聚乙二醇修饰蛋白药物研究进展

迄今。利用生物技术已开发成功了70多种重要治疗药物,年销售额已超过300亿美元,生物技术药物中大多为蛋白质类药物。在临床上,蛋白质类药物既有作用位点专一、疗效明确的优点,也有多项缺点,如在胃肠道内极易被蛋白酶水解,一般仅限注射给药;血浆半衰期较短,需反复多次注射,抗原性较强,易引起过     

聚乙二醇化天花粉蛋白注射液中天花粉蛋白的HPLC测定

建立了HPLC法检测聚乙二醇化天花粉蛋白注射液中天花粉蛋白的含量。采用硅胶基TSK-G3000SW凝胶柱，0．02mol／L磷酸二氢钠溶液-0．15mol／L氯化钠溶液-95％乙醇（45：45：10）为流动相，检测波长222nm。天花粉蛋白在5～50μg／ml范围内线性关系良好。平均回收率为100．0％，RSD为1．8％。     

蛋白药物聚乙二醇修饰技术研究进展

近年来,聚乙二醇修饰技术已经成为改良蛋白药物最有效的技术之一,得到越来越广泛的应用。目前已经有十余种聚乙二醇修饰的蛋白药物上市,在临床疗效和安全方面有优良表现。此文综述了蛋白药物聚乙二醇修饰技术的发展,特别是聚乙二醇定点修饰的新技术和新方法,并展望了聚乙二醇修饰技术的发展方向。     

聚乙二醇衍生物及其蛋白药物修饰研究进展

聚乙二醇及其衍生物因其出色的亲水性、生物相容性、生物学惰性等特性而被广泛应用于蛋白药物修饰,其修饰可有效降低蛋白药物的免疫原性并延长体内半衰期。聚乙二醇衍生物的发展经历了第一代随机修饰,第二代特异性和功能性修饰,以及第三代分支型结构的应用。其应用也从简单的药物修饰扩展到生物传感、药物传输等方面。     

天花粉蛋白的核糖体灭活活体部位

目的：天花粉蛋白核糖体灭活活性部位的定位。方法：羟胺特异裂解天花粉蛋白唯一Ａｓｎ－Ｇｌｙ肽键。制备性凝胶电泳获ＨＡＴｆ１和ＨＡＴｆ２二片段。免疫印迹确定天花粉蛋白上不同表位并筛选抗体。兔网织红无细胞系统测定天花粉蛋白及片段对蛋白合成的抑制活性。结果：ＨＡＴｆ１和ＨＡＴｆ２纯度各达９６．６％和８０．５％。ＨＡＴｆ１和ＨＡＴｆ２纯度各达９６．６％和８０．５％。ＨＡＴｆ１保留完整天花粉蛋白的抑制活性。第     

天花粉蛋白中期引产孕妇心理护理几点体会

中期引产术已成为贯彻计划生育政策的必要的补救措施,我站自1993年1月—1994年1月中,妊娠在12—24周要求中止妊娠而无禁忌者行天花粉蛋白引产共68例,其中年龄最大的是37岁,最小的年龄20岁,未婚3例,初孕妇8例,根据心理学分析,孕妇中出现愤怒、恐惧、焦虚、忧郁、悲伤等不良情绪,均可使孕妇产生或诱发各种疾病,导致剧烈或     

结晶天花粉蛋白宫颈注射引产125例临床分析及病理探讨

结晶天花粉蛋白是从瓜蒌的鲜根中提炼出的植物蛋白,有良好的抗生育作用。我院于1989年1月～1990年7月甩结晶天花粉蛋白注射宫颈引产125例,效果满意,现介绍如下。一、一般资料本组年龄最小14岁,最大42岁,平均27.1岁。在125例中,初孕妇26例,经孕妇99例;怀孕8～11周20例,12～15周93例,16～20周12例;哺乳期8例,剖宫产8例,近期人流19例,多     

