孕马血清促性腺激素提取与纯化的研究

为了获取可用于制备抗体的高纯度孕马血清促性腺激素(PMSG)蛋白,试验采用偏磷酸-乙醇沉淀法从孕马血清中提取PMSG粗品,并在此基础上进一步探讨通过阴、阳离子柱层析提取高纯度PMSG的方法。结果表明:从1 000 mL孕马血清中平均可获得约为12.25 g的PMSG粗品,其平均PMSG比活为153.63 IU/mg;将1 000 mg PMSG粗品经阴离子柱层析后,纯化的PMSG的回收率为54.8%,但其PMSG比活仅能提高至173.08 IU/mg;再将阴离子柱层析纯化的PMSG经阳离子柱层析纯化可获得3个洗脱峰,从第3峰中可回收获得1.26 mg蛋白,其PMSG比活可达926.41 IU/mg。     

SP-SephadexC-50柱层析法纯化孕马血清促性腺激素(PMSG)

应用HPO_3沉淀-硫酸铵浓缩-乙醇分部沉淀法,从孕马血清中粗提PMRG(平均比活1026IU/mg.平均回收率75%),用SP-Scphadex C-50一次柱层析纯化PMSG粗品,获得平均比活11847IU/mg的高活性PMSG精品,对粗品的平均回收率为88.5%.     

PMSG的简易提取法

1930年英国科学家Cole和Hart发现,在孕马血液中存在促性腺激素(PMSG).PMSG是一种糖蛋白,它由胚胎滋养层衍化的特殊细胞所分泌,具备卵泡刺激素(FSH)及黄体生成素(LH)的双重性质.国外PMSG的提取研究已取得引人注目的成就.Denis等用层析技术获得了15800IU/mg的PMSG.国内对PMSG的提取起步较晚,但成绩可喜.邓瑞春用反亲和层析技术获得了630IU/mg的PMSG,丁若愚用HPO_3沉淀-超滤浓缩一乙醇部分沉淀-超滤截留SE-Sephadexc-50柱层析一羟基磷灰石柱层析获得了9000IU/mg的PMSG.     

PMSG免疫学检测方法的建立

为了建立一种马属动物孕马血清促性腺激素(pregnant mare serum gonadotropin,PMSG)浓度的检测方法,试验使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)检测商用PMSG抗原纯度,斑点印迹杂交(Dot-blot)判断PMSG和抗体是否结合,蛋白免疫印迹杂交(Western-blot)确定抗体和PMSG抗原的结合方式,应用酶联免疫吸附试验(ELISA)建立PMSG浓度-吸光度值标准曲线,并拟合方程。结果表明:PMSG抗原纯度符合要求,抗体与PMSG抗原采用空间表位方式结合;PMSG浓度与OD_(450)值的标准曲线拟合方程为y=0.004 5x+0.020 2,R~2=0.998,在0～4×10~2IU/m L范围内,PMSG浓度与OD_(450)值之间高度相关,R~20.99。说明PMSG抗原可与抗体通过空间表位方式特异性结合,所建立的PMSG免疫学检测方法具有较高的准确性。     

水牛超排处理后血清PMSG残留量的酶联免疫吸附测定

应用孕马血清促性腺激素(PMSG)纯品主动免疫家兔制备抗血清,用辣根过氧化物酶标记葡萄球菌A蛋白(SPA-HRP)代替酶标IgG抗体,建立了PMSG抗原-抗体-SPA-HRP酶联免疫吸附测定方法。方法学鉴定证明,该法灵敏度高(1.8miu/孔),重复性好(板内和板间变异系数分别为4.7％和8.6％,样本数均为27);PMSG抗血清的特异性和方法的准确性均达到放射免疫测定中世界卫生组织所要求的质量控制指标。应用该法测定水牛注射PMSG2000iu后每天的PMSG残留量,证明第一天最高,平均为69.4±1.8miu/ml,以后逐渐降低到第11天的9.5±0.7miu/ml。     

孕马血清促性腺激素(PMSG)提取方法的研究

应用生物化学制备技术,对PMSG的提取进行了研究,发现偏磷酸氢氧化钠pH分离-低温乙醇沉淀-透析法的脱杂蛋自效果优于硫酸胺盐析-透析法,更优于偏磷酸pH分离-离子交换法,而氯化钠盐析法则达不到脱杂蛋白的效果;血清中PMSG的效价损失随着脱去杂蛋白的增多而增大;在所采用的冻干曲线下,脱去杂蛋白愈多,则冻于过程中PMSG的损失就愈小。     

鲤鱼卵透明带糖蛋白分离纯化研究

温度、PH值对ZP粗品制备的影响研究，得出ZP粗品制备最佳条件为：在温度为75℃、PH值9．0－10．2的溶液中提取。将40％饱和（NH4）2SO4去除部分杂蛋白的ZP组份用Q－SepharoseFF层，分别用不同浓度的NaCl、不同PH值的Tris-HCl缓冲液作洗脱液纯化ZP粗品即可得ZP－1、ZP－2单组份。     

用于ELISA的牛白血病病毒抗原的制备

人工感染牛白血病病毒(BLV)的绵羊血清IgG与溴化氰活化的Sepharose4B偶联,制成免疫吸附柱Ⅰ:用兔抗牛血清IgG与之偶联,制成免疫吸附柱Ⅱ.将BLV粗提抗原经柱Ⅰ和柱Ⅱ亲和层析后获得无色透明抗原。该抗经AGID试验表明具有gp和P抗原活性,回收率分别为38.8％和51.8％;经SDS-PAGE分析表明,含有5种蛋白组分,分子量分别为15、24、64、70和80K道尔顿;经薄层扫描,测得5种组成的百分含量之和为72.08。将提纯抗原同提纯各阶段不同纯度的抗原同时进行ELISA测定,结果以提纯抗原最理想,本底很浅,工作浓度仅为5μg/ml。     

犬干扰素α制备工艺的优化

根据Genbank的犬干扰素仪序列,经密码子优化后,设计并合成了编码CalFN-仪蛋白的基因片段。CaIFN-0l基因与pBV220温度诱导表达载体连接,转化大肠杆菌BL21。经培养、升温诱导表达,目的蛋白以包涵体的形式存在,蛋白分子量约为18000,蛋白表达量为25％。菌体经超声破碎、变性、复性后,目的蛋白纯度为69％,收率为28％;疏水层析后,目的蛋白纯度87％,收率为81％;分子筛层析后,目的蛋白纯度为96％,收率为90％。纯化的CalFN-o蛋白的生物学比活性为3×10’IU／mg,内毒素≤10EU／mg,符合兽用药要求。此制备工艺总产率高达20％、重现性好,适用于大规模生产。     

饲粮铜和维生素A及其互作效应对肉仔鸡生长性能及抗氧化功能的影响

本研究旨在探讨铜和维生素A及其互作效应对内仔鸡生长性能及抗氧化功能的影响.试验选用1日龄艾维茵肉仔鸡448只,随机分为8组,每组4个重复,每个重复14只鸡.采用4×2(铜×维生素A)完全随机设计,饲粮铜的添加量分别为0、8、150、225 mg/kg,维生素A的添加量分别为1 500、5 000 IU/kg,分为2个生长阶段,前期为1～4周龄,后期为5～7周龄.结果表明:1)高铜( 150、225mg/kg)抑制了全期肉仔鸡生长性能,提高了全期血清铜蓝蛋白(Cp)和后期血清总超氧化物歧化酶(T-SOD)活性(P＜0.05).2)5 000 IU/kg维生素A组获得较好的生长性能,提高了全期血清T-SOD活性,降低了前期血清Cp活性(P＜0.05).3)铜和维生素A互作效应对前期体增重、血清Cp和T-SOD活性及全期料重比有显著影响(P＜0.05),且二者间存在互补作用.铜(8 mg/kg)×维生素A(5 000 IU/kg)组与铜(0 mg/kg)×维生素A(5 000 IU/kg)组对前期肉仔鸡获得良好生长性能和提高血清T-SOD活性均有促进作用,但对血清Cp活性有显著降低作用(P＜0.05).由此可见,在基础饲粮铜水平为16～23mg/kg时,铜的适宜添加量为前期8 mg/kg,后期0～8 mg/kg;全期维生素A的适宜添加量为5 000 IU/kg.     

