目的:建立一种检测大鼠血清中聚乙二醇化重组蛋白（PEG-SOP55）浓度的ELISA方法,并进行该蛋白的药代动力学初步研究。方法:以该蛋白的多抗包被,标记生物素的单抗检测,生物素-链霉亲和素-辣根过氧化物酶两级酶联放大检测信号,构建双抗夹心ELISA法进行浓度检测。通过对包被抗体和检测抗体工作浓度的优化实验,血清基质的干扰实验,不同结构聚乙二醇分子的比较实验,建立该方法的基本实验条件。并参考相关法规,对其方法特异性、准确度、精密度、耐用性、检测限度以及样品稳定性进行验证。分别用PEG-SOP55 30kDa 、PEG-SOP55 40kDa 、PEG-SOP55 40kDa branched 进行Lewis大鼠腹腔单次给药然后测定血清浓度,获得该蛋白用不同分子结构聚乙二醇分子修饰后的药代动力学参数。结果:实验证实聚乙二醇修饰降低了检测抗体和目标分子的结合效率,且大鼠血清和聚乙二醇分子结构变化对检测有干扰。通过提高包被抗体浓度,获得对目标分子较好的捕获效率。通过将血清样品稀释液从PBS替换为咪唑缓冲液,且保持标准曲线和待测样品血清稀释倍数一致,克服了血清基质所带来的干扰。通过保持标准曲线和待测样品聚乙二醇分子结构一致,克服了因聚乙二醇分子结构不同带来的干扰。方法验证结果表明该方法特异性良好,回收率为（100±15.2）%,实验内精密度和实验间精密度验证数据RSD均不超过20%,不同操作人员对检测结果无显著影响,标准曲线线性良好,标准曲线的最高和最低浓度能够准确测定。将大鼠血清样品反复冻融3次,在室温放置4 h其稳定性均符合要求,满足该方法检测条件。通过对Lewis大鼠血清样品进行浓度测定,获得PEG-SOP55 30kDa 、PEG-SOP55 40kDa 、PEG-SOP55 40kDa branched 的药代动力学参数。其C max 分别为31.9、54.4、67.7 mg·m L -1 ,AUC 0-∞ 分别为823.0、1 978.0、3 502.6 mg·h·m L -1 ,t 1/2 分别为15.6、22.3、30.6 h。结论:成功建立检测PEG-SOP55在大鼠血清中浓度的ELISA方法。通过方法验证表明:该方法专属性、准确度、精密度、线性以及耐用性良好,测定范围为5~80 ng·m L -1 。大鼠血清样品稳定性满足该方法检测要求,该方法可作为PEG-SOP55药代动力学研究的检测方法,也为类似制品的血清浓度检测提供借鉴。初步药代动力学研究表明,随着聚乙二醇分子对蛋白的屏蔽作用增强,能够显著延长其半衰期,提高药物在体内的暴露量。

Objective To construct a fully human single-chain variable fragment (scFv) DNA library and select anti-epidermal growth factor receptor (EGFR) scFvs using ribosome display technology.Methods A fully human scFv library was constructed with peripheral blood samples collected from 52 advanced cancer patients.Eukaryotic ribosome display was used to screen anti-EGFR scFvs from the library.The selected scFvs were then expressed in E.coli and purified.Their binding affinities were validated by ELISA.Results The size of constructed human scFv DNA library was 4.3×1013.After 3 rounds of selection,49 unique clones were recovered.The top two binders were further purified and their binding affinities were determined by ELISA to be 108-107mol/L.Conclusion Ribosome display is an efficient screening method to obtain high affinity scFvs that may serve as potential therapeutic candidates.

Objective To optimize and validate the enzyme linked immunosorbent assay(ELISA)for hamster-susceptible viruses used for production of live attenuated Japanese encephalitis(JE) vaccine.Methods The ELISA was optimized,and used for determination of the reovirus 3(Reo3),pneumonia virus of mice(PVM),Sendai virus(SV)and lymphocytic choriomeningitis virus(LCMV) in sera of hamster to validate the reproducibility,intermediate precison and specificity,based on which the acceptable criteria for validation parameters was established according to the requirements in WHO /VSQ / 97.02.Results The optimized ELISA showed high reproducibility,intermediate precison and specificity,of which the various validation parameters met the designed acceptable criteria.Conclusion The optimized ELISA may be used for the quality control of live attenuated JE vaccine.