聚乙二醇修饰药物技术的研究进展

聚乙二醇修饰技术已经成为改善蛋白质药物和非蛋白质药物临床效果的重要手段。此文综述了国内外聚乙二醇修饰药物技术的研究现状和发展趋势。     

蛋白质药物的聚乙二醇修饰

蛋白质药物经聚乙二醇修饰后其性质会发生多种变化。本文综述了该项技术的国内外研究现状，包括聚乙二醇修饰的原理与方法，修饰对蛋白质性质的影响，修饰方法的生物优化，有关的鉴定与检测方法及修饰产品的开发与临床应用状况。     

蛋白药物聚乙二醇化修饰技术的研究进展

蛋白质药物主要有蛋白质激素和干扰素、血浆蛋白质、蛋白质类生长调节因子和神经营养因子、黏蛋白、胶原蛋白、碱性蛋白、蛋白酶抑制剂和植物凝集素、白细胞介素、集落刺激因子等[1].蛋白质药物由于具有作用专一、高效等特点,对人类健康发挥着重要作用,其原料有动植物细胞和微生物细胞等非人体来源.     

聚乙二醇对溶菌酶和粒细胞集落刺激因子的初步化学修饰

目的考察聚乙二醇 (PEG)修饰对溶菌酶和粒细胞集落刺激因子 (G CSF)活性的影响。方法以不同方法活化的PEG修饰溶菌酶 ,通过正交试验和单因素考察确定合适的修饰条件 ;溶菌酶活力测定采用溶壁小球菌法 ,抗原性测定采用试管沉淀法 ;G CSF活性测定以小鼠血浆中中性粒细胞数目增殖情况反映。结果当偶联上一个PEG分子时 ,溶菌酶的活性保留 13% ,抗原性显著减弱 ;G CSF经PEG修饰后对小鼠中性粒细胞数目的增加有显著促进作用 ,且作用时间明显延长。结论PEG修饰对G CSF血循环半衰期的延长有显著作用     

甲氧基聚乙二醇苯并三唑修饰对淋巴细胞功能的影响

目的　研究甲氧基聚乙二醇 苯并三唑 (mPEG BTC)修饰淋巴细胞表面人类白细胞抗原 (HLA)对细胞有无损伤。方法　利用微量淋巴细胞毒性实验和单向混合淋巴细胞培养来检测mPEG BTC的修饰效果 ;利用电镜观察修饰前后淋巴细胞的形态 ;通过淋巴细胞转化实验及培养上清液的IL 2含量的检测对淋巴细胞的增殖、分化及分泌功能进行评价 ;检测淋巴细胞表面CD分子评价淋巴细胞的抗原识别功能 ;体外保存淋巴细胞检测其寿命 ;对淋巴细胞染色体进行分析 ,评价mPEG对细胞遗传物质的影响。结果修饰后淋巴细胞的微量淋巴细胞毒性实验结果 ,淋巴细胞转化率 ,淋巴细胞相对转化指数 ,IL 2分泌含量分别由修饰前的 (8.0± 0 )分 ,(6 3.6± 7.8) % ,(1.0 7±0 .2 9) ,(38± 11)ng·L- 1降至 (1.1± 0 .3)分 ,(0 .2±1.6 ) % ,(0 .3± 0 .11) ,(11± 3)ng·L- 1。CD2 +,CD4 +,CD8+,CD5 8+分子荧光强度亦有不同程度降低 ,而mPEG BTC修饰对淋巴细胞的形态、寿命及遗传物质无明显改变。结论　mPEG BTC修饰可阻断HLA介导的特异性免疫反应 ,降低淋巴细胞的增殖、抗原分泌能力及分泌 ,但对其形态、结构、遗传物质及寿命无明显影响。     

Antibodies against polyethylene glycol in healthy subjects and in patients treated with PEG-conjugated agents

In contrast to the accepted general assumption that polyethylene glycol (PEG) is non-immunogenic and non-antigenic, animal studies clearly showed that uricase, ovalbumin and some other PEGylated agents can elicit antibody formation against PEG (anti-PEG). In humans, anti-PEG may limit therapeutic efficacy and/or reduce tolerance of PEG-asparaginase (PEG-ASNase) in patients with acute lymphoblastic leukemia and of pegloticase in patients with chronic gout, but did not impair hyposensitization of allergic patients with mPEG-modified ragweed extract or honeybee venom or the response to PEG-IFN in patients with hepatitis C. Of major importance is the recent finding of a 22 – 25% occurrence of anti-PEG in healthy blood donors, compared with a very low 0.2% occurrence two decades earlier. This increase may be due to an improvement of the limit of detection of antibodies during the years and to greater exposure to PEG and PEG-containing compounds in cosmetics, pharmaceuticals and processed food products. These results raise obvious concerns regarding the efficacy of PEG-conjugated drugs for a subset of patients. To address these concerns, the immunogenicity and antigenicity of approved PEGylated compounds should be carefully examined in humans. With all these data in hand, patients should be pre-screened and monitored for anti-PEG prior to and throughout a course of treatment with a PEGylated compound. Finally, protein conjugates with the poorly immunogenic hydroxy-PEG sequence or other hydrophilic polymers are in early phases of development and may represent an alternative to immunogenic PEGylated proteins.     

天花粉蛋白对人宫颈癌HeLa细胞增殖和细胞调亡的影响

天花粉蛋白(Trichosanthin,TCS)是从葫芦科植物栝(蒌)的块根中提取出来的一种单链核糖体失活蛋白(ribosome-in-activating protein,RIP),主要引发中期流产,抑制爱滋病病毒(HIV)增殖,调节人体免疫功能等多方面的作用[1].     

Conformation similarities of ricin A-chain and trichosanthin

The conformation of ricin A-chain from castor bean was studied by circular dichroism at pH 4.7, 7 and 9 and compared with that of trichosanthin from the Chinese herb Tianhuafen. The CD spectra of ricin A-chain and trichosanthin were nearly identical at each of the three pHs. Analysis of the data indicated that, like trichosanthin, ricin A-chain had about 29%α-helix and 42%β-sheet but no β-turn. However, there was a subtle difference in the CD spectra in 20 mm sodium dodecyl sulfate, the addition of which at pH 7 slightly increased the helicity and decreased the content of β-sheet of ricin A-chain in contrast to a larger increase in helicity at the expense of β-sheet for trichosanthin, thus indicating a different stability against the surfactant. Native ricin A-chain and trichosanthin had about the same amount of secondary structure, which supports the belief that a high degree of sequence homology of the two proteins [Zhang & Wang (1986) Nature 321, 477–478] may lead to a conformational similarity between them, even though the two proteins are not taxonomically related.     

High level synthesis of biologically active recombinant trichosanthin in Escherichia coli

Two forms of recombinant trichosanthin (rTCS) were synthesized in high levels in Escherichia coli by putting the TCS cDNA under the control of a T7 RNA polymerase-directed promoter. Purification schemes were developed to isolate the recombinant protein from both soluble and insoluble fractions. Form I rTCS possessed the mature TCS sequence and had similar biological activities as the natural protein. Its IC₅₀ was approximately 0.13 nm in an in vitro rabbit reticulocyte translational system and a dose of around 35 μg protein per 25 g body weight was sufficient to induce complete abortion in mice. Form II rTCS had a propeptide of 19 aa at the C-terminus and was five times less active than Form I in inhibiting protein synthesis by a rabbit reticulocyte lysate.     

Quantitation of free polyethylene glycol in PEGylated protein conjugate by size exclusion HPLC with refractive index (RI) detection

In this study, size exclusion high performance liquid chromatography was evaluated for its application in separation and quantitation of free polyethylene glycol (PEG) and its PEGylated-protein-conjugate (PEG-conjugate). Although the large mass of the free PEG (2-fold greater than the protein) made separation difficult, chromatographic conditions were identified enabling resolution and quantitation of the free PEG, PEG-conjugate and non-PEGylated protein with Shodex Protein KW803 and KW804 columns in series and refractive index detection. The optimum resolution of 1.7 and 2.0 was achieved for the free PEG and PEG-conjugate as well as the free PEG and non-PEGylated protein using 20 mM HEPES buffer at pH 6.5. Under this condition, the plot of log₁₀MW of all the pertinent analytes against retention time showed a linear relationship with a correlation coefficient of 1. Limited assay performance evaluation demonstrated that the method was linear in the concentration range of 10 to 250 μg/mL of free PEG with correlation coefficients of ≥0.99. When free PEG in this concentration range was spiked into PEG-conjugate samples at 1 mg/mL, the recovery was in the range of 78%–120%. Detection and quantitation limits were determined to be, respectively, 10 and 25 μg/mL for free PEG. The R.S.D. for intra- and inter-day precision was 0.09% or less for retention time measurements and 2.9% or less for area count measurements. Robustness testing was performed by deliberately deviating ±0.2 pH units away from the desired pH as well as by increasing the flow rate. These deviations resulted in no significant impact on area percent distribution of all species. However, separation was found to be sensitive to high ionic strength and buffer species.     

肿瘤微环境双重响应性的智能型蛋白毒素给药系统的构建和表征

目的 利用蛋白重组技术和PEG定点修饰技术,制备具有肿瘤微环境双重响应性的智能型蛋白毒素给药系统。方法 利用基因重组技术,在天花粉蛋白(trichosanthin,TCS)的C端引入天冬酰胺内肽酶(legumain)的底物天冬酰胺肽段和半胱氨酸残基,将所构建的突变体转化到大肠杆菌中表达目的蛋白并纯化。进一步将重组蛋白末端的半胱氨酸与具有巯基反应性的mPEG-Hz-Mal偶联合成TCS-Asn10-Hz-PEG,采用弱阳离子交换柱纯化TCS-Asn10-Hz-PEG,并在体外考察了TCS-Asn10-Hz-PEG的酸敏感性和酶敏感性。结果 成功制备、分离和纯化得到了TCS-Asn10-Cys,完成了mPEG-Hz-Mal与TCS-Asn10-Cys的定点偶联,得到智能型蛋白毒素给药系统TCS-Asn10-Hz-PEG。TCS-Asn10-Hz-PEG在体外pH 5.6的介质中和天冬酰胺内肽酶的作用下,能够水解或酶解释放出TCS,具有酸敏感特性和酶敏感特性。结论 本实验设计的蛋白毒素给药系统,具有酸敏感和酶敏感双重响应特性。     

A Sixty-Year Research and Development of Trichosanthin,a Ribosome-Inactivating Protein

Tian Hua Fen, a herbal powder extract that contains trichosanthin (TCS), was used as an abortifacient in traditional Chinese medicine. In 1972, TCS was purified to alleviate the side effects. Because of its clinical applications, TCS became one of the most active research areas in the 1960s to the 1980s in China. These include obtaining the sequence information in the 1980s and the crystal structure in 1995. The replication block of TCS on human immunodeficiency virus in lymphocytes and macrophages was found in 1989 and started a new chapter of its development. Clinical studies were subsequently conducted. TCS was also found to have the potential for gastric and colorectal cancer treatment. Studies on its mechanism showed TCS acts as an rRNA N-glycosylase (EC 3.2.2.22) by hydrolyzing and depurinating A-4324 in α-sarcin/ricin loop on 28S rRNA of rat ribosome. Its interaction with acidic ribosomal stalk proteins was revealed in 2007, and its trafficking in mammalian cells was elucidated in the 2000s. The adverse drug reactions, such as inducing immune responses, short plasma half-life, and non-specificity, somehow became the obstacles to its usage. Immunotoxins, sequence modification, or coupling with polyethylene glycerol and dextran were developed to improve the pharmacological properties. TCS has nicely shown the scientific basis of traditional Chinese medicine and how its research and development have expanded the knowledge and applications of ribosome-inactivating proteins.