PMSG profiles in superovulated and anti-PMSG antiserum treated mice and heifers with enzymeimmunoassay

A sandwich enzymeimmunoassay (EIA) for pregnant mare serum gonadotropin (PMSG) using a microtiter plate was developed. Sensitivity of the assay to PMSG was 15.6 mIU/ml (0.2 ng/well). The PMSG levels in serum were measured with the EIA in superovulated and anti-PMSG rabbit antiserum treated mice and heifers. In mice, the PMSG blood level was measurable in the serum 4-6 days after intraperitoneal injection of 5-30 IU of PMSG. The administration of anti-PMSG antiserum at the same dose level as PMSG caused a rapid decrease in the PMSG blood level, declining to undetectable levels within 17 hours. In heifers, the PMSG level was measurable at 10-11 days after the injection of 2500 or 3000 IU of PMSG. When antiserum was injected 48 hours after the PMSG injection, the clearance rate of PMSG was affected by the route of the administration. The administration of 3000 units of anti-PMSG antiserum intravenously caused a rapid decline and the disappearance of circulating PMSG within 17 hours. When 3000 units of anti-PMSG antiserum was injected intra-muscularly, the PMSG blood level also decreased and became unmeasurable 24 hours after administration; however, it was still detectable for up to 17 hours. These results indicate that the administration of anti-PMSG antiserum at the proper timing and dosage could lead to successful superovulation through the improvement of hormonal conditions.     

Studies on the effect of the HCG injection date on the fertility of ovulation-synchronized gilts

The time gap between pregnant mare serum gonadotropine injection (PMSG) and subsequent human chorionic gonadotropine injection (HCG) had major effects, within 72 to 80 hours, on the number of animals with recorded toleration reflex to deadline-oriented insemination as well as on actual fertility. Delay of injections within the above limits always led to higher farrowing and piglet rates. HCG injection was round to be optimally timed when 78-80 hours were allowed to elapse from the preceding PMSG injection.     

THE INDUCTION OF OESTRUS AND OVULATION IN THE BITCH USING PREGNANT MARE SERUM GONADOTROPHIN AND HUMAN CHORIONIC GONADOTROPHIN

Seventeen bitches (11 anoestrous, 4 prepubertal, 1 pregnant and 1 postpartum) were treated with pregnant mare serum gonadotrophin (110 iu/kg) at weekly intervals up to the occurrence of oestrus or a maximum of 3 treatments. Oestrus was induced in 8/11 anoestrous bitches only. The between bitch ovarian response was very variable. Ovulation occurred in 7/8 PMSG induced oestrous bitches. The 3 oestrous bitches treated with human chorionic gonadotrophin (500 iu) at the start of oestrus had a better ovulatory response than those treated with PMSG alone.     

孕马血清促性腺激素的提纯研究

以比活100 IU/mL以上的孕马和孕驴血清为原料,应用HPO2沉淀-超滤浓缩(或用聚 乙二醇浓缩)-乙醇分部沉淀-超滤截留-SE-SephadexC-50柱层析-羟基磷灰石柱层析,进 行PMSG提纯试验,获得了比活9 000 IU/mg左右的高纯度产品,经PAG圆盘电泳只出现一条带.     

Superovulation in the cow with pregnant mare serum gonadotrophin: effects of dose and antipregnant mare serum gonadotrophin serum.

The effects of pregnant mare serum gonadotrophin (PMSG) dose and PMSG antiserum on superovulation in crossbred beef cows were studied. In experiment I, three groups were treated with 1200, 2400 or 3600 IU of PMSG and 48 h later with prostaglandin (PGF). The mean numbers of corpora lutea (CL), unovulated follicles, and total ova/embryos collected increased as the PMSG dose increased. The percent of fertilized ova and transferable embryos was lowest in the highest dose group (p < 0.05). In experiment II, all cows received 2500 IU of PMSG; groups 1 and 2 were treated with sheep anti-PMSG serum at 48 h or 60 h after PGF; group 3 cows were PMSG-only controls. The number of CL was lowest and the number of unovulated follicles highest in the PMSG-only group (p < 0.05). The number of CL was higher in group 2 (anti-PMSG at 60 h) than in the control group, with the anti-PMSG at 48 h not different from the other groups. Numbers of total ova/embryos, fertilized ova, and transferable embryos were higher (p < 0.05) in both antiserum-treated groups relative to the PMSG-only group. We conclude that superovulation of beef cows with PMSG and treatment with PMSG antiserum will induce a higher superovulatory response and will result in higher CL numbers and fewer unovulated follicles. Further, the variability in the superovulatory response to PMSG treatment was still evident when PMSG antiserum was administered.     

Serum progesterone and estradiol-17beta concentrations, and lapaloscopic observations of the ovary in the cheetah (Acinonyxjubatus) with pregnant mare serum gonadotropin and human chorionic gonadotropin treatments.

In 3 adult female cheetahs, induced-superovulation treatment was conducted, by means of 200 IU of pregnant mare serum gonadotropin (PMSG) and 100 IU of human chorionic gonadotropin (hCG) 80 hr after PMSG. The administration of PMSG created a sharp increase in the estradiol-17beta concentration, resulting in 232 pg/ml 8 hr later in one specimen out of three. The hCG administration showed an increase in the progesterone concentration of 2.29 ng/ml 46 hr later. In addition, after direct observation of the ovary surface by laparoscopy, 5 follicles in the right ovary over 2 mm in diameter, and 7 corpora lutea (5 in the right ovary and 2 in the left) were found. It is assumed that ovulation can be induced with hCG after 80 hr on PMSG during a cheetah's diestrus or proestrus.     

昆明鼠超数排卵影响因素研究

为探讨昆明鼠超数排卵的最佳条件,提高超排效果,比较促性腺激素使用剂量、孕马血清促性腺激素(PMSG)与人绒毛膜促性腺激素(hCG)注射间隔时间、超排小鼠所处发情周期阶段及小鼠周龄等因素对昆明鼠超数排卵结果的影响。PMSG 8IU+hCG 8IU、间隔46h～48h注射组超排效果最好;3周龄、8周龄～10周龄雌鼠较4周龄～7周龄雌鼠超排效果好,小鼠发情间期或发情前期开始超排的效果显著高于发情周期的其他时期。     

Changes in plasma testosterone and testicular transferrin concentration, testicular histology and semen quality after treatment of testosterone-depot plus PMSG to 3 dogs with asthenozoospermia

Three dogs diagnosed as having asthenozoospermia were given three intramuscular injections of 50 mg testosterone(T)-depot plus 250 IU pregnant mare serum gonadotropin (PMSG) at 2-week intervals, and their plasma T and testicular transferrin (Tf) concentrations, testicular histology, and semen quality were examined during the period of hormone therapy. Plasma T concentrations temporarily increased, and there was a slight improvement in spermatogenesis. Increased Tf concentrations suggested that Sertoli cell function in all three dogs was promoted by hormone treatment. The results showed that semen quality, especially the percentages of motile sperm and abnormal sperm, were improved between 1 and 5 weeks after the start of T-depot plus PMSG treatment.     

Orientation studies of ovulation release in mice to test gonadotropic preparations. 2. FSH, PMSG and Gn-RH activity

The doses of FSH (follicle-stimulating hormone), PMSG (pregnant mare serum gonadotrophin), and gn-RH (gonadotrophin-releasing hormone) effective in terms of triggering ovulation were determined in a mouse ovulation test. Varying doses of the above preparations were subcutaneously injected, 48 hours after overstimulation by injection of 0.5 or 1.0 IU of PMSG. The animals were sacrificed for examination, after another 18-20 hours had passed. Roughly 50 per cent of all animals treated (threshold) in one and the same dosage group (n = 5) had ovulated in response to 0.02-0.1 IU of FSH per animal. The following FSH and PMSG dosages are recommended: 0.02, 0.04, 0.06, 0.08, and 0.1 IU of FSH, 0.6, 1.0, 1.4, 1.8, 2.2, 2.6, 3.0 IU of PMSG. When mouse ovulation tests were used in orientation studies, ovulation was regularly induced by Gn-RH doses per animal between 0.01 and 1.0 micrograms. Dosage spacings or increments should be specified with higher accuracy by further studies.