【作者】 刘宇；

【导师】 魏泓；

【作者基本信息】 西南师范大学 ， 动物学， 2003， 硕士

【摘要】 本课题围绕3000Da以下低分子量羊胎免疫调节因子（goat embryo immunoregulatory factor，GEIF）的质量标准进行了初步的研究，着重对其指纹图谱指标进行色谱条件的确定和方法的建立，并在此基础上对主要成分峰及其稳定性进行了初步分析。 1、原料羊胎标准：根据山羊各期特征的比较确定了二月龄左右的山羊胚胎，胎长为5.0-10.0cm，胎重8-40g，头长1.5-3cm，颈长0.5-2.0cm，尾长1-2.5cm，前肢长1.5-3cm，耳廓长0.2-0.5cm。 2、主要工艺标准：通过初步比较透析和超滤工艺，选择获取成分较多且活性相对较高的超滤作为主要工艺。 3、鉴别和检查指标标准：通过理化性质、紫外光谱、分子量指标、指纹图谱、水解氨基酸指标五部分进行确定。 （1）理化性质指标：按照《中华人民共和国药典》（2000版）附录和《生物制品规程》（2000版）所载方法对连续10批低分子量羊胎免疫调节因子的颜色、澄明度、pH值、多肽含量及核酸含量、蛋白定性反应进行鉴定。 结果表明：低分子量羊胎免疫调节因子为微黄色澄明液体，具有可透析性，可超滤性：pH值位于6.5～7.0之间；其中所含多肽含量＞600ug／ml；核酸含量＞600ug／ml；蛋白质定性反应确定低分子量羊胎免疫调节因子呈现蛋白阴性特征。 （2）紫外光谱指标：在230nm～330nm之间作紫外扫描分析，发现10批次低分子量羊胎免疫调节因子的特征吸收峰均在251±2nm处，而且A_260nm／A_280nm＞2.0。 （3）分子量指标：利用凝胶色谱法确定低分子量羊胎免疫调节因子分子量范围，10批次低分子量羊胎免疫调节因子中均检测不到3000Da以上的物质。 （4）指纹图谱指标：通过反相高效液相色谱（Reversed-Phase High-performance Liquid Chromatography，RP-HPLC）多种参数的比较，探讨 理学硕士学位论文：低分子公羊胎免疫调节因子质量标准的初步研究了流动相、梯度、流速、温度、波长各种条件对未知混合成分分离的影响，建立了最佳色谱测定方法；并对连续10批样品进行指纹图谱分析；在此前提下，对主要成分峰进行进一步分析：用二极管阵列检测器检测到各峰不同时间段的光谱扫描图，同时结合部三酮反应的结果，对各主要成分峰的纯度和性质作了初步判断；另外，通过指纹图谱，初步分析温度及放置时间对样品稳定性的影响。 获得的 17个主要成分峰中，其中大于总面积 5％的峰有 7个，方法精密度实验说明各峰保留时间的相对标准偏差均可控制在0．3％以下，峰面积和峰高则在5．0％和生0％以内：并且10批次样品指纹图谱相似度都与1接近；进一步的主要成分峰分析发现17个分高峰中有8个是纯峰，其中可初步辨别8种成分为多肽类，9种为核酸类；稳定性研究发现七0oC及20eC下放置2小时、4小时、6小时、8小时、12小时、24小时的样品在相同色谱条件下指纹图谱相似度均高于0．995，出峰没有显著变化。 ，（5）水解氨基酸指标：利用氨基酸自动分析仪对水解氨基酸进行分析，证实了低分子量羊胎免疫调节因子中含十七种水解氨基酸。 4、免疫活性试验：通过活E玫瑰花结和淋巴转化实验初步验证了低分子量羊胎免疫调节因子具有细胞免疫调节功能。 5、安全性标准：选择无菌试验、过敏试验、异常毒性试验和热原质反应对其进行评价，说明低分子量羊胎免疫调节因子对实验动物并无任何毒副作用，。 综上所述，通过原料、工艺的选择和一系列定性定量指标的确定，为制定完整低分子量羊胎免疫调节因子的质量标准提供了必要的实验数据；同时建立了控制质量标准较精确的定性定量指标——指纹图谱，也能为类似动物生化药物提供质量控制的方法学参考：另外，实验显示出低分子量羊胎免疫调节因子是安全、有效、可控的，为其开发为一种新型生物制品提供了必要的依据。更多还原

【Abstract】 In this article, how to control the quality standard of the goat embryo immunoregulatory factor(GEIF) of low molecular weight has been systematic studied. And we have developed a Chromatographic conditions and ways to detect the Chromatographic Fingerprints, We also have analysed attributes and stabilities of main componentes.1 , The standard of raw material : according to the comparative study of the characteristics of different stage of goat embryo development , the goat embryo about 2 month old has been identified, That is: embryo length for 5.0-15.0 cm, embryo weight for 10-50 g, head length for 1.5-3.5 cm, neck length for 0.4-2.0 cm, tail length for 1-2.5 cm, foreleg length for 1.5-3.5 cm, pinna length for 0.3-0.6 cm.2, The standard of main technology: through comparing the results of dialysis with ultrafiltration ,we found the latter may obtain more compositions with high activity. So the ultrafiltration has been choosed as the main craft.3, The standard of identification: it has been identified according to physiochemistry , UV spectrum , molecular weight , Chromatographic Fingerprints and amino acid analysis.(1) The standard of physiochemistry: According to methods from the Chinese pharmacopoeia (2000 version) and discipline of biological substance (2000 version) , we identified the colon diaphaneity, pH, the content of polypeptide and nucleic acid, protein reactionThe results showed that the GEIF is a clear and yellow liquid , pH is 6.5~ 7.0. and has the characteristic of dialysis and ultrafiltration, the content of polypeptides and nucleic acids are more than 600 ug / ml respectively. Sulfosalicylic acid Reaction appeared no protein.(2) The standard of UV spectrum: GEIF of ten samples obtained from different time have the character absorption peak at 251 ? nm and the ratio of A260nm to A28onm is higher than 2.0.(3) The standard of the molecular weight: the molecular weight of GEIF of ten samples obtained from different time was all under 3000 Da.(4) The standard of the Chromatographic Fingerprints: We have analysed GEIF by Reversed-Phase High-performance Liquid Chromatography (RP-HPLC) and studied the influence of mobile phase, gradient flow-rate, temperature and wavelength to separate unknown composition and developed the best condition of HPLC; Under this condition, we have analysised the Chromatographic Fingerprints of ten samples; The purity and characteristic of main components was analysised through spectrum scanning diagrams detected by Diode Array Detector(DAD), and Ninhydrin Reaction; Moreover, the Chromatographic Fingerprints at different times(2 h,4 h,6 h,8 h,12 h,24 h) under two temperatures(-20癈and 20癈) were analysised, respectively.Among the 17 separated peaks, we got the area above 5% of total area have 7 peaks. As to the precision , all the standard deviation of reserve-time can be controlled below 0.3%, The area and the height of peaks below 5.0% and 4.0% respectively ; Comparing the Chromatographic Fingerprints of ten samples , the similarities are near to 1; We found 8 pure peaks among these. Moreover, 8 peaks were recognized to have the compositions of peptide, 7 peaks have the nucleic acid and 2 peaks have nucleic acid peptide. Similarities of the Chromatographic Fingerprints obtained by stability experiments are all higher than 0.995.(5) The standard of amino acids: though analysed the amino acids of GEIF by Amino acids Analysis Meter, we found it contained 17 kinds of hydrolysed amino acides, including Asp, Thr, Ser, Glu, Pro, Glu, Ala, Val, Cys, Met, lle, Leu, Tyr, Phe, His, Lys and Arg.4, The standard of bioactivity: The cell-mediated immunoregulatoryfunction was identified by E rosette formation and T lymphocyte transformation5, The standard of reliability: according to the sterile experiment, theallergy experiment, the excrescent poison experiment, the pyrogen respondexperiment, proved that the GEIF of ten samples had no poison side effects.In conclusion, the experiment data for更多还原