利用固定化酵母细胞转化反式肉桂酸生产L—苯丙氨酸

研究了深红酵母（Ｒｈｏｄｏｔｏｒｕｌａｒｕｂｒａ）的培养基成分,培养固定化及转化条件,实验表明最佳基成分（％）葡萄糖０．５,胰蛋白胨０．５,酵母膏０．５,磷酸二氢钾０．０５,Ｌ－Ｐｈｅ０．０５,ｐＨ７．０,３０℃２０Ｌ发酵罐中培养１５～１７ｈ,最佳固定化条件为：用２．５％卡拉胶包埋１８％的湿菌体,最佳转化条件为：１．０％反式肉桂酸,４ｍｏｌ／Ｌ铵离子,ｐＨ０．５,３０℃,用卡拉胶固定化深红酵母（Ｒ     

Long-lasting adenovirus transgene expression in mice through neonatal intrathymic tolerance induction without the use of immunosuppression.

The major barrier to the clinical application of adenovirus gene therapy for diseases that require stable transgene expression is the immunogenicity of recombinant adenovirus, which ordinarily limits the duration of its effects to a period of about 2 weeks. We postulated that tolerance to adenovirus could be induced and transgene expression could be prolonged if T lymphocytes underwent thymic selection in the presence of adenovirus antigens. Mice were inoculated in the thymus with a recombinant adenovirus containing the lacZ marker gene during the neonatal period at a time before T-cell maturation had occurred. When the virus was administered intravenously to these mice in adulthood, they were found to have an impaired adenovirus-specific cytotoxic T-lymphocyte response which allowed prolonged hepatic lacZ expression, for up to 260 days. The ability to achieve unresponsiveness to a recombinant adenovirus after inoculation of the thymus in neonates extends the paradigm of intrathymic tolerance induction. Furthermore, this model will enable the study of stable adenovirus transgene expression in vivo without the use of immunosuppression and ultimately may have clinical utility.     

Screening and characterization of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp.

Summary Three screening methods were used to isolate GL-7-ACA acylase-producing strains. Three positive isolates were identified with Pseudomonas nitroreducens CCRC 11041 possessing the highest activity, against GL-7-ACA and GL-7-ADCA. No activity was detected when Ceph C or succinyl-7-ACA was used as substrate; glutaric acid was found to be inhibitory. CCRC 11041 could produce maximal GL-7-ACA acylase activity when cultivated on meat extract medium II. The enzyme had a pH optimum of 5.0 and a temperature optimum of 42°C.     

软骨血管生成抑制因子抑制血管生成的研究

小牛气管软骨经盐酸胍抽提,丙酮分级沉淀,膜超滤,柱层析等步骤得到软骨血管生成抑制因子(cartilage angiogenesis inhibiting factor,CAIF).SDS-聚丙烯酰胺凝胶电泳显示CAIF由单一组分组成,分子量为27700.通过[ ³H]-TdR掺入,活细胞检测等方法测定CAIF对内皮细胞、Hela细胞、QGY7703细胞与小鼠骨髓细胞、人皮肤成纤维细胞等的DNA合成的影响,以及细胞毒作用.采用鸡胚绒毛尿囊膜实验测定CAIF对血管生成的抑制效应.结果显示:CAIF对内皮细胞产生强的抑制作用,对Hela细胞抑制很弱,对QGY7703细胞、小鼠骨髓细胞、人皮肤成纤维细胞均无抑制作用;对鸡胚绒毛尿囊膜的血管生成产生明显的抑制作用.提示CAIF能较特异地抑制血管生成,CAIF达到电泳纯,是专一性较强的血管生成抑制因子.     

对分子置换法中积分半径选取方法的探讨

通过对晶胞中帕特逊向量的统计分布的研究,从理论上阐述了根据模型结构单元的线度确定旋转函数的积分半径这种作法的合理性,并且指出了估算积分半径取值范围的具体方法,以及自身旋转函数与交叉旋转函数的积分半径取值范围的区别。经过我们将此方法应用于酚胰岛素Ｂ链羰端六肽胰岛素的旋转函数求解,计算结果证实了这种积分半径的估算方法的可靠性。     

Production and characterization of antibody against aflatoxin Q1.

Antibodies against aflatoxin Q1 (AFQ1) were obtained from rabbits after immunization of either AFQ1-hemisuccinate or AFQ2a conjugated to bovine serum albumin. Both radioimmunoassay and enzyme-linked immunosorbent assaY (ELISA) were used for the determination of antibody titers and specificities. Antibodies obtained from rabbits after immunization with AFQ1-hemisuccinate-bovine serum albumin had the highest affinity to aflatoxin B1, whereas antibodies obtained from rabbits after immunization with AFQ2a-bovine serum albumin bound most effectively with AFQ2a. AFQ2a antibody was selected for the subsequent direct and indirect ELISA for the detection of AFQ1 in biological fluids. When AFQ2a-peroxidase and AFQ2a antibody were used, direct ELISA was able to detect as low as 2 ppb (ng/ml) of AFQ1 spiked in the urine samples that had been subjected to a Sep-Pak cleanup treatment. In indirect ELISA in which the antigen (AFQ2a-bovine serum albumin) was coated to the solid phase followed by reaction with rabbit antibody and goat anti-rabbit immunoglobulin G-peroxidase conjugate, 50-fold less antibody was used without sacrificing sensitivity. Recoveries of AFQ1 added to urine samples (2 to 40 ppb) were 46.3 to 73% and 65.8 to 85.8% for direct and indirect ELISA, respectively.     

Production and characterization of monoclonal antibodies against aflatoxin M1.

By using an indirect enzyme-linked immunosorbent assay, four monoclonal antibodies were selected after fusion of mouse P3-NS1-Ag4-1 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with aflatoxin M1 (AFM1) conjugated to bovine serum albumin. Two of these antibodies were found to be specific for AFM1 and were designated AMW-1 and AMW-4. The specificities of AMW-1, which had higher affinity to AFM1, were determined by a competitive direct enzyme-linked immunosorbent assay with peroxidase-AFM1 as the marker. The relative cross-reactivity of each toxin (relative to AFM1) with AMW-1, as determined by the amount of aflatoxin necessary to cause 50% inhibition of enzyme activity, was 12, greater than 40, 12, and greater than 40 for B1, B2, G1, and G2, respectively.     

Immunologically reactive tryptic fragments of human prostatic acid phosphatase.

Three peptide fragments (designated II, III and IV) of human prostatic acid phosphatase (PAP) were isolated to homogeneity from a limited tryptic hydrolysate of PAP by gel filtration on Sephadex G-100, followed by chromatography on DEAE-cellulose and Sephadex G-75. The homogeneity was confirmed by disc poly-acrylamide-gel electrophoresis. The Mr values were 32 500, 25 000 and 11 000 as estimated by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Immunoprecipitation study revealed that only fragment II formed an immune precipitate with anti-PAP antibodies. Fragment II exhibited 45% of maximum inhibitory activity on the reaction between PAP and goat anti-PAP IgG (immunoglobulin G) antibodies (or rabbit anti-PAP antibodies), whereas fragments III and IV demonstrated 24% (or 23%) and 29% (or 27%) inhibition respectively. A mixture of these three tryptic fragments of PAP result in 96% (for goat anti-PAP antibodies) and 94% (for rabbit anti-PAP antibodies) inhibitory activities, which were equivalent to the sum of maximum inhibitory activity of the three fragments individually. The results demonstrated that these three tryptic peptide fragments carried all the antigenic active sites of the native PAP, and suggested that the entire molecule of human PAP comprised a minimum of four distinguishable, nonoverlapping antigenic determinants. These three fragments also were shown to retain all the disulphide bonds of the native PAP, and thus were useful reagents for the elucidation of PAP molecular structure.     

Production of antibody against T-2 toxin.

Antibody against T-2 toxin was obtained after immunization of rabbits with bovine serum albumin-T-2 hemisuccinate conjugate. The antibody had greatest binding efficiency for T-2 toxin, less efficiency for HT-2, and least for T-2 triol. Cross-reaction of antibody with neosolaniol, T-2 tetraol, and 8-acetyl-neosolaniol was very weak. Diacetoxyscirpenol, trichodermin, vomitoxin, and verrucarin A essentially gave no cross-reaction with the antibody. The sensitivity of the binding assay for T-2 toxin detection was in the range of 1 to 20 ng per assay. Detailed methods for the preparation of the conjugate and the production of immune serum and methods for antibody determination are described.