同型半胱氨酸转甲基酶的提纯和活性测定
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摘要
目的
1.对含有同型半胱氨酸转甲基酶外源性基因的大肠埃希氏菌培养、收集含有同型半胱氨酸转甲基酶的母液。
2.分离提纯同型半胱氨酸转甲基酶。
3.判断提纯的同型半胱氨酸转甲基酶是否有活性,初步确定其活性的大小。
方法
1.将含有同型半胱氨酸转甲基酶外源性基因的大肠埃希氏菌接种至血平板,37℃培养过夜后接种于含氨苄青霉素的LB培养液中,37℃在摇床上培养至对数中期,加入IPTG诱导8小时之后离心弃上清留沉淀。用缓冲液混匀沉淀后用电动搅拌机5000转／分钟以破坏细菌,冰浴匀浆2分钟,15000g离心15分钟,弃沉淀取上清。上清液经过Sephadex CL-6B层析柱,DEAE Sepharose Fast Flow层析柱,Sephadex G-75层析柱之后得到提纯的同型半胱氨酸转甲基酶。
2.用Western Blot和考马斯亮蓝染色验证层析后的蛋白峰是否含有同型半胱氨酸转甲基酶,根据Mark的条带大概判断其kD值。
3.用金斯尔G系列同型半胱氨酸检测试剂盒测定20份血清的同型半胱氨酸浓度,再用提纯的同型半胱氨酸转甲基酶与谷氨酸脱氢酶组成的混合液替换试剂2,重新检测相同的20份血清同型半胱氨酸的浓度,根据两种试剂盒检测的数值来判断提纯的同型半胱氨酸转甲基酶是否有活性以及活性的大小。
结果
1.将提纯后的酶做Western Blot验证,证明是所需要的同型半胱氨酸转甲基酶。
2.与Western Blot和考马斯亮蓝染色上Mark的条带相比较,同型半胱氨酸转甲基酶单体的分子量为45kD。
3.利用原试剂盒和替换试剂2后的试剂盒分别测定相同的20份标本,对两组数据进行统计,用配对t检验,原试剂盒测定的均数为16.48±3.47,标准误为0.77；试剂2替换后的测定的均数为17.19±4.10,标准误为0.92。原试剂盒和试剂2替换之后的试剂盒测定值的相关系数为0.92,两种方法相关系数假设检验的P值<0.05,表明试剂2替换前后同型半胱氨酸的检测高度相关。配对t检验的P值>0.05,无统计学意义,说明替换前后两种试剂盒检测同型半胱氨酸无差异。
结论
1.成功提纯出了同型半胱氨酸转甲基酶,并建立了一整套提纯同型半胱氨酸转甲基酶的方案,可供科研、生产使用。
2.提纯同型半胱氨酸转甲基酶单体为45kD。
3.提纯的同型半胱氨酸转甲基酶有活性,可用于同型半胱氨酸浓度的检测。
Objective
1 To culture escherichia coli which contain exogenous genes of homocysteine methyltransferase and collect supernatant which contain homocysteine methyltransferase.
2 To separate and purify the homocysteine methyltransferase
3 To judge whether the purified homocysteine methyltransferase has activity or not; determining its activity preliminarily.
Methods
1. The escherichia coli which containing exogenous genes of homocysteine methyltransferase were inoculated in the blood agar. the blood agar were culture at 37℃overnight, then the escherichia coli were inoculated to Medium LB which contain ampicillin and cultured to mid-log phase in shake at 37℃. After adding IPTG to Medium LB for 8 hours, the liquid were centrifuged for obtaining precipitation. Precipitation were dissolved with buffer, then mixed with electric mixer(5000 r/min,2min) to destroy bacteria. The supernatant obtained after centrifugation (15000g,15min) were loaded on to the Sephadex CL-6B column, the DEAE Sepharose Fast Flow column and the Sephadex G-75 column for obtaining the purified homocysteine methyltransferase.
2. To verify whether protein peaks after chromatography contain homocysteine methyltransferase by applying Western Blot and Coomassie brilliant blue staining, then determine roughly the molecular weight according to Mark strip.
3. To determine the homocysteine concentration in serum by using Homocysteine Test Kit, replace reagent 2 with the mix liquor which is consisted of the purified homocysteine methyltransferase and glutamate dehydrogenase, then re-testing the homocysteine concentration of the same sample, at last to determine if the purified homocysteine methyltransferase has activity according to the result of two kinds of kits.
Results
1. The enzyme purified is what we needed through Western blot verification.
2. Compared with the Western Blot and Coomassie brilliant blue staining bands on Mark, the molecular weight of homocysteine methyltransferase monomer is 45kD.
3. we determined the homocysteine concentration in serum(n=20) separately by using the Homocysteine Test Kit and the kit that reagent 2 has been replaced; analyzed the result with matched t-test. Data are shown as mean±SD, the result of the Homocysteine Test Kit is 16.48±3.48, standard error is 0.77. the result of the kit that reagent 2 has been replaced is 17.19±4.10, standard error is 0.92. The correlation coefficient of the two method is 0.92, the P value of the correlation coefficient<0.05,so we concluded that the two method correlated extremely. the two method wasn't regarded significant because the P value of matched t-test>0.05.
Conclusion
1. The homocysteine methyltransferase was purified successfully, we established the method of purification that can be used on research and production.
2. The molecular weight of homocysteine methyltransferase that we purified monomer is 45kD.
3. The homocysteine methyltransferase that we purified has activity. It can be used to detect the concentration of homocysteine.
1.对含有同型半胱氨酸转甲基酶外源性基因的大肠埃希氏菌培养、收集含有同型半胱氨酸转甲基酶的母液。
2.分离提纯同型半胱氨酸转甲基酶。
3.判断提纯的同型半胱氨酸转甲基酶是否有活性,初步确定其活性的大小。
方法
1.将含有同型半胱氨酸转甲基酶外源性基因的大肠埃希氏菌接种至血平板,37℃培养过夜后接种于含氨苄青霉素的LB培养液中,37℃在摇床上培养至对数中期,加入IPTG诱导8小时之后离心弃上清留沉淀。用缓冲液混匀沉淀后用电动搅拌机5000转／分钟以破坏细菌,冰浴匀浆2分钟,15000g离心15分钟,弃沉淀取上清。上清液经过Sephadex CL-6B层析柱,DEAE Sepharose Fast Flow层析柱,Sephadex G-75层析柱之后得到提纯的同型半胱氨酸转甲基酶。
2.用Western Blot和考马斯亮蓝染色验证层析后的蛋白峰是否含有同型半胱氨酸转甲基酶,根据Mark的条带大概判断其kD值。
3.用金斯尔G系列同型半胱氨酸检测试剂盒测定20份血清的同型半胱氨酸浓度,再用提纯的同型半胱氨酸转甲基酶与谷氨酸脱氢酶组成的混合液替换试剂2,重新检测相同的20份血清同型半胱氨酸的浓度,根据两种试剂盒检测的数值来判断提纯的同型半胱氨酸转甲基酶是否有活性以及活性的大小。
结果
1.将提纯后的酶做Western Blot验证,证明是所需要的同型半胱氨酸转甲基酶。
2.与Western Blot和考马斯亮蓝染色上Mark的条带相比较,同型半胱氨酸转甲基酶单体的分子量为45kD。
3.利用原试剂盒和替换试剂2后的试剂盒分别测定相同的20份标本,对两组数据进行统计,用配对t检验,原试剂盒测定的均数为16.48±3.47,标准误为0.77；试剂2替换后的测定的均数为17.19±4.10,标准误为0.92。原试剂盒和试剂2替换之后的试剂盒测定值的相关系数为0.92,两种方法相关系数假设检验的P值<0.05,表明试剂2替换前后同型半胱氨酸的检测高度相关。配对t检验的P值>0.05,无统计学意义,说明替换前后两种试剂盒检测同型半胱氨酸无差异。
结论
1.成功提纯出了同型半胱氨酸转甲基酶,并建立了一整套提纯同型半胱氨酸转甲基酶的方案,可供科研、生产使用。
2.提纯同型半胱氨酸转甲基酶单体为45kD。
3.提纯的同型半胱氨酸转甲基酶有活性,可用于同型半胱氨酸浓度的检测。
Objective
1 To culture escherichia coli which contain exogenous genes of homocysteine methyltransferase and collect supernatant which contain homocysteine methyltransferase.
2 To separate and purify the homocysteine methyltransferase
3 To judge whether the purified homocysteine methyltransferase has activity or not; determining its activity preliminarily.
Methods
1. The escherichia coli which containing exogenous genes of homocysteine methyltransferase were inoculated in the blood agar. the blood agar were culture at 37℃overnight, then the escherichia coli were inoculated to Medium LB which contain ampicillin and cultured to mid-log phase in shake at 37℃. After adding IPTG to Medium LB for 8 hours, the liquid were centrifuged for obtaining precipitation. Precipitation were dissolved with buffer, then mixed with electric mixer(5000 r/min,2min) to destroy bacteria. The supernatant obtained after centrifugation (15000g,15min) were loaded on to the Sephadex CL-6B column, the DEAE Sepharose Fast Flow column and the Sephadex G-75 column for obtaining the purified homocysteine methyltransferase.