Objective To develop a simple and reliable method for monitoring the free thiol concentration during refolding of recombinant human tumor necrosis factor receptorⅠ(TNFRⅠ)protein to guide and monitor the refolding process and the judgment of endpoint. Methods The linear ranges and correlation coefficients of three methods were determined,based on which a method suitable for determination of free thiol concentration in refolded TNFRⅠ solution was developed.The thiol concentration in TNFRⅠ during refolding as well as its correlation to the biological activity of refolded protein were monitored by the method. Samples were taken from protein 48,96 and 114 h after refolding and evaluated for stability after storage at 4 ℃ for 3 and 6 d. Results The standard error value(STDEV)of test results by method 3 was the lowest in the three methods,while the linear range was 0 ~ 12. 5 mmol / L,R2= 0. 999 9,indicating that the method was more suitable for monitoring the thiol concentration in refolded protein solution. The thiol concentration showed a sig-nificantly decreasing tendency with the increasing hours for refolding(R2= 0. 989 1). The biological activity of refolded protein increased rapidly during decrease of thiol concentration from 2 to 1 mmol / L,while reached a peak value when the thiol concentration decreased to 1 mmol / L or below. The cell activity was stable 144 h after refolding of protein.Conclusion A simple and reliable method for determination of thiol concentration was developed,which provided an important reference for increasing the refolding efficiency of inclusion body.

目的:以重组肿瘤坏死因子-α受体1（sTNFα-R1）的基因序列为模板,构建同义密码突变库,在不改变原氨基酸序列的同时筛选获得目标蛋白产量提高的突变菌株。方法:分段设计并合成同义密码突变兼并引物,通过优化PCR反应条件,构建目的基因的完整同义密码突变库。突变库经双酶切插入到pET11（b）载体上,并电转至E.coli BL21（DE3）细胞中。阳性克隆经PCR鉴定后,细胞表达目标蛋白,应用SDS-PAGE等方法分析目标蛋白产量。结果:目的基因同义突变库的PCR扩增产物可见330bp的特征条带,突变库测序结果显示整个基因序列的第三位碱基发生预期的同义突变;完成了445株克隆筛选,针对其中产量高于对照组的45株克隆进行测序,获得42株基因序列不同但氨基酸序列未发生变化的克隆菌株,其同义密码突变阳性率高达93%;选择其中2株初筛产量较高的菌株进行放大验证,结果显示7#突变株的包涵体收率及目标蛋白相对百分含量明显高于对照株,其每升发酵液的目标蛋白总产量比对照株提高2.44倍,405#突变株比对照株提高1.44倍。结论:通过优化PCR反应条件成功构建了目标基因全序列的同义密码突变库,采用同义密码突变库技术途径,在不改变原氨基酸序列的前提下可以快速优化蛋白表达产量。

目的 采用DoE设计方法对人可溶性肿瘤坏死因子Ⅰ型受体(soluble tumour necrosis factor αreceptors Ⅰ,sTNFα-RⅠ)的聚乙二醇(PEG)修饰工艺参数进行优化,以期获得稳定、可控的工艺参数.方法 采用UNICORN(DoE)软件中Central Composite Face Centered(CCF)模型对sTNFα-RⅠ的3个PEG修饰工艺参数,即溶液pH值(pH)、反应时间(Time)和PEG与蛋白质量比(PEGrate)进行优化,设置4个响应值(多PEG修饰蛋白峰面积、单PEG修饰蛋白峰面积、未修饰蛋白峰面积及单PEG修饰蛋白峰面积与多PEG修饰蛋白峰面积比值)揭示其化学反应过程,确定参数操作空间;应用蒙特-卡罗模拟法(Monte-Carlo Simulation)对优化参数进行风险评估.结果 3因子17组试验结果的重复性较好,中心点重复组响应值介于最低值和最高值之间,且随机分布,符合DoE试验设计的要求;4个响应值所对应的回归系数(R2)均＞0.75,预测准确性(Q2)＞0.5,模型有效性(Model Validity)＞0.25,重复性(Reproducibility)＞0.5,模型预测与实验值之间偏差较小,具有可靠的预测准确性;17组试验结果标准残差在允许范围(-4～4)之间,线性较好,预测值与观测值之间相关性较好,建立的模型拟合度高,可较为准确地预测试验结果.溶液pH值对多PEG修饰产量影响较小,随着Time和PEGrate的升高,多PEG修饰产物增加,单PEG修饰产物经二次或多次修饰形成多PEG修饰产物,减少Time和PEG用量可减少多PEG修饰产物;单PEG修饰产物随着pH上升,产量有所增加,与Time呈二次项关系,在Time· PEGrate交互作用中,减少PEG用量可增加单PEG修饰产物量;减少Time和降低PEG用量可提高单PEG修饰产物与多PEG修饰产物的比值,有利于下游纯化.确定了工艺参数的操作空间,当pH为5.5时,反应时间控制在30 h以上,PEG与蛋白质量比控制在3.7以下,符合预设值;pH在6.5时,反应时间控制在22～35 h之间,PEG与蛋白质量比控制在2.5 ～4.0之间,符合预设值;蒙特-卡罗模拟法对在最佳操作参数(反应时间34.1h,溶液pH值6.5,PEG与蛋白质量比2.5)时模型预测的响应值(单PEG修饰产物峰面积值5 635.82和单PEG修饰产物与多PEG修饰产物比值3.226 5)的风险评估,10万次模拟试验可信度分别为99.825％和99.982％,试验风险较小,工艺可控.结论 确定了sTNFd-R Ⅰ蛋白PEG修饰稳健的工艺参数.

Objective: To optimize the technological parameters for PEGylation of soluble tumor necrosis factor α receptor I (sTNFα-R I) by Design of Experiment (DoE) so as to obtain stable and controllable parameters. Methods: Three technological parameters of PEGylation of sTNFα-R I , pH, time and PEGrate, were optimized by using the Central Composite Face Centered (CCF) model in UNICORN (DoE) software, based on which the chemical reaction process was indicated by four response values, i.e. peak area of multiple PEGylation product, peak area of single PEGylation product, peak area of unmodified protein as well as ratio of peak area of single PEGylation product to that of multiple PEGylation product, and the control space of the parameters were determined. The risks of optimized parameters were evaluated by Monte-Carlo Simulation method. Results: The results of 17 groups of tests on three technological parameters showed high reproducibility. The response value of central point repeat group was between the minimum and maximum and distributed randomly, which met the requirement for DoE. All the regression coefficients (R2) corresponding to the four response values were more than 0.75, while the predicted accuracy (Q2) was more than 0.5, the model validity was more than 0.25, and the reproducibility was more than 0.5. The deviations between the predicted and measured values were small, indicating a reliable accuracy. The residual standard deviations of 17 groups of test results were within the permitted range (-4-4), and good linearity was observed. The predicted value showed high relationship to the measured value, indicating high degree of fitting of the established model which was suitable for accurate prediction of test result. The pH value of solution showed no significant effect on multiple PEGylation rate. However, along with the increasing time and PEGrate, multiple PEGylation rate increased, and single PEGylation product formed multiple PEGylation product by PEGylation for 2 times or more. Decreasing time and PEGrate decreased multiple PEGylation product. Single PEGylation product increased with the increasing pH value, which showed a quadratic term relationship to the time. In Time • PEGrate interaction, decreasing PEG used increased the single PEGylation product, while decreasing time and PEG used increased the ratio of single PEGylation product to multiple PEGylatioin product, which was beneficial to downstream purification. The control spaces of the parameters were determined. At pH 5.5, the reaction time was controlled at more than 30 h, while PEGrate at less than 3.7, which were consistent with the designed values. However, at pH 6.5, the reaction time was controlled at 22-35 h, while PEGrate at 2.5-4.0, which were consistent with the designed values. The optimal reaction time, pH value and PEGrate were 34.1 h, 6.5 and 2.5 respectively. Under the optimal condition, the peak area of single PEGylation product was 5 635.82, while its ratio to that of multiple PEGylation product was 3.226 5. The reliabilities of the two response values evaluated by Monte-Carlo Simulation method in 100 000 simulation tests were 99.825% and 99.982% respectively, indicating low risk of test and controllability of procedure. Conclusion: Stable technological parameters for PEGylation of s TNFα-R I were determined.