丝状真菌斜卧青霉JUA-10具有启动子功能的DNA片段的克隆与表达

将斜卧青霉JUA－10染色体DNA的BamHI或PstI片段连接到大肠杆菌的启动子探针型载体pSUPV4上，克隆了7个不同大小的具有启动子功能的DNA片段。卡那霉素抗性试验表明，含重组质粒的大肠杆菌均能耐受100μg/ml以上的卡那霉素，最高可达1100μg/ml。说明斜卧青霉的某些DNA片段能在大肠杆菌中有铲地启动基因表达。取抗性水平最高的pPdsuI进一步进行限制酶谱分析，表明插入片段的分子量为2.4Kb.Southern杂交分析结果表明该插入DNA片段与斜卧青霉染色体DNA具同源性。     

A recombinant chimeric antigen toward a standardized serodiagnosis of Taenia solium neurocysticercosis

Neurocysticercosis (NC) invokes formidable neurological problems worldwide. Previous proteomic analyses revealed most of the low‐molecular‐weight proteins might derive from two macromolecules of 120 kDa (consisting of 14–38 kDa subunits) and 150 kDa (7–15 kDa subunits) of Taenia solium metacestode (TsM) cyst fluid (CF). We characterized serological properties of these two proteins and established an immunopotent chimera. The 120 and 150 kDa proteins harbored 54–81 and 94–98% of the antibody‐binding activity of the crude CF with minimal antigenic cross‐reactivity to each other. The expression and immune recognition of the 150 kDa subunits were relatively constant, regardless of the different geographical origins of the CF collected, while those of the 120 kDa subunits varied by their origins (Asia vs. America). We cloned four representative proteins (one from the 120 kDa and three from the 150 kDa) that showed different epitope specificities, generated a chimera, and demonstrated that this regimen may bolster serodiagnostic reliability. Overall sensitivity and specificity, against sera from active‐/mixed‐stage NC and those from other infections, and healthy‐controls, were determined to be 97.5% (156/160 samples) and 97.8% (265/271 cases). Patient sera from adult taeniases, sparganosis, and fascioliasis showed weak cross‐reactions. Micro‐ELISA showed similar results. This chimera may prove useful in the construction of standardized platform for NC serodiagnosis.     

UTILIZATION OF DYNAMIC RESPONSES OF RECOMBINANT CELLS TO IMPROVE CSTBR OPERATIONS

Stable recombinant strains containing different copy numbers (1, 2, and 10) of a multiple integration vector were constructed and their dynamic responses were studied. The dynamic responses of the host strain are faster than those of the recombinant strains. For the recombinant strains the higher the copy numbers, the slower the responses. Unstructured dynamic models were developed for the host and stable recombinant strains. These dynamic models were combined with a two-population segregational model to mimic a segregationally unstable system. The model suggests that cyclic shifts in dilution rate and feed substrate concentration can stabilize the segregationally unstable plasmid system in a continuous bioreactor over a long period of time. The experimental results of cyclic shifts in dilution rate and feed substrate concentration show similar trends indicated by the simulation results. Experimental cyclic shifts in feed substrate concentration between 10.0 and 0.0 g/l, stabilized the bioreactor containing an unstable plasmid-harboring strain.     

重组人成骨蛋白-1复性条件的优化

目的优化重组人成骨蛋白-1(rhOP-1)包涵体蛋白的复性条件。方法将表达rhOP-1的大肠杆菌菌体在冰浴下超声裂解,分离提取包涵体,用8mol/L尿素溶解,纯化后进行梯度透析复性。利用TotalLab软件分析目的蛋白二聚体的含量,用体内法和体外法测定其生物学活性。结果经纯化后,目的蛋白纯度达97%以上。最佳复性条件为4℃,pH9.0,蛋白浓度为0.4～0.8mg/ml,尿素浓度为2mol/L,L-Arg浓度为0.4mol/L;目的蛋白复性率达75%以上,复性后蛋白具有较高的生物学活性。结论已确定了rhOP-1包涵体梯度透析复性的最佳条件。     

重组乙型肝炎疫苗不同剂量免疫效果观察

应用重组痘苗病毒表达的乙肝疫苗共免疫2150人。10μg和5μg组免后24个月，抗体阳转率分别为98.36％和96．33％，二组无明显差异；抗体水平分别为176．70mIU／ml和132．00mIU／ml，两组间有明显差异。2．5μg组抗体阳转率为76．47％，抗体水平为88.98mIU／ml，均明显低于上述两组。与相同剂量的血源疫苗组比较，各组间无统计学差异。     

B→O血型转换批量制备通用型红细胞的研究

目的建立B→O血型转换红细胞(通用型红细胞)批量制备工艺,为临床提供通用型红细胞制品。方法用不同pH值的缓冲液对1个使用单位(200ml)的B型红细胞进行预处理。在专门设计的转型密闭处理系统中,用α半乳糖苷酶对B型红细胞表面B抗原进行酶解,并检测酶解后红细胞ABO血型抗原及结构、功能。结果酶解前用pH4.2缓冲液预处理组上清游离Hb含量显著低于pH2.8缓冲液处理组;经α半乳糖苷酶(100Uml红细胞)在pH5.6,26℃条件下酶解60min,红细胞B型抗原被完全清除,转换为O型;红细胞形态、变形性、ATP、2,3DPG、胆固醇含量、乙酰胆酐酯酶活性、渗透脆性、pH、上清游离Hb、K+、Na+含量与对照组(正常红细胞)比较差异无显著意义,均在正常范围。结论建立了B→O血型转换通用型红细胞的批量制备工艺。通用型红细胞结构、功能、形态均正常,细菌、热原实验合格。     

重组人尿激酶原质控方法和质量标准的研究

目的建立重组人尿激酶原的质控方法和质量标准。方法以纤维蛋白平板法测定重组人尿激酶原的生物学活性,S-2444发色底物法测定单双链比例,还原型SDS-PAGE测定相对分子质量,SDS-PAGE和分子筛色谱测定纯度,毛细管电泳法测定等电点,胰蛋白酶酶切后分析肽图,其余检测项目按《中国药典》三部(2005版)规定进行。结果用建立的方法对原液和成品进行了检定,各项指标均符合《人用重组DNA制品质量控制技术指导原则》和《中国药典》三部(2005版)的要求。结论所建立的质控方法和质量标准可用于重组人尿激酶原产品的常规检定。     

抗肿瘤重组鸡痘病毒体外抑制骨肉瘤细胞S180的效应

目的探讨Apoptin、HN和IL18融合基因的重组鸡痘病毒(vFV3)对骨肉瘤细胞S180的抑制效应。方法以vFV3感染人骨肉瘤细胞S180,应用MTT法检测细胞活性,流式细胞仪分析vFV3感染致S180细胞周期变化,并用Rhodamine123染色结合FCM检测线粒体跨膜电位,从细胞生物学角度探讨vFV3致S180肿瘤细胞凋亡的途径。结果vFV3感染可有效抑制S180肿瘤细胞,杀伤率达46.8%。感染使S180肿瘤细胞线粒体膜电位下降,导致S180肿瘤细胞凋亡。结论vFV3可诱导S180肿瘤细胞凋亡,并主要通过线粒体途径诱导凋亡。     

SENSITIVITY OF RECOMBINANT FERMENTATION WITH RUN-AWAY PLASMIDS: A STRUCTURED ANALYSIS OF THE EFFECT OF DILUTION RATE

A recently validated four-compartment model is used to describe intra-cellular dynamics during fermentations with recombinant microorganisms containing temperature-sensitive (run-away) plasmids. The time-dependent sensitivities with respect to the dilution rate have been computed for normal and run-away replication. During normal replication the cells are most sensitive in the early stage of fermentation, and the intra-cellular compartment containing RNA and ribosomes is more sensitive than the other three lumped components. This is explained through a buffering effect. During run-away replication the sensitives are either constant or increase slowly with time, and there is a shift in the relative sensitivities of the intra-cellular components. All sensitivities except that of the substrate increase as the dilution rate increases.     

硼中子俘获治疗癌症的基础性实验研究

分别利用²⁴¹Am放射源和HI-13串列加速器产生的α粒子和⁷Li离子来模拟硼中子俘获治疗中的核反应产物，对DNA水溶液进行辐照，然后利用原子力显微镜(AFM)对DNA碎片进行观测，最后通过大量的统计分析获取DNA碎片长度、DNA形态的实验数据。实验结果表明：DNA碎片的平均长度随剂量的增大逐渐减小；线性和开环的DNA分子所占的比例随着剂量的增大逐渐增多；⁷Li离子比α粒子具有更强的相对生物学效应。 