精制狂犬病疫苗纯化方法的比较研究

地鼠肾细胞狂犬病疫苗原液经１００ ｋＤ 膜浓缩 ３０ 倍,分别选用（１）ＤＥＡＥ Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ离子交换层析法;（２）Ｓｅｐｈａｃｒｙ１ Ｓ－２００ ＨＲ 分子筛选层析法;（３）二次蔗糖等密度区带离心法对其进行纯化。用此三种方法各试制３ 批精制疫苗,结果表明,经ＤＥＡＥ Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ离子交换层析纯化后疫苗总蛋白含量减少９９％ 以上,抗原比活性提高１５９ 倍,抗原回收率达５０％ ,纯化疫苗以ＮＩＨ 法效力测定平均为５．４ ＩＵ／２ｍ ｌ;经Ｓｅｐｈａｃｒｙ１ Ｓ－２００ＨＲ 分子筛层析纯化后疫苗总蛋白含量减少 ９８％ 以上,抗原比活性提高４１ 倍,抗原回收率达６３％ ,纯化疫苗效力平均为６．２５ ＩＵ／２ｍ ｌ;经一次蔗糖等密度区带离心法纯化后疫苗总蛋白含量减少９８％ 以上,抗原比活性提高３２１ 倍,抗原回收率达４３％ ,纯化疫苗效力平均为６．１８ ＩＵ／２ｍ ｌ,三种纯化疫苗均符合Ｗ ＨＯ 规程要求。     

链球菌溶血素“O”纯化的研究

乙型链球菌３２１７２ 株接种于含有月示蛋白胨的牛肉水培养基中培养。得到含有溶血素“Ｏ”的培养液，其溶血效价２５６ ＩＵ／ｍ ｌ。比活为１１．６ ＩＵ／ｍ ｇ 蛋白。经过超滤除去９５％ 的非目的蛋白，所得超滤截留液的溶血效价为２０４８ ＩＵ／ｍ ｌ，比活为３３５．７ ＩＵ／ｍ ｇ 蛋白。在此基础上用离子交换柱层析纯化，所得活性峰溶血效价为８１９２ ＩＵ／ｍ ｌ，比活为４３１１．５ ＩＵ／ｍ ｇ 蛋白。通过以上两步的分级纯化，溶血素“Ｏ”的比活提高了３７１ 倍。此纯化蛋白在ＳＤＳ－ＰＡＧＥ电泳中呈现一条染色带。     

抗人α2a型基因工程干扰素单克隆抗体及亲和层析柱的研制

应用鼠－鼠杂交瘤细胞技术建立了三系稳定分泌抗重组人α２ａ型干扰素（ｒＨｕ－ＩＦＮ－α２ａ）单克隆抗体细胞系１Ａ５、１Ｂ５和２７Ａ７。细胞连续传代和在液氮中冻存后复苏,分泌抗体能力不变,并且维持在较高水平,细胞培养上清效价１∶２５６～１∶４０９６,腹水１∶２６×１０５～１∶２７×１０８。Ｉｇ亚类测定,１Ａ５和１Ｂ５ＭｃＡｂ为ＩｇＧ１,２７Ａ７为ＩｇＧ２ａ。三系ＭｃＡｂ均识别ｒＨｕ－ＩＦＮ－α２ａ和－α２ｂ,与ｒＨｕ－ＩＦＮ－α１和正常大肠菌体裂解液无反应。竞争ＥＬＩＳＡ试验,三系ＭｃＡｂ分别针对ｒＨｕ－ＩＦＮ－α２ａ上三个不同表位。同ＭｃＡｂ建立双抗体夹心ＥＬＩＳＡ对ｒＨｕ－ＩＦＮ－α２ａ和－α２ｂ均可检测到１５０ｐｇ／ｍｌ,约１０ＩＵ／ｍｌ的敏感度。用提纯的单抗制备亲和层析柱,单抗偶联率９５％以上。三系单抗亲和层析柱均可将粗制ｒＨｕ－ＩＦＮ－α２ａ提纯到９７％以上纯度。平均回收率为：１Ａ５单抗柱９０２％,１Ｂ５单抗柱９５３％,２７Ａ７单抗柱９４７％。比活性平均值依次为１９４×１０８ＩＵ／ｍｇ,１９７×１０８ＩＵ／ｍｇ和１６４×１０８ＩＵ／ｍｇ,残余鼠ＩｇＧ量均符合规程要求。纯化ｒＨｕ－ＩＦＮ?     

