点状产气单胞菌脯氨酰内肽酶在大肠杆菌中的高效表达

采用ＰＣＲ分段克隆法将点状产气单胞菌点状亚种（Ａｅｒｏｍｏｎａｓ ｐｕｃｔａｔａ ｓｕｂｓｐ．ｊｐｕｃｔａｔａ）脯氨酰内肽酶（ｐｒｏｌｙｌ ｅｎｄｏｐｅｐｔｉｄａｓｅ,ａｐＰＥＰ）的编码区基因分成３段扩增并拼接成编码６９０个氨基酸的完整基因ａｐＰＥＰ,将其克隆在表达载体ｐＢＬ和ｐＫＫＨ上,构建成温度和ＩＰＴＧ诱导型高效表达ａｐＰＥＰ的重组大肠杆菌ＢＬ２１／ｐＢＬ－ＰＥＰ和ＢＬ２１／ｐＫＫＨ－ＰＥＰ     

大鼠催乳素基因真核细胞可表达性质粒的构建及应用研究

７３５ｂｐ的ＰＲＬｃＤＮＡ片段从质粒ＰＲＬ－ＳＰ６５＃１中回收后,用粘性末端连接法将其重组到真核表达载体ｐｃＤＮＡ３上,筛选出正向连接重组体ｐｃＤＮＡ３－ＰＲＬＳ和反向连接重组体ｐｃＤＮＡ３－ＰＲＬＡＳ。将重组体ｐｃＤＮＡ３－ＰＲＬｓ和空载体ｐｃＤＮＡ３分别转入ＮＩＨ３Ｔ３细胞系,用Ｇ４１８筛选出阳性细胞后与未转染的ＮＩＨ３Ｔ３细胞在加Ｅ２和不加Ｅ２的情况下,用原位杂交的方法,分别用ＰＲＬｃＤＮＡ探针和原癌基因ｃ－Ｈ－ｒａｓｃＤＮＡ探针进行检测,未转染的ＮＩＨ３Ｔ３细胞在加Ｅ２和不加Ｅ２时都几乎无催乳素基因的表达,同样,转入空载体的ＮＩＨ３Ｔ３细胞也无ＰＲＬ的表达,而转入重组体ｐｃＤＮＡ３－ＰＲＬＳ的ＮＩＨ３Ｔ３细胞则有大量的ＰＲＬ基因的表达,与对照组相比有显著差异（Ｐ＜０．０１）。正常和转入空载体的ＮＩＨ３Ｔ３细胞有一定程度的原癌基因ｃ－Ｈ－ｒａｓ的表达,当分别加入Ｅ２和转入重组体ｐｃＤＮＡ３－ＰＲＬＳ后,ＮＩＨ３Ｔ３细胞中的ｃ－Ｈ－ｒａｓ基因表达水平都显著升高（Ｐ＜０．０５）。     

人神经生长因子低亲和力受体（p~（75）NGFR）cDNA的克隆及其在神经细胞中表达活性的初步研究

利用酸性异硫氰酸胍－酚－氯仿一步法从人胎儿基底前脑中提取总ＲＮＡ,用逆转录与聚合酶链反应相结合的ＲＴ－ＰＣＲ法,扩增出人神经生长因子低亲和力受体ｐ~（７５）ＮＧＦＲ基因ｃＤＮＡ,在限制性内切酶ＳｍａⅠ存在下的连接体系中,将扩增出的ｃＤＮＡ片段克隆入ｐＵＣ１２的ＳｍａⅠ位或,经限制性内切酶ＥｃｏＲⅠ和ＰｓｔⅠ酶切鉴定是否插入以及ＨｉｎｄⅢ酶切鉴定方向。将重组质粒中的ｐ~（７５）ＮＧＦＲ的ｃＤＮＡ再次亚克隆至ｐＵＣ１２载体中后,以其双链ＤＮＡ为模板,用末端终止法测出其全部核苷酸顺序,证实其核苷酸编码的ｐ~（７５）ＮＧＦＲ除两个碱基突变外,其余与文献完全一致。完整的ｐ~（７５）ＮＧＦＲ的ｃＤＮＡ分两步克隆到逆转录病毒表达载体ｐＸＴ－１,经ＰＡ３１７包装细胞株体外包装后、收集病毒上清转染条件不死性大鼠小脑神经细胞系Ｒ２．初步结果表明转染了ｐ~（７５）ＮＧＦＲ的Ｒ２细胞株去除ＮＧＦ培养时出现程序化死亡的典型特征梯型ＤＮＡ带。     

