一种东亚钳蝎碱性神经毒素的纯化和初步晶体学研究

经ＳｅｐｈａｄｅｘＧ－５０和ＳＰ－ＳｅｐｈａｄｅｘＣ－２５两次柱层析,从河南淅川马氏钳蝎毒素中分得一种碱性神经毒素ＢｍＫＭＩ８。等电聚聚焦和ＳＤＳ电泳显示单一组分,其ＰＩ为９．１,Ｍｒ为７１００,毒性测试结果表明,该组分对小白鼠有较强的毒性,对昆虫也有一定的毒性。已获得该毒素的两种晶全型的大单晶,并测定其空间群为Ｐ２１２１２１,晶胞参数为：ＢｍＫＭＩ８－Ａ：ａ＝３６．７Ａ,ｂ＝２６．６Ａ,ｃ＝５２     

具有中等毒性的马氏钳蝎神经毒素的纯化和初步晶体学研究

运用柱层析技术对产自淅川和常德的马氏钳蝎毒素进行分离纯化,得到８种哺乳动物神经毒素。运用制备型等电聚焦电泳技术,对常德样品中具有中等毒性的蝎神经毒素（ＢｍＫ５）进一步纯化,获得了高纯度样品。两个产地的蝎毒素ＢｍＫ５均已经成功地获得了大晶体。空间群均为Ｐ２１２１２１,晶胞参数分别为：ａ＝３８．４６埃,ｂ＝３７．２８埃,ｃ＝３６．９７埃（淅川）;ａ＝３８．４４埃,ｂ＝３７．５５埃,ｃ＝３６．８３埃（常德）。对两个产地的晶体分别收集了２．１埃（淅川）和１．６２埃（常德）分辨率的衍射数据。     

具有中等毒性的马氏钳蝎神经毒素的纯化和初步晶体学…

运用柱层析技术对产自淅川和常德的马氏钳蝎毒素进行分离纯化,得到８种哺乳动物神经毒素。运用制备型等电聚焦电泳技术,对常德样品中具有中等毒性的蝎神经毒素进一步纯化,获得了高纯度样品。两个产地的蝎毒素ＢｍＫ５均已经成功地获得了大晶体。空间群均为Ｐ２１２１２１,晶胞参数分别为：ａ＝３８．４６埃,ｂ＝３７．２８埃,ｃ＝３６．９７埃;ａ＝３８．４４埃,ｂ＝３７．５５埃,ｃ＝３６．８３埃。对两个产地的晶体分别收     

一种强毒性中华马氏钳蝎哺乳动物神经毒素新晶型的初步晶体学研究

经ＳｅｐｈａｄｅｘＧ－５０和Ｓｐ－ＳｅｐｈａｄｅｘＣ－２５两次柱层析,从常德马氏钳蝎毒素中分离得到一种强毒性的哺乳动物神经毒（Ｂｍｋｌ）,等电聚焦及ＳＤＳ－电泳显示它为单一组份,其ｐＩ为９．４４,ＭＷ为７．１ＫＤ．毒性试验结果表明,该组份对小白鼠的最小致死刘量为０．５μｇ／ｇ小白鼠,对昆虫及甲壳动物也有毒性．已获得该毒素的一种新晶型的大晶体。其空间群为Ｐ２１２１２,晶胞参数为：α＝８３．４６Ａ,ｂ＝４０．３６Ａ,ｃ＝２４．００Ａ,单位晶胞体积为８０８４２．６９Ａ,一个不对称单位含１个分子,已收集了分辨率为１．７５Ａ的数据．     

蒿属植物的染色体数目和核型研究（一）

本文报道了我国蒿属６种的染色体核型,它们的核型公式分别为：牡蒿（ＡｒｔｅｍｉｓｉａｊａｐｏｎｉｃａＴｈｕｎｂ．）２ｎ＝１８＝１２ｍ＋６ｓｍ（２ＳＡＴ）;西南牡蒿（Ａ．ｐａｒｖｉｆｌｏｒａＢｕｃｂ．－Ｈａｍ．ｅｘＲｏｘｂ．）２ｎ＝３６＝３０ｍ＋４ｓｍ＋２ｓｔ（６ＳＡＴ）;多花蒿（Ａ．ｍｙｒｉａｎｔｈａＷａｌｌ．ｅｘＢｅｓｓ．）２ｎ＝３６＝３０ｍ＋６ｓｍ：牛尾蒿（Ａ．ｄｕｂｉａＷａｌｌ．ｅｘＢｅｓｓ．）２ｎ＝３６＝２８ｍ＋８ｓｍ（２ＳＡＴ）;野艾蒿（Ａ．ｌａｖａｎｄｕｌａｅｆｏｌｉａＤＣ．）２ｎ＝５４＝４２ｍ＋１２ｓｍ;荚毛蒿（Ａ．ｖｅｌｕｔｉｎａＰａｍｐ．）２ｎ＝５４＝３６ｍ＋１８ｓｍ．     

吉林省产5种百合的核型研究

报道了吉林省产５种百合科植物的染色体数目和核型：①毛百合Ｌｉｌｉｕｍ ｄａｕｒｉｃｕｍ Ｋｅｒ．－Ｇｅｗ１．２ｎ＝２４＝２ｍ（２ＳＡＴ）＋２ｓｍ（２ＳＡＴ）＋８ｓｔ（２ＳＡＴ）＋１２ｔ（２ＳＡＴ）;②有斑百合Ｌ．ｃｏｎｃｏｌｏｒ Ｓａｌｉｓｂ．ｖａｒ．ｂｕｓｃｈｉａｎｕｍ（Ｌｏｄｄ．）Ｂａｋｅｒ ２ｎ＝２４＝２ｍ（２ＳＡＴ）＋４ｓｍ（４ＳＡＴ）＋６ｓｔ（２ＳＡＴ）＋１２ｔ;③兰州百合Ｌ．ｄａｖｉｄ     

B链氨端去－肽（B1）羧端去五肽（B26—30）猪胰岛素的初步晶体学研究

应用悬滴气相扩散法在含６％氯化钠和１２．５％丙酮的柠檬酸缓冲体系中,获得可供Ｘ射线结构分析用的Ｂ链氨端去－肽（Ｂ１）羧端去五肽（Ｂ２６－３０）猪胰岛素单晶体。晶体属四方晶系,空间群为Ｐ４１２２或Ｐ４３２２,晶胞参数为：ａ＝ｂ＝３６．０Ａ,ｃ＝１２０．０Ａ,α＝β＝γ＝９０度。单位晶胞中每个结晶学不对称单位含有２个ＤｅｓＢ１－ＤＰＩ分子。     

系统感染TMV的番茄叶胞外β-1,3-葡聚糖酶

系统感染ＴＭＶ （ｔｏｂａｃｃｏ ｍ ｏｓａｉｃ ｖｉｒｕｓ）的番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．）叶胞外蛋白提取液经冰冻干燥浓缩、－ ２０℃丙酮沉淀、ＣＭ－Ｓｅｐｈａｄｅｘ Ｃ－２５离子交换层析、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析和Ｓｅｐｈａｄｅｘ Ｇ－７５凝胶层析纯化,获得ＰＡＧＥ均一的β－１,３－葡聚糖酶．ＳＤＳ－ＰＡＧＥ证明,它包含分子量为３６ ｋＤ 和２７ ｋＤ的两个同工酶．以昆布多糖为底物,酶的最适ｐＨ 在４．８—５．２之间,在ｐＨ ４—８稳定;酶的最适温度在３０—４０℃之间,在４０℃保温１ｈ 后酶活性不变;Ｋｍ 值为９．２ ｍ ｇ／ｍ Ｌ．在系统感染ＴＭＶ 的番茄叶胞外蛋白提取液中,有分子量为２２ ｋＤ、２７ ｋＤ和３６ ｋＤ的３个β－１,３－葡聚糖酶同工酶     