2. To verify whether protein peaks after chromatography contain homocysteine methyltransferase by applying Western Blot and Coomassie brilliant blue staining, then determine roughly the molecular weight according to Mark strip.
3. To determine the homocysteine concentration in serum by using Homocysteine Test Kit, replace reagent 2 with the mix liquor which is consisted of the purified homocysteine methyltransferase and glutamate dehydrogenase, then re-testing the homocysteine concentration of the same sample, at last to determine if the purified homocysteine methyltransferase has activity according to the result of two kinds of kits.
Results
1. The enzyme purified is what we needed through Western blot verification.
2. Compared with the Western Blot and Coomassie brilliant blue staining bands on Mark, the molecular weight of homocysteine methyltransferase monomer is 45kD.
3. we determined the homocysteine concentration in serum(n=20) separately by using the Homocysteine Test Kit and the kit that reagent 2 has been replaced; analyzed the result with matched t-test. Data are shown as mean±SD, the result of the Homocysteine Test Kit is 16.48±3.48, standard error is 0.77. the result of the kit that reagent 2 has been replaced is 17.19±4.10, standard error is 0.92. The correlation coefficient of the two method is 0.92, the P value of the correlation coefficient<0.05,so we concluded that the two method correlated extremely. the two method wasn't regarded significant because the P value of matched t-test>0.05.
Conclusion
1. The homocysteine methyltransferase was purified successfully, we established the method of purification that can be used on research and production.
2. The molecular weight of homocysteine methyltransferase that we purified monomer is 45kD.
3. The homocysteine methyltransferase that we purified has activity. It can be used to detect the concentration of homocysteine.
引文
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[26]Finkelstein JD, Mudd SH. Trans-sulfuration in mammals. The methionine-sparing effect of cystine. J Biol Chem,1967,242(5):873-880
[27]Kamely D, Littlefield JW, Erbe RW. Regulation of 5-methyltetrahydrofolate:homocysteine methyltransferase activity by methionine, vitamin B12, and folate in cultured baby hamster kidney cells. Proc Natl Acad Sci U S A,1973,70(9):2585-2589
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[29]Lee MB, Blunt JW, Lever M, et al. A nuclear-magnetic-resonance-based assay for betaine-homocysteine methyltransferase activity. Anal Biochem,2004,330(2):199-205
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[31]Uthus EO, Yokoi K, Davis CD. Selenium deficiency in Fisher-344 rats decreases plasma and tissue homocysteine concentrations and alters plasma homocysteine and cysteine redox status. J Nutr,2002,132(6):1122-1128
[32]Emmert JL, Webel DM, Biehl RR, et al. Hepatic and renal betaine-homocysteine methyltransferase activity in pigs as affected by dietary intakes of sulfur amino acids, choline, and betaine. J Anim Sci,1998,76(2):606-610
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[49]Lawson-Yuen A, Levy HL. The use of betaine in the treatment of elevated homocysteine. Mol Genet Metab,2006,88(3):201-207
[50]Yang Q, Botto LD, Erickson JD, et al. Improvement in stroke mortality in Canada and the United States,1990 to 2002. Circulation,2006,113(10):1335-1343
[51]Spence JD, Bang H, Chambless LE, et al. Vitamin Intervention For Stroke Prevention trial: an efficacy analysis. Stroke,2005,36(11):2404-2409
[52]Singh RH, Kruger WD, Wang L, et al. Cystathionine beta-synthase deficiency: effects of betaine supplementation after methionine restriction in B6-nonresponsive homocystinuria. Genet Med,2004,6(2):90-95
[53]Heil SG, Lievers KJ, Boers GH, et al. Betaine-homocysteine methyltransferase (BHMT): genomic sequencing and relevance to hyperhomocysteinemia and vascular disease in humans. Mol Genet Metab,2000,71(3):511-519
[54]Weisberg IS, Park E, Ballman KV, et al. Investigations of a common genetic variant in betaine-homocysteine methyltransferase (BHMT) in coronary artery disease. Atherosclerosis, 2003,167(2):205-214
[55]van Vlijmen-van Keulen CJ, Vahl AC, Hennekam RC, et al. Genetic linkage of candidate genes in families with abdominal aortic aneurysms? Eur J Vasc Endovasc Surg, 2003,26(2):205-210