凝血酶激活的单链尿激酶型纤溶酶原激活剂的构建、表达、纯化和性质研究

用Ｋｕｎｋｅｌ突变法,将单链尿激酶型纤溶酶原激活剂（ｓｃｕ－ＰＡ）ｃＤＮＡ基因中编码Ｐｒｏ１５５—Ｌｙｓ１５８的片段定点突变,并将此突变的ｓｃｕ－ＰＡ（ｔｓｃｕ－ＰＡ）的ｃＤＮＡ克隆到表达载体ｐＣＭ－β－ｎｅｏ中,与ｐＣＭ－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ－细胞．获得的稳定表达株在无血清培养基中２４ｈ的表达量为６２０ＩＵ／１０６细胞．经锌离子螯合Ｓｅｐｈａｒｏｓｅ亲和层析得到ｔｓｃｕ－ＰＡ纯品．ＳＤＳ－ＰＡＧＥ显示ｔｓｃｕ－ＰＡ分子量为５３ｋＤ左右,与预期的结果相符．ｔｓｃｕ－ＰＡ是由凝血酶激活而不是由纤溶酶激活,但激活后也能转变为双链分子（ｔｃｕ－ＰＡ）．ｔｓｃｕ－ＰＡ仍保持了ｓｃｕ－ＰＡ的血纤维蛋白亲和性．酶动力学研究表明,激活后的ｔｓｃｕ－ＰＡ水解Ｓ２４４４的Ｋｍ和Ｋｃａｔ值与高分子量尿激酶（ＨＵＫ）相似．体外溶栓实验结果表明,ｔｓｃｕ－ＰＡ可以选择性地溶解富含凝血酶的血凝块,对贫凝血酶的血凝块作用不大     

鹅源腺病毒基因组DNA物理图谱的构建

将鹅源腺病毒Ｙ８１Ｇ４株全基因组ＤＮＡ的ＨｉｎｄⅢ酶切片段分别插入质粒ｐＵＣ１８,成功构建了全基因组ＤＮＡ文库。在此基础上,将重组质粒携带的插入片段切出、回收并分别用地高辛标记后作为探针,与经限制酶ＢａｍＨⅠ、ＥｃｏＲⅠ、ＰｓｔⅠ、ＥｃｏＲⅤ消化的病毒基因组ＤＮＡ进行Ｓｏｕｔｈｅｒｎ Ｂｌｏｔｔｉｎｇ,杂交结果经比较综合后获得了该病毒基因组ＤＮＡ的ＨｉｎｄⅢ、ＢａｍＨⅠ、ＥｃｏＲⅠ、ＰｓｔⅠ、Ｅｃ     

小鼠金属硫蛋白 Ⅰ cDNA编码序列的克隆及其在昆虫细胞中的表达

将质粒ｐＢＸ－ＭＴ上的小鼠ＭＴ－ⅠｃＤＮＡ片段切下作为模板,通过ＰＣＲ方法删除该片段的非编码序列,将编码序列克隆到质粒ｐＢＳ－ＳＫ中,经ＤＮＡ序列测定后证明其克隆序列正确．再将ＭＴ－ⅠｃＤＮＡ编码序列插入到转移载体ｐＢａｃＰＡＫ８的ＢａｍＨⅠ和ＥｃｏＲⅠ位点之间,通过磷酸钙／ＤＮＡ共转染方法将其导入昆虫细胞Ｓｆ９中,以Ｗｅｓｔｅｒｎｂｌｏｔ和ＤｏｔＥＩＡ方法对表达产物进行了检测,表达量为１ｍｇ／Ｌ     