B链氨端去－肽（B1）羧端去五肽（B26—30）猪胰岛素的初步晶体学研究

应用悬滴气相扩散法在含６％氯化纳和１２．５％丙酮的柠檬酸缓冲体系中,获得可供Ｘ射线结构分析用的Ｂ链氨端去一肽（Ｂ１）羧端去五肽（Ｂ２６—３０）猪胰岛素（ＤｅｓＢ１－ＤＰＩ）单晶体。晶体属四方晶系,空间群为Ｐ４１２２或Ｐ４３２２,晶胞参数为：ａ＝ｂ＝３６．０Ａ,ｃ＝１２０．０Ａ,ａ＝β＝γ＝９０°。单位晶胞中每个结晶学不对称单位含有２个ＤｅｓＢ１—ＤＰＩ分子。     

东亚钳蝎神经毒素在大肠杆菌中的表达

以山西风陵渡东亚钳蝎（ＢｕｔｈｕｓｍａｒｔｅｎｓｉＫａｒｓｃｈ）的尾腺总ＲＮＡ为模板,根据已知的蝎神经毒素保守氨基酸序列设计引物,利用ＰＣＲ技术,扩增并克隆了两个蝎神经毒素基因．序列分析表明,由两个基因导出的氨基酸序列（ＢｍＫＭｍ１和ＢｍＫＭｍ２）与已知的蝎神经毒素ＢｍＫⅠ、ＢｍＫⅡ、ＢｍＫⅢ、ＢｍＫＭ１、ＢｍＫＭ９有很高的同源性．将ＢｍＫＭｍ２基因重组到大肠杆菌分泌型表达载体ｐＥｘＳｅｃ１中进行表达．ＳＤＳ－ＰＡＧＥ证明表达产物被分泌到细胞周间质及培养液中．经ＩｇＧ－Ｓｅｐｈａｒｏｓｅ纯化后的蛋白质注射小白鼠表明表达产物有生物学活性     

马氏钳蝎毒昆虫类神经毒素的分离、纯化及性质研究

经Sephadex G-50,sp-Sephadex C-25二步柱层析法,从山东马氏钳蝎(Bu-thus martensii Karch)粗毒中分离出四种对美洲(虫非)蠊有强直麻痹反应的毒性蛋白组份。其中二个组分在SDS聚丙烯酰胺电泳和等电聚焦电泳上均呈现单一区带,命名为BmK IT-Ⅰ,BmK IT-Ⅱ其pI分别为8.2和8.4,分子量分别为8400和7560。同时还分析了二个组份的氨基酸组成。经DABITC/PITC双偶合法测定了BmKIT-Ⅰ和BmK IT-Ⅱ的N端部份氨基酸排列顺序,它们分别为H_2NVal.Arg.Asp.Ala……H_2NVal.Arg.Asp.Gly……。 电生理学研究表明,纯化的BmK IT-I(1×10~(-5)g/ml)对(虫非)蠊腹Ⅵ神经节的突触传递有阻断作用,阻断后用生理溶液洗,则突触传递可恢复。从同一蝎毒粗毒中分离纯化的哺乳动物类神经毒素BmKⅢ在浓度高出100倍(1×10~(-3)g/ml)时也可以阻断(虫非)蠊腹Ⅵ神经节的突触传递,但用生理溶液冲洗没有观察到恢复。     

Binding characteristics of BmK I, an alpha-like scorpion neurotoxic polypeptide, on cockroach nerve cord synaptosomes.

In this study, the binding characteristics of BmK I, an alpha-like neurotoxic polypeptide purified from the venom of the Chinese scorpion Buthus martensi Karsch, were investigated on rat brain and cockroach nerve cord synaptosomes. The results showed that BmK I can bind to a single class of noninteracting binding sites on cockroach nerve cord synaptosomes with medium affinity (Kd = 16.5 +/ - 4.4 nM) and low binding capacity (Bmax = 1.05 +/- 0.23 pmol/mg protein), but lacks specific binding on rat brain synaptosomes. BmK AS, BmK AS-1 (two novel sodium channel-blocking ligands), BmK IT (an excitatory insect-selective toxin) and BmK IT2 (a depressant insect-selective toxin) from the same venom were found to be capable of depressing BmK I binding in cockroach nerve cord synaptosomes, which might be attributed to either allosteric modulation of voltage-gated Na+ channels by these toxic polypeptides or partial overlapping between the receptor binding sites of BmK I and these toxins. This thus supported the notion that alpha-like scorpion neurotoxic polypeptides bind to a distinct receptor site on sodium channels, which might be similar to the binding receptor site of alpha-type insect toxins, and also related to those of BmK AS type and insect-selective scorpion toxins on insect sodium channels.     

BmK AS在大鼠脑和蟑螂神经索突触体上的药理结合特性

用ＩＮＤＯＧＥＮ法对一个新型东亚钳蝎活性多肽ＢｍＫ ＡＳ进行了＾１２５Ｉ标记。分别在哺乳动物和昆虫标本上观察了标记物的药理结合特性,结果表明ＢｍＫ ＡＳ大大鼠脑突触体标本上有单一非协同结合位点,其平衡解离常数Ｋｄ与最大结合容量Ｂｍａｘ分别为１．４９ｎｍｏｌ／Ｌ,１．３９ｎｍｏｌ／ｇ。此外,ＢｍＫ ＡＳ在蟑螂神经索突触体标本同样也只有单一非协同结合位点,其平均解离常数Ｋｄ与最大结合容量Ｂｍａｘ分别为     

Expression and Purification of the BmK M1 Neurotoxin from the Scorpion Buthus martensii Karsch

The gene encoding a neurotoxin (BmK M1) from the scorpion Buthus martensii Karsch was expressed in Saccharomyces cerevisiae at a high level with the alcohol dehydrogenase promoter. SDS–PAGE of the culture confirmed expression and showed secretion into medium from yeast. Recombinant BmK M1 was purified rapidly and efficiently by ion exchange and gel filtration chromatography to homogeneity, produced a single band on tricine–SDS–PAGE, and processed the homologous N-terminus. Amino acid analysis and N-terminal sequencing demonstrated that the recombinant toxin was processed correctly from the α-mating factor leader sequence and was chemically identical to the native form. The expressed recombinant BmK M1 was toxic for mice, which indicated that it was biologically active. Quantitative estimation showed that recombinant BmK M1 had an LD₅₀ similar to that of the native toxin.     

Cloning and high-level expression of scorpion toxin BmKITa1 in Escherichia coli and insect cells

BmK ITa1 cDNA was cloned and highly expressed in E. coli and insect cell. SDS-PAGE and western blot analysis revealed that subunit molecular weight of expression products is about 40 kDa and 10 kDa respectively. The expression product purified by a Ni(2+)-IDA-sepharose 6B column was toxic for insect, which indicated that it was biologically activity. Furthermore, Quantitative estimation show that the biological activity of recombinant BmK ITa1 from Tn cells was more powerful than from E. coli.     

Cloning,co-expression with an amidating enzyme,and activity of the scorpion toxin BmK ITa1 cDNA in insect cells

BmK ITa1 is an insect-specific neurotoxin from the Chinese scorpion Buthus martensi Karsch (Bmk). We succeeded in obtaining biologically active recombinant BmK ITa1 protein by simultaneous expression in insect cells of BmK ITa1 cDNA with an amidating enzyme expressed by the rat peptidylglycine α-amidating monooxygenase (PAM) gene. We investigated the insecticidal efficacy of recombinant BmK ITa1/W (without coexpression of PAM), and of BmK ITa1/A (with coexpression of PAM) in 5th instar Bombyx mori, by injecting these recombinant toxins into larvae. The lethal time for 50% of larvae (LT₅₀) was 9 h for BmK ITa1/A and 17 h for BmK ITa1/W. At 19 h after injection all of the larvae exposed to BmK ITa1/A had been killed, whereas only half of the larvae exposed to BmK ITa1/W had been killed. These results show that the simultaneous expression of an amidating enzyme can result in apparently higher insecticidal activity of BmK ITa1.     

Isolation of Multiple Subspecies of Bacillus thuringiensis from a Population of the European Sunflower Moth, Homoeosoma nebulella

Five subspecies of Bacillus thuringiensis were isolated from dead and diseased larvae obtained from a laboratory colony of the European sunflower moth, Homoeosoma nebulella. The subspecies isolated were B. thuringiensis subspp. thuringiensis (H 1a), kurstaki (H 3a3b3c), aizawai (H 7), morrisoni (H 8a8b), and thompsoni (H 12). Most isolates produced typical bipyramidal crystals, but the B. thuringiensis subsp. thuringiensis isolate produced spherical crystals and the B. thuringiensis subsp. thompsoni isolate produced a pyramidal crystal. Analysis of the parasporal crystals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the crystals from the B. thuringiensis subsp. kurstaki and aizawai isolates contained a protein of 138 kDa whereas those from B. thuringiensis subsp. morrisoni contained a protein of 145 kDa. The crystals from B. thuringiensis subsp. thuringiensis contained proteins of 125, 128, and 138 kDa, whereas those from B. thuringiensis subsp. thompsoni were the most unusual, containing proteins of 37 and 42 kDa. Bioassays of purified crystals conducted against second-instar larvae of H. nebulella showed that the isolates of B. thuringiensis subspp. aizawai, kurstaki, and thuringiensis were the most toxic, with 50% lethal concentrations (LC(inf50)s) of 0.15, 0.17, and 0.26 (mu)g/ml, respectively. The isolates of B. thuringiensis subspp. morrisoni and thompsoni had LC(inf50)s of 2.62 and 37.5 (mu)g/ml, respectively. These results show that a single insect species can simultaneously host and be affected by a variety of subspecies of B. thuringiensis producing different insecticidal proteins.     

Expression and purification of the BmK M1 neurotoxin from the scorpion Buthus martensii Karsch

The gene encoding a neurotoxin (BmK M1) from the scorpion Buthus martensii Karsch was expressed in Saccharomyces cerevisiae at a high level with the alcohol dehydrogenase promoter. SDS-PAGE of the culture confirmed expression and showed secretion into medium from yeast. Recombinant BmK M1 was purified rapidly and efficiently by ion exchange and gel filtration chromatography to homogeneity, produced a single band on tricine-SDS-PAGE, and processed the homologous N-terminus. Amino acid analysis and N-terminal sequencing demonstrated that the recombinant toxin was processed correctly from the alpha-mating factor leader sequence and was chemically identical to the native form. The expressed recombinant BmK M1 was toxic for mice, which indicated that it was biologically active. Quantitative estimation showed that recombinant BmK M1 had an LD(50) similar to that of the native toxin.     

Purification,crystallization and initial structural solution of a new alpha-like toxin with cardiac toxicity from scorpion Buthus martensii Karsch

An alpha-like toxin named BmK M7 active on both mammals and insects has been purified from the venom of scorpion Buthus martensii Karsch (BmK) recently. The electrophysiological experiments showed that M7 can bind to human cardiac Na+-channel and modify its normal properties, hence can be considered as a cardiotoxin. Single crystals of M7 have been obtained by hanging-drop vapor diffusion method using ammonium sulfate as precipitant in Tris-HCl buffer at pH 8.5. A data set to 1.40 A resolution was collected using synchrotron radiation and CCD detector in Photon Factory in Japan. Data analysis showed that the crystals belonged to space group P3(1)21/P3(1)21, with cell dimensions a=b=32.76 A, c=176.82 A. Assuming two molecules per asymmetric unit, the Vm value is 1.92 A3/Da. The initial structural analysis was carried out by molecular replacement, which showed the correct space group (P3(1)21), and the orientations and positions of the two molecules in the asymmetric unit.