrolyzes arachidonic acid, and other polyunsaturated fatty acids, from the sn-2 position to release free arachidonic acid and a lysophospholipid. Previous studies reported that the PLA2 in invertebrate organisms participates in lipid signaling molecules like arachidonic acid release in immune-associated tissues like hemo- cytes and fat bodies. In the present study, we cloned the BmPLA2 gene from fat body tissue of silkworm Bombyx mori, which has a total sequence of 1.031 kb with a 31.90 kDa pro- tein. In silico results of BmPLA2 indicated that the protein has a putative WD40 conserved domain and its phylogeny tree clustered with Danaus plexippus species. We investigated the transcriptional expression in development stages and tissues. The highest expression of BmPLA2 was screened in fat body among the studied tissues of third day fifth instar larva, with a high expression on third day fifth instar larva followed by a depression of expression in the wandering stage of the fifth instar larva. The expression of BmPLA2 in female pupa was higher than that of male pupa. Our RNAi-mediated gene silencing results showed highest reduction of BmPLA2 expression in post-24 h followed by post-48 and post-72 h. The BmPLA2-RNAi larvae and pupa could be characterized by pharate adult lethality and underdevelopment. The phenotypic characters of fat body cells in RNAi-induced larva im- plied that BmPLA2 affects the metabolic functions of fat body tissue in silkworm Bombyx mori.