枯草芽孢杆菌中性内切β-甘露聚糖酶的纯化及性质

三草芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＢＭ９６０２产生的中性内切β－甘露聚糖酶（ｅｎｄｏ－β－１,４－Ｄ－ｍａｎｎａｎ ｍａｎｎａｎｏｈｙｄｒｏｌａｅｓ,ＥＣ,３．２．１．７８）经硫酸铵分级沉淀、ＤＥＡＥ－纤维素（ＤＥ２２）离子交换柱层析,得到电泳纯的样品,提纯了４５．５倍,收率为５．９％。用ＳＤＳ－ＰＡＧＥ测得该酶的分子量为３５ｋＤ。用ＰＡＧＥＩＥＦ测得其等电点ｐⅠ为４．５。酶反应的     

牛病毒性腹泻病毒基因组cDNA文库的构建

以牛病毒性腹泻病毒（ＢＶＤＶ）┥ｎＬ株的基因组ＲＮＡ为模板,经逆向转录酶作用,合成第一链ｃＤＮＡ,再以ＲＮａｄｓｅ／Ｈ与ＤＮＡ聚合酶Ｉ联合作用合成ｄｓｃＤＮＡ,并以ｄＣ同聚物尾化。ｐＵＣ８ＤＮＡ在Ｐｓｔ Ｉ酶解后,以ｄＧ同聚物尾化,两者退火构成重组质粒,转化到Ｅ．ｃｏｌｉ ＪＭ１０１受体菌中,另以γ－＾３２Ｐ－ＡＴＰ标记ＢＶＤＶ ＲＮＡ制备探针,通过菌落原位杂交筛选重组子。酶切分配表明重组质粒插入     

日本血吸虫26kD抗原基因在BCG中的表达

研究了外源基因日本血吸虫２６ｋＤ抗原（Ｓｊ２６ＧＳＴ）在卡介苗（ｂａｃｉｌｕｓＣａｌｍｅｔｅ－Ｇｕｅｒｉｎ,ＢＣＧ）、耻垢分枝杆菌（Ｍ．ｓｍｅｇｍａｔｉｓ）和大肠杆菌（Ｅ．ｃｏｌｉ）中的表达．运用重组ＤＮＡ和聚合酶链反应（ＰＣＲ）等分子生物学技术,以表达Ｓｊ２６ＧＳＴ的Ｅ．ｃｏｌｉｐＧＥＸ衍生质粒为模板,经ＰＣＲ得到编码Ｓｊ２６ＧＳＴ的全长ｃＤＮＡ片段．将其按正确的阅读框顺序,克隆到人结核杆菌热休克蛋白（ｈｅａｔｓｈｏｃｋｐｒｏｔｅｉｎ,ＨＳＰ）７０的启动子下游,再将ＨＳＰ７０启动子和Ｓｊ２６ＧＳＴ基因一起亚克隆到Ｅ．ｃｏｌｉ－分枝杆菌穿梭质粒ｐＢＣＧ－２０００中,得到Ｅ．ｃｏｌｉ－分枝杆菌穿梭表达质粒ｐＢＣＧ－Ｓｊ２６．ｐＢＣＧ－Ｓｊ２６电转化入ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓｍｃ２１５５中表达Ｓｊ２６ＧＳＴ抗原,所表达的天然重组Ｓｊ２６ＧＳＴ（ｒＳｊ２６ＧＳＴ）为可溶性蛋白,在ＳＤＳ－ＰＡＧＥ上分子量为２６ｋＤ处可见明显的表达蛋白带．其表达量分别占ＢＣＧ和Ｍ．ｓｍｅｇｍａｔｉｓ菌体总蛋白的１５％和１０％．可见,Ｓｊ２６ＧＳＴ基因能在ＢＣＧ中高效表达．     