高分子量和低分子量尿激酶的分离纯化及动力学性质研究

人尿激酶粗品经苯甲脒亲和柱纯化和Ｐｒｏｔｅｉｎ－ＰａｋＳＰ柱分离后,得到两种分子量的尿激酶（ＵＫ）,即高分子量尿激酶（ＨＵＫ）和低分子量尿激酶（ＬＵＫ）,采用民斯亮蓝法测定蛋白质浓度,纤维蛋白板法测定活力,测得ＨＵＫ比活为２．９×１０＾５ＩＵ／ｍｇ蛋白,ＬＵＫ为３．５１０＾５ＩＵ／ｍｇ蛋白,活力回收为７０％以上,经ＳＤＳ－ＰＡＧＥ鉴定,ＨＵＫ和ＬＵＫ均呈单一条带,分子量分别为５４ｋＤ和３３ｋＤ,Ｈ     

重组干扰素γ的中间试制

用带有表达质粒ＰＢＶ２２０（含有γ干扰素基因）的大肠杆菌ＤＨ５α株进行发酵培养,３批中试的菌产量平均为１４．１克／Ｌ,γ干扰素的表达量平均为１．０２±１０＾９ＩＵ／Ｌ,收集的菌体经高压匀质破菌后收集包涵体,用７ｍｏｌ／Ｌ盐酸胍提取干扰素,此过程中去除７８．４％菌体蛋白,而干扰素活性仅损失１０．４１％,粗制干扰素经复性可使干扰素活性提高４０５％,比活也有明显提高,３批平均为１．７６×１０＾７ＩＵ／ｍ     

刺激大鼠视上核后电针“足三里”对蓝斑灌流液中去甲肾上腺素含量的影响

运用行为测痛结合蓝斑（ＬＣ）灌流液去甲肾上腺素（ＮＥ）的高压液相（ＨＰＬＣ）测定；观察 大鼠痛阈（ＰＴ）与ＬＣ灌流液中去甲肾上腺素含量变化间的相互关系，结果表明：（ｌ）视上核 （ＳＯＮ）内注射 １０μｍＬ－谷氨酸（Ｌ－ｇｌｕｔａｍｉｃａｃｉｄ， Ｌ－Ｇｌｕ）后 ３０分钟，大鼠ＰＴ较注射前增加１３３． ２± ２１．４％，此时ＬＣ 灌流液中ＮＥ含量从注射前的４３７．３±２０．４ｎｇ／ｍｌ降到２２９．２±１１．９ｎｇ／ｍｌ，注射 后６０分钟ＰＴ仍比注射前高８３．９±１４．７％，而灌流液中ＮＥ的含量为３２８．６±２８．０ｎｇ／ｍｌ，与人工 脑脊液（ＡＣＳＦ）对照组相比有非常明显的差别（Ｐ＜０．０５～０．００１）。（２） ＳＯＮ注射Ｌ－Ｇｌｕ后，电 针足三里３０分钟（Ｌ－ＧＩＵ＋ＥＡ组）增加到注射前的１８８．２±２３．９％，同ＡＣＳＦ电针组（ＡＣＳＦ＋ ＥＡ）的 ９４．９±７．１％相比有明显差异（Ｐ＜０．０１）。此时ＬＣ灌流液中ＮＥ的含量虽较注射前都明显 降低、分别为１３７．６±７．５ｎｇ／ｍｌ和 １５１，１±１１．５ｎｇ／ｍｌ，但两者相比无明显差异。停针后３０分钟Ｌ－ Ｇｌｕ＋ＥＡ组的ＰＴ仍比注射前高１３３．８±２７．９％，明显高于     

亲和层析纯化HepG2细胞分泌的PAI—1

本文报道用抗ＰＡＩ－１单克隆抗体（ＭｃＡｂ）亲和层析建立了纯化ＰＡＩ－１的简便方法。经免疫亲和层析,Ｓｅｐｈａｃｒｙｌ Ｓ２００凝胶过滤,从ＨｅｐＧ２细胞培液中分离到糖基化和非糖基化两种形式的ＰＡＩ－１,回收率为８４％,ＰＡＩ－１比活性为６．１×１０＾４ＩＵ／ｍｇ。糖基化ＰＡＩ－１分子量为５０ｋＤ,比活性５．８×１０＾４ＩＵ／ｍｇ。非糖基化ＰＡＩ－１分子量４３ｋＤ,占总ＰＡＩ－１３０％,仍具有ＰＡ     

人重组IL6／IL2融合蛋白的变化,复性及纯化

经超声破碎,分离已表达ＣＨ９２５包涵体,较系统地研究变性剂浓度、融合蛋白浓度对蛋白折叠的影响,在还原型及氧化型谷胱复性条件下,成功地将融合蛋白ＣＨ９２５折叠成具有ＩＬ６及ＩＬ２双活性蛋白,ＩＬ６的比活为２．３×１０＾８Ｕ／ｍｇ,ＩＬ２比活为２．２×１０６Ｕ／ｍｇ。经阴离子交换,凝过滤层析,获得一定纯度的ＣＨ９２５,配合反相ＨＰＬＣ,洗脱收集蛋白峰,ＣＨ９２５纯度为９８％。     

啤酒花茎段培养及快速繁殖技术研究

以啤酒花的幼嫩茎段作为外植体,研究出理想的一次性成苗培养基：ＭＳ＋６－ＢＡ０．０１ｍｇ／Ｌ＋ＩＡＡ０．１ｍｇ／Ｌ＋琼脂６．５ｇ／Ｌ＋葡萄糖２％。试管苗月增殖率４．２,生根率１００％,成苗率９０％。采用试管苗微扦插的方式进行快速繁殖,经锻炼后直接移入经改造后的土壤中,移栽成活率达９３．８％。     

单克隆抗体亲和层析纯化人白细胞干扰素的研究

本文报道了一种单克隆抗体亲和层析纯化人白细胞干扰素的方法．我们采用自己研制的α－干扰素单克隆抗体亲和层析柱进行了人白细胞干扰素较大规模纯化的研究,结果表明,其活性回收率平均达１０６．９％以上,经ＳＤＳ－ＰＡＧＥ银染法鉴定,白细胞干扰素的大多数活性成分均被吸附和回收,所得的干扰素蛋白成分主要位于分子量１５０００～２１０００Ｄ的干扰素活性区域,产品的纯度大大提高,比活可达８×１０６ＩＵ／ｍｇ;ＥＬＩＳＡ夹心法测定,每毫升洗脱样品中鼠源ＩｇＧ含量小于４ｎｇ,我们所建立起的亲和层析法操作简便,亲和吸附和洗脱条件温和,可适用于大规模的白细胞干扰素的纯化．     

Influence of absorption promoters on pulmonary insulin bioactivity

The purpose of this research was to enhance the bioactivity of insulin by the pulmonary route using a combination of absorption promoters. Aliquots (100 μL) containing 1.0 IU/kg to 7.0 IU/kg doses of porcine insulin solutions with different classes of absorption promoters and combinations of these at 3 concentration levels were instilled intratracheally to the anesthetized rats. Blood concentrations of glucose were measured at specific time points. Out of 3 concentration levels of each of the absorption promoters used, the formulations having the leastconcentration with the maximum percentage of blood glucose reduction were selected for combining absorption promoters, and their pharmacodynamic parameters related to insulin absorption were determined. The pharmacodynamics of porcine insulin following subcutaneous administration of increasing doses were also determined. The relative pulmonary bioactivity of insulin in phosphate buffer pH 7.4 and citrate buffer pH 3.5 was 11.36%±1.27% and 43.20% ±2.48%, respectively, compared to subcutaneous administration. Relative pulmonary bioactivity of 155.60%±5.19% was obtained when oleic acid sodium salt, sodium tauroglycocholate, bestatin, and chymostatin were coadministered in citrate buffer pH 3.5 solution. However, only 61.91%±3.21, 67.09%±3.23%, 67.24%±2.11%, and 69.84%±3.02% were obtained, respectively, upon incorporation of these absorption promoters individually. Absorption promoters in combination have significant potential for increasing the pulmonary bioactivity of insulin. These studies support the argument that pulmonary administration of insulin is a viable alternative to subcutaneous administration for diabetic patients.     

Proximate,vitamins A and E,and mineral composition of free‐ranging cotton mice (Peromyscus gossypinus) from St. Catherines Island,Georgia

Although rodents are an integral part of numerous carnivore diets, there is little published information regarding nutrient composition in free‐ranging mice for comparison with laboratory‐reared prey. Cotton mice (Peromyscus gossypinus, n=6) were captured on St. Catherines Island, Georgia, and analyzed for water, ash, protein, and fat content (proximate constituents), as well as minerals and the fat‐soluble vitamins A and E. The overall body composition (mean±SD: 65.8%±1.9% water, 10.9%±2.2% ash, 56.4%±4.1% protein, and 27.2%±3.9% fat) was similar to published values for adult laboratory mice (Mus domesticus). The macro‐ (Ca, K, Mg, Na, and P) and trace (Cu, Fe, Mn, and Zn) mineral levels were also similar to previously reported values for laboratory mice, and in general met or exceeded the established nutrient requirements for domestic carnivores. Vitamin E ranged from 77 to 170 IU/kg dry matter (DM) in these mice–again, similar to values previously quantified in laboratory mice. However, the vitamin A concentrations (21,947±6,893 IU/kg DM) in the free‐ranging mice were consistently and substantially lower than values reported for whole laboratory mice. To our knowledge, this is the first quantification of vitamin A in free‐ranging mice used as prey by carnivores. While other nutrients measured were similar between captive‐reared and free‐ranging mice, the current data suggest the need for further investigation of vitamin A nutrition in the development of optimal diets for carnivores in captivity. Zoo Biol 23:253–261, 2004. © 2004 Wiley‐Liss, Inc.     

AngⅡ和PKC对心肌细胞AngⅡ 1型受体的转录调节

利用体外培养的心肌细胞,观察血管紧张素Ⅱ（ＡｎｇⅡ）和蛋白激酶Ｃ（ＰＫＣ）在诱导ＡｎｇⅡ１型受体（ＡＴ１）基因表达及蛋白质代谢中的作用．研究结果表明：ＡｎｇⅡ可诱导ＡＴ１ｍＲＮＡ水平一过性下调,呈时间及剂量依赖性,１０ｎｍｏｌ／ＬＡｎｇⅡ刺激细胞６ｈ,引起ＡＴ１ｍＲＮＡ水平降低幅度最大,降至对照的５１．６％±９．５％,然后逐渐回升,２４ｈ恢复至对照水平．３０μｍｏｌ／ＬＨ－７（ＰＫＣ抑制剂）能阻断ＡｎｇⅡ诱导的ＡＴ１ｍＲＮＡ水平的下调．０．３μｍｏｌ／Ｌ的ＰＭＡ（ＰＫＣ激活剂）单独应用可诱导ＡＴ１ｍＲＮＡ水平下调达对照的４３％±８％,加入ＡＴ１拮抗剂ＤＭＰ８１１及Ｄｕｐ７５３均可阻断ＡｎｇⅡ诱导的ＡＴ１ｍＲＮＡ水平的下调．１０ｎｍｏｌ／Ｌ的ＡｎｇⅡ刺激心肌细胞９６ｈ可使蛋白含量降低至对照的７３．４％±５．６％,而加药持续刺激１４４ｈ可使蛋白含量较对照增加３３．８％±６．３％,Ｈ－７不能阻断ＡｎｇⅡ诱导的蛋白含量降低,但可有效地抑制蛋白含量的增加．以上结果提示：ＡｎｇⅡ对心肌细胞ＡＴ１基因的转录和细胞的蛋白代谢有调节作用,而ＰＫＣ则参与了ＡｎｇⅡ的这种调节作用     

Beneficial effects of sustained activity on the use of dietary protein and carbohydrate traced with stable isotopes 15N and 13C in gilthead sea bream (Sparus aurata)

To determine the effects of sustained swimming on the use and fate of dietary nutrients in gilthead sea bream, a group of fish were forced to undertake moderate and sustained swimming (1.5 BL s^?1) for 3 weeks and compared with a control group undertaking voluntary activity. The exercise group showed a significant increase in specific growth rate (C: 1.13 ± 0.05; E: 1.32 ± 0.06 % day^?1, P < 0.05) with no significant change in food intake (C: 3.56 ± 0.20; E: 3.84 ± 0.03 % of body weight). The addition of ¹³C-starch and ¹⁵N-protein to a single meal of 1 % ration allowed analysis of the fate of both nutrients in several tissues and in their components, 6 and 24 h after force-feeding. In exercised fish improved redistribution of dietary components increased the use of carbohydrates and lipid as fuels. Gilthead sea bream have a considerable capacity for carbohydrate absorption irrespective of swimming conditions, but in trained fish ¹³C rose in all liver fractions with no changes in store contents. This implies higher nutrient turnover with exercise. Higher retention of dietary protein (higher ¹⁵N uptake into white muscle during the entire post-prandial period) was found under sustained exercise, highlighting the protein-sparing effect. The combined effects of a carbohydrate-rich, low-protein diet plus sustained swimming enhanced amino acid retention and also prevented excessive lipid deposition in gilthead sea bream.     

Biochemical values of blood serum in the female silvered leaf-monkey,Presbytis cristatus

Thirteen days after capture, the blood serum of eight anesthetized female leaf-monkeys,Presbytis cristatus (3.86 kg of mean body weight), were analyzed for hematocrit (35.6±6.7 %), total protein (6.7±0.8 g/dl), albumin (3.61±0.77 g/dl), α-1 globulin (0.13±0.04 g/dl), α-2 globulin (0.73±0.18 g/dl), β globulin (0.87±0.27 g/dl), γ globulin (1.36±0.55 g/dl), A/G ratio (1.23±0.38), Na (161±6.14 mEq/l), K (5.61±0.74 mEq/l), LDH (575±257 IU/l), GOT (93±67 IU/l), GPT (34±33 IU/l), CPK (250±200 IU/l), ALP (613±633 IU/l), LAP (115±68 IU/l), γ-GTP (28±30 IU/l), TG (47±24 mg/dl), T-Cho (141±31 mg/dl), BUN (29.0±5.7 mg/dl), T-bil (0.21±0.07 mg/dl), and IP (3.4±2.0 mg/dl).     

重组人组织型纤溶酶原激活剂(rht-PA)及其突变体的纯化

稳定高效表达重组人组织型纤溶酶原激活剂 (rht PA)的CHO细胞株和表达组合突变体的细胞株进行了 3L转瓶培养 .将培养上清分别进行了Lys Sepharose 4B亲和层析和Zn2 + Sepharose 4B层析两步纯化 ,rht PA纯度提高了 5 34倍 ,比活达 2 5× 10 5IU mg ,产率为 73% ;突变体纯度提高了1119倍 ,比活达 5 9× 10 5IU mg ,产率为 6 9% .纯化产物SDS PAGE分析显示 ,rht PA和突变体基本都呈单一条带 ,扫描分析均达到 98%以上纯度 .rht PA和突变体在纯化系统中的行为作对照分析发现 ,突变体的构建思想在Lys Sepharose 4B亲和层析过程中有充分体现 .这两步层析组合是很好的纯化t PA及其突变体的方法 ,尤其是Lys Sepharose 4B纯化突变体效果更好     

Stability study of stavudine-loaded O-palmitoyl-anchored carbohydrate-coated liposomes

The purpose of this study was to evaluate the physicochemical stability of carbohydrate-anchored liposomes. In the present study, carbohydrate (galactose, fucose, and mannose) was palmitoylated and anchored on the surface of positively charged liposomes (PL). The stabilities of plain neutral liposomes (NL), PL, and O-palmitoyl carbohydrate-anchored liposomes were determined. The effects of storage conditions (4°C±2°C, 25°C±2°C/60%±5% relative humidity [RH], or 40°C±2°C/75%±5% RH for a period of 10, 20, and 30 days) were observed on the vesicle size, shape, zeta potential, drug content, and in vitro ligand agglutination assay by keeping the liposomal formulations in sealed ambercolored vials (10-mL capacity) after flushing with nitrogen. The stability of liposomal formulations was found to be temperature dependent. All the liposomal formulations were found to be stable at 4°C±2°C up to 1 month. Storage at 25°C±2°C/60%±5% RH and 40°C±2°C/75%±5% RH adversely affected uncoated liposomal formulations. Carbohydrate coating of the liposomes could enhance the stability of liposomes at 25°C±2°C/60%±5% RH and 40°C±2°C/75%±5% RH. Published: May 18, 2007     

Selenium Distribution in Camel Blood and Organs After Different Level of Dietary Selenium Supplementation

Eight young female camels shared in four groups of two 2 years received a basal diet enriched respectively with 0, 2, 4, and 8 mg selenium under sodium selenite form for 64 days. Feed intake was assessed daily; blood samples were taken on weekly basis. One camel from each group was killed at the end of the experiment. Se concentration in serum was increased significantly in the supplemented groups with an average of 176.3 ± 18.0 ng/mL in the control group, 382.7 ± 107.6 in the group receiving 2 mg Se, 519.8 ± 168.4 in the group receiving 4 mg Se, and 533.4 ± 158.6 in the group receiving 8 mg Se daily. For glutathione peroxidase (GSH-Px) activity, the control group (51.0 IU/g Hb) and the group receiving 2 mg (50.5 IU/g Hb) were significantly different than groups receiving 4 and 8 mg (respectively, 65.9 and 76.1 IU/g Hb). No significant variation occurred for vitamin E (mean, 0.56 ± 0.23 ng/mL). Significant correlation between serum Se and GSH-Px was reported. Kidney was the richest organ in selenium followed by lung, spleen, and liver, but the increase in supplemented groups was more marked in liver and kidney. The hair seemed to be the best indicator of selenium intake in camel.     

Design and development of hydrogel beads for targeted drug delivery to the colon

The purpose of this research was to develop and evaluate a multiparticulate system of chitosan hydrogel beads exploiting pH-sensitive property and specific biodegradability for colon-targeted delivery of satranidazole. Chitosan hydrogel beads were prepared by the cross-linking method followed by enteric coating with Eudragit S100. All formulations were evaluated for particle size, encapsulation efficiency, swellability, and in vitro drug release. The size of the beads was found to range from 1.04±0.82 mm to 1.95±0.05 mm. The amount of the drug released after 24 hours from the formulation was found to be 97.67%±1.25% in the presence of extracellular enzymes as compared with 64.71%±1.91% and 96.52%±1.81% release of drug after 3 and 6 days of enzyme induction, respectively, in the presence of 4% cecal content. Degradation of the chitosan hydrogel beads in the presence of extracellular enzymes as compared with rat cecal and colonic enzymes indicates the potential of this multiparticulate system to serve as a carrier to deliver macromolecules specifically to the colon and can be offered as a substitute in vitro system for performing degradation studies. Studies demonstrated that orally administered chitosan hydrogel beads can be used effectively for the delivery of drug to the colon. Published: July 13, 2007