This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous nitron 1 sequence. hFIX minigene was obtained with middle sequence truncated nitron 1 inserted into the relative site ...This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous nitron 1 sequence. hFIX minigene was obtained with middle sequence truncated nitron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5i’IX, retroviral vector GINaCi’IX were constructed. These vectors were transduced into target cells of PA317, C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/pKG5i’IX, 151 "g/106 cells/24h; PA317/GINaCi’IX, 308ng/106 cells/24 h; C2C12/G1 NaCi’IX, 186 ng/106 cells/24 h; RSF/GINaCi’IX, 1929 ng/106 cells/24 h; HSF/GlNaCi’IX, 1646 ng/106 cells/ 24 h. These results indicated that hFIX minigene with nitron 1 is able to increase the expression level to about 3 times of that of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from nitron splicing during viral production, a retroviral vector GlNaCi’IXR with reversely inserted hFIX minigene expression cassette was constructed. The expression level of reverse constructor in PA317 cells was 390 ng/106 cells/24 h with 79% of bioactivity. PCR detection of HT/GlNaCi’IXR cells infected with PA317/ClNaCi’IXR supernatant confirmed the existence of nitron 1 sequence. These results suggested that expression vector with forward-inserted intronl-carrying hFIX expression cassette can be used in directed gene Human factor IX expression with nitron transfer, but when using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying hFIX expression cassette should be considered.展开更多
Fluvoxamine maleate is an excellent antidepressive drug. In the literatures, it was synthesized by the use of 4-（trifluoromethyl） aniline or 4-（trifluoromethyl） benzonitrile as starting materials and 5-methoxy-4＇...Fluvoxamine maleate is an excellent antidepressive drug. In the literatures, it was synthesized by the use of 4-（trifluoromethyl） aniline or 4-（trifluoromethyl） benzonitrile as starting materials and 5-methoxy-4＇-（trifluoromethyl-phenyl） valerophenone as a key intermediate. However, the methods in literatures have some disadvantages, such as the use of expensive materials, heavy pollution of environment, long reaction time and low yield of the product （only 30-40% overall yield）. We herein report an environmentally friendly synthetic method of fluvoxamine maleate, which used 4-（trifluoromethyl） benzoic acid and tetrahydrofuran as starting materials and FeCI3 as catalyst for the coupling of acid chloride with Grignard reagent. The fluvoxamine maleate was synthesized in 46% overall yield, through the oximation, etheration and salification of the intermediate successively.This method has some advantages, such as the use of commercially available materials, low cost, short production period （12-14 h）, high yield and light pollution.展开更多
Syntheses of 3-substituted coumarins by the condensation reaction of salicylaldehyde with activated methylene compounds （ethyl cyanoacetate, malonic acid） are discussed in media of ionic liquids--（2-hydroxyethyl）a...Syntheses of 3-substituted coumarins by the condensation reaction of salicylaldehyde with activated methylene compounds （ethyl cyanoacetate, malonic acid） are discussed in media of ionic liquids--（2-hydroxyethyl）ammonium carboxylates （formates, acetates, lactates） without utilization of any other catalysts. The dependence of yields of the reaction product is investigated on the structure of ionic liquids （their cations, anions, total polarity and pseudo-pH values）. 3-Substituted coumarins are prepared in high yields in media of these environmentally friendly ionic liquids which serve simultaneously as reaction media and as catalysts. The mentioned ionic liquids are prepared by reactions of corresponding hydroxyethyl-amines with carboxylic acids, and their quantitative analyses being made by potentiometric titration of ionic liquids with perchloric acid in 100% acetic acid.展开更多
文摘This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous nitron 1 sequence. hFIX minigene was obtained with middle sequence truncated nitron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5i’IX, retroviral vector GINaCi’IX were constructed. These vectors were transduced into target cells of PA317, C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/pKG5i’IX, 151 "g/106 cells/24h; PA317/GINaCi’IX, 308ng/106 cells/24 h; C2C12/G1 NaCi’IX, 186 ng/106 cells/24 h; RSF/GINaCi’IX, 1929 ng/106 cells/24 h; HSF/GlNaCi’IX, 1646 ng/106 cells/ 24 h. These results indicated that hFIX minigene with nitron 1 is able to increase the expression level to about 3 times of that of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from nitron splicing during viral production, a retroviral vector GlNaCi’IXR with reversely inserted hFIX minigene expression cassette was constructed. The expression level of reverse constructor in PA317 cells was 390 ng/106 cells/24 h with 79% of bioactivity. PCR detection of HT/GlNaCi’IXR cells infected with PA317/ClNaCi’IXR supernatant confirmed the existence of nitron 1 sequence. These results suggested that expression vector with forward-inserted intronl-carrying hFIX expression cassette can be used in directed gene Human factor IX expression with nitron transfer, but when using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying hFIX expression cassette should be considered.
文摘Fluvoxamine maleate is an excellent antidepressive drug. In the literatures, it was synthesized by the use of 4-（trifluoromethyl） aniline or 4-（trifluoromethyl） benzonitrile as starting materials and 5-methoxy-4＇-（trifluoromethyl-phenyl） valerophenone as a key intermediate. However, the methods in literatures have some disadvantages, such as the use of expensive materials, heavy pollution of environment, long reaction time and low yield of the product （only 30-40% overall yield）. We herein report an environmentally friendly synthetic method of fluvoxamine maleate, which used 4-（trifluoromethyl） benzoic acid and tetrahydrofuran as starting materials and FeCI3 as catalyst for the coupling of acid chloride with Grignard reagent. The fluvoxamine maleate was synthesized in 46% overall yield, through the oximation, etheration and salification of the intermediate successively.This method has some advantages, such as the use of commercially available materials, low cost, short production period （12-14 h）, high yield and light pollution.
文摘Syntheses of 3-substituted coumarins by the condensation reaction of salicylaldehyde with activated methylene compounds （ethyl cyanoacetate, malonic acid） are discussed in media of ionic liquids--（2-hydroxyethyl）ammonium carboxylates （formates, acetates, lactates） without utilization of any other catalysts. The dependence of yields of the reaction product is investigated on the structure of ionic liquids （their cations, anions, total polarity and pseudo-pH values）. 3-Substituted coumarins are prepared in high yields in media of these environmentally friendly ionic liquids which serve simultaneously as reaction media and as catalysts. The mentioned ionic liquids are prepared by reactions of corresponding hydroxyethyl-amines with carboxylic acids, and their quantitative analyses being made by potentiometric titration of ionic liquids with perchloric acid in 100% acetic acid.