中国大陆若干群体的黑果蝇的线粒体DNA多态性研究

本文研究了果蝇Ｄ．ｖｉｒｉｌｉｓ种群Ｄ．ｖｉｒｉｌｉｓ线粒体ＤＮＡ（ｍｉｔｏｃｈｏｎｄｒｉａｌＤＮＡ,ｍｔＤＮＡ）的多态性。用９种限制性内切酶ＸｂａⅠ,ＥｃｏＲⅠ,ＰｓｔⅠ,ＨｉｎｄⅢ,ＢｇｌⅡ,ＳａｃⅠ,ＳｃａⅠ,ＥｃｏＲＶ和ＰｕｖⅡ,对青岛、南京、上海、宁波与泉州５个Ｄ．ｖｉｒｉｌｉｓ群体的ｍｔＤＮＡ进行了限制性片段长度多态性（ｒｅｓｔｒｉｃｔｉｏｎｆｒａｇｍｅｎｔｓｌｅｎｇｔｈｐｏｌｙｍｏｒｐｈｉｓｍ,ＲＦＬＰ）的研究。在５群体中,发现５种不同的酶切图谱,它们彼此之间的遗传差异π为０．４６％－１．７６％,群体内遗传差异πｉｊ为０．００％－０．３３％,群体间的差异ｄｘｙ,为０．００％－０．８２％。分布于中国大陆的Ｄ．ｖｉｒｉｌｉｓ的群体间遗传差异在总遗传差异中所占比例γｓｔ值为２４．６２％。我们发现,Ｄ．ｖｉｒｉｌｉｓ的栖息环境对ｍｔＤＮＡ的遗传变异有十分明显的影响,而不同地理纬度的群体之间其遗传距离并无倾群（ｃｌｉｎｅ）表现。     

Xanthones from Swertia punctata

Isolation of 1-O-primeverosyl-3,8-dihydroxy-5-methoxyxanthone and 1-O-gentiobiosyl-3,7-dimethoxy-8-hydroxyxanthone, along with five known xanthones, isobellidifolin, methylbellidifolin, isoswertianin, methylswertianin and norswertianin-1-O-beta-D-glucoside, from the roots of Swertia punctata is reported. In the aerial parts four xanthones, bellidifolin, methylbellidifolin, swertianolin and mangiferin, and flavone-C-glucoside, isoorientin were identified. The chemotaxonomic and pharmacological significance of these results is discussed.     

X-Linked dominant chondrodysplasia punctata

Summary X-linked dominant chondrodysplasia punctata is a syndrome consisting of skeletal, ocular, and cutaneous anomalies with asymmetric involvement of the body. The skin lesions, the hallmark of this condition, are distributed in a linear or blotchy pattern and include congenital ichthyosiform erythroderma, systematized atrophoderma mainly involving the hair follicles, and circumscribed alopecia. The remaining scalp hair is in part normal and in part irregularly twisted and coarse. The eyebrows and lashes are sparse. The nails may be flattened and split into layers. Thirty-five cases of this new syndrome are reviewed, and an additional observation is reported. The ratio of females to males is 36:0. The concept of X-linked dominant chondrodysplasia punctata has been suggested, and it has been postulated that there is a connection between the mosaic phenotype and the limitation to the female sex. Both facts would be explained by an X-linked gene giving rise to a pattern of lyonization in females, and lethal in hemizygous males. The classification of chondrodysplasia punctata thus includes three forms: the rhizomelic type, the Conradi-Hünermann type, and the X-linked dominant type. Two of these, the rhizomelic type and the X-linked dominant type, are well-defined entities. Whether the Conradi-Hünermann type, after separation of the X-linked form, is still heterogeneous, remains to be determined.     

Interrenal responses to high ambient temperature in soft-shelled turtle, Lissemys punctata punctata

An exposure to ambient temperature of 25 degrees C had no perceptible effect on interrenal function but further increase of temperature to 35 degrees C caused nuclear hypertrophy with increase of nuclear diameter, RNA concentration, acid phosphatase and alkaline phosphatase activities, accompanied by quantitative depletions of cholesterol (free, esterified and total) and ascorbic acid levels in the interrenal gland of the soft-shelled turtle Lissemys p. punctata. Similar manifestations of stimulation, except in the nucleus, were marked after exposure to 38 degrees C, but the degree of response in respect of esterified and free cholesterol levels was higher at 38 degrees C than at 35 degrees C. Moreover, withdrawal of 38 degrees C temperature and subsequently maintaining at 25 degrees C for 15 days showed reverse manifestations to those of 35 degrees C/38 degrees C, leading to a tendency towards normalcy. It is suggested that high a ambient temperature of 35 degrees C significantly stimulates interrenal function of Lissemys turtles, but further increase of 38 degrees C does not cause further overall stimulation, and withdrawal of higher temperature (38 degrees C) shows a tendency towards normalcy. It is also suggested that (a) high ambient temperature causes thermal stress, (b) it is reversible and (c) it acts on interrenal activity presumably via CRF-ACTH-axis in turtles.     

Thyroid responses to altered photoperiod in the soft-shelled turtle Lissemys punctata punctata bonnoterre

The aim of the current investigation was to investigate the effect of photoperiod on thyroid activity in soft-shelled turtles (Lissemys punctata punctata). Thirty days exposure of short photoperiod with 2L:22D increased relative weight, follicular epithelial height and peroxidase activity of the thyroid gland; whereas exposure of long photoperiod with 22L:2D for 30 days showed reversed changes to those of the short photoperiod in adult female turtles. These findings indicate that short photoperiod stimulates thyroid activity and long photoperiod inhibits its activity in soft-shelled turtles. It is suggested that photoperiod exerts its action on thyroid activity presumably via gonads and/or pineal-gonadal axis in turtles.     

Seasonal Patterns of Adrenomedullary Hormones and Glycemia in the Soft-Shelled Turtle Lissemys punctata punctata

The aim of the present study was to acertain the seasonal pattern of adrenomedullary hormones and of glycemia in Lissemys turtles. Both the norepinephrine and epinephrine concentrations as well as blood glucose levels varied seasonally which began to rise from February, became maximum during April-May (early summer), declined during June-September (late summer) and were extremely low subsequently (October-January). The seasonal adrenomedullary hormonal and glycemic cycles however do not coincide with the annual ovarian cycle, thereby indicating that the adrenomedullary and glycemic cycles are out of phase with the ovarian cycle in turtles. The possible mechanisms of seasonality of the adrenal medulla and glycemia are discussed.     

Thyroidal modulation following hypo- and hyper-thermia in the soft-shelled turtle Lissemys punctata punctata Bonnoterre

The aim of the current study was to investigate the effects of ambient temperature on thyroid activity of the soft-shelled turtle (Lissemys punctata pucntata). Turtles exposed to low ambient temperature (10 degrees C for 15 days) showed a significant decrease in relative thyroid weight, follicular cell size (cell became squamous from cuboidal type) and epithelial height in both the peripheral and central follicles of the gland, with the appearance of homogeneous colloid materials in the follicular lumen. Thyroid peroxidase activity declined significantly. In contrast, high ambient temperatures (32/34 degrees C for 15 days) caused reverse changes to those observed after exposure with low ambient temperature. No significant difference was marked in thyroid activity between 32 and 34 degrees C temperature treatments. The findings provide evidence that low ambient temperature inhibits thyroid activity and high ambient temperature stimulates the gland activity in soft-shelled turtles. Ambient temperature acts presumably via the hypothalamo-hypophysial (TRF-TSH) axis which in turn alters thyroid function in turtles.     

Seasonal Patterns of Adrenomedullary Hormones and Glycemia in the Soft-Shelled Turtle Lissemys punctata punctata

The aim of the present study was to acertain the seasonal pattern of adrenomedullary hormones and of glycemia in Lissemys turtles. Both the norepinephrine and epinephrine concentrations as well as blood glucose levels varied seasonally which began to rise from February, became maximum during April–May (early summer), declined during June–September (late summer) and were extremely low subsequently (October–January). The seasonal adrenomedullary hormonal and glycemic cycles however do not coincide with the annual ovarian cycle, thereby indicating that the adrenomedullary and glycemic cycles are out of phase with the ovarian cycle in turtles. The possible mechanisms of seasonality of the adrenal medulla and glycemia are discussed.     

Lysimachia punctata L. in Schlesien

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