计算机辅助设计使重组ricin-A链在E.coli中的高效表达

基于对螺旋区随机堆积的ＲＮＡ二级结构的预测和密码子偏性计算，建立了ｐＢＶ２２０载体中外源基因高效表达的判别模型。该模型认为，外源基因的表达水平，与其５′端３０３９区域，以及３′端３０３９区域的二级结构自由能具有显著的统计学意义；其次与３′端９ｂｐ的局部密码子偏性相关。据此模型，我们设计了用于克隆表达ｒｉｃｉｎＡ链的引物。将ＰＣＲ扩增的基因克隆入ｐＢＶ２２０载体中，在ＤＨ５α宿主中获得了高效表达（表达水平在２０％以上），而且在ｐＥｘＳｅｃＩ载体系统中亦获得了高效表达。

家蚕核糖体的提取及其Sarcin/Ricin结构域的初步鉴定

用超速离心结合密度梯度离心法从蚕蛹中提取家蚕80S核糖体,经尿素变性聚丙烯酰胺凝胶电泳,结果表明家蚕核糖体的RNA同大鼠核糖体RNA在序列长度上有明显差异,家蚕5.8S rRNA高于大鼠5.8S rRNA,而家蚕5S rRNA的序列长度则略低于大鼠。用核糖体失活蛋白R ic in分析鉴定家蚕核糖体的Sarc in/R ic in结构域,结果是家蚕Sarc in/R ic in结构域比大鼠Sarc in/R ic in结构域离28S rRNA的3′末端更近。

IgY-Ricin A诱导癌细胞程序化死亡的研究

研究了抗癌导向药物IgY-Ricin A杀伤癌细胞的作用机理。人胃低分化粘液腺癌MGC-803细胞经IgY-Ricin A处理,细胞的增殖明显受到抑制,而同样处理的人胚正常肺细胞2BS,其生长不受这种药物的影响。FCM实验结果表明,IgY-Ricin A处理的MGC-803细胞,在8h开始出现细胞程序化死亡峰Apo,同样处理的2BS细胞则无Apo峰出现。经lgY-Ricin A处理的MGC-803细胞核由均一状态变为浓缩凝集状,经激光共聚焦显微镜进行光切片和三维重组,发现MGC-803细胞核由原来的球形变成高度浓缩凝集的点状结构。DNA凝胶电泳分析显示,IgY-Ricin A处理的MGC-803细胞DNA被降解,呈现梯状电泳条带这一典型的细胞程序化死亡指标。研究结果表明,IgY-Ricin A通过诱导细胞程序化死亡来控制MGC-803细胞的增殖,最终杀死癌细胞。

抗ricin中和性单抗4C13单链抗体的真核表达及生物活性检测

目的:实现基于中和性单抗4C13单链抗体的真核表达,并对其生物学活性进行评价.方法:用TRIzol提取抗ricin中和性单抗杂交瘤细胞4C13总RNA,扩增其轻、重链可变区基因,登陆GenBank;用Linker(GGGGS)3将VH和VL连接,用重叠延伸PCR克隆入真核表达载体pCDNA3.1,用脂质体转染293T细胞,对瞬时表达产物进行生物学活性检测.结果:中和性单抗杂交瘤细胞4C13轻、重链可变区基因在GenBank中的登录号为DQ389248和DQ389245,ScFv与ricin可特异性结合,培养上清原液和2倍稀释液的抑制率分别为33.7%和29%;ScFv在ricin浓度为0.01～0.005ng/mL时可中和ricin对SP2/0细胞的细胞毒,随着ricin浓度逐渐增大,细胞存活率逐渐减低.结论:研究结果为研制基于中和性单抗的其他ricin拮抗剂奠定了重要的理论和实践基础.

深海热液区管状蠕虫Ridgeia piscesae中Ricin B-lectin型结构域基因的克隆与表达

作为一种重要的模式识别因子,凝集素在多个领域中均有着广泛的应用价值,因此,开发和利用新型凝集素将具有重要意义.本研究以生活在深海热液区极端环境条件下的管状蠕虫Ridgeia piscesae为材料,首次从管状蠕虫中克隆获得Ricin B-lectin基因rgal.序列分析表明,RGAL与已知序列的相似性较低,其含有2个Ricin B-lectin型结构域,并且该结构域具有该家族所特有的β-三叶草形三维结构.多方面的分析结果显示RGAL可能是一个新颖的凝集素蛋白.对RGAL进行了原核重组表达,并对其复性条件进行了摸索优化,成功获得了其可溶性表达产物,为后续RGAL的生物活性分析奠定了坚实的基础.

Ricin引起外周神经跨越神经节溃变的超微结构研究

为了探索神经毒剂Ricin对神经元溃变过程中的效用及最佳用量的效果,将10%Ricin注射于坐骨神经分支中,按梯次剂量将实验动物分组,分别于术后不同存活时间观察后根节神经元的溃变状态及过程,并在电镜下观察骶髓后连合核内的溃变终末像。结果表明注射6μl的10%Ricin、存活5-7天的材料,初级传入终末溃变的超微结构像最佳。Ricin所致的跨节溃变终末呈现多型性变化。

四种植物25S rRNA sarcin/ricin结构域甲基化核苷的分析

应用不同浓度的dNTP逆转录反应,分析了苦瓜、丝瓜、蓖麻和水稻的25S rRNA sarcin/ricin区域中的甲基化核苷,发现了这4种植物25S rRNA的第3 023、2 966和2 962位的腺嘌呤、胸腺嘧啶和腺嘌呤是2’-O-甲基化核苷;在丝瓜和苦瓜25S rRNA的第2 992、2 990位的胞嘧啶和鸟嘌呤是葫芦科植物所特有的两个2’-O-甲基化核苷;第2 944位点的尿嘧啶是苦瓜、丝瓜、蓖麻和水稻的一个碱基甲基化核苷;而且,植物25S rRNA第3 023位甲基化腺嘌呤核苷刚好位于sarcin/ricin结构域的茎环上,与核糖体失活蛋白切割位点相距仅5个核苷酸,而在哺乳动物和酵母的rRNA相同位点上没有甲基化修饰的存在,根据这一结果推测,RIPs来源植物核糖体RNA的自我保护可能与这一位点的修饰有关。

rRTA:DS27,ricin:DS27两种免疫毒素的初步比较

目的:用重组的蓖麻毒素A链与抗CD5的单克隆抗体DS27交联为免疫毒素rTRA:DS27,与完整蓖麻毒素制备的ricin:DS27进行对比研究.方法:分析它们对靶细胞的特异性杀伤,对骨髓造血细胞增殖的影响,内化情况,以及NH_4cl对rRTA:DS27毒性的增强作用.结果:rRTA:DS27具有更好的导向特异性,对骨髓造血功能影响较小,NH_4cl能明显增强rRTA:DS27的毒性.结论:rTRA:DS27免疫毒素具有更多的优点.

Interstitial chemotherapy with ricin-loaded thermosensitive hydrogel in pancreatic cancer xenograft

BACKGROUND: Pancreatic cancer is one of the most aggressive malignancies, and has a poor prognosis. Despite efforts made in multiple fields, there has been little success in improving the disease-free survival rate of patients. This study was undertaken to investigate the effectiveness and feasibility of using intra-tumoral injection of ricin-loaded thermosensitive hydrogel for treatment of pancreatic cancer xenografts, attempting to develop a new treatment for human pancreatic cancer. METHODS: BALB/c-(nu/nu) nude mice were inoculated subcutaneously in the right flank with the human pancreatic cancer cells, SW1990. Fourteen days after inoculation, 32 mice, bearing tumors of volume 1.5-2.0 cm(3), were randomly assigned to one of four groups, and given an intra-tumoral injection of: (1) saline; (2) 23% w/w thermosensitive hydrogel alone; (3) ricin, 10 mu g/kg; or (4) 10 mu g/kg ricin loaded in thermosensitive hydrogel. On day 14 after administration, the tumors were excised to calculate the inhibition rate of tumor growth and perform histopathological examination. Tumor cell apoptosis was detected by flow cytometry, and RT-PCR was performed to evaluate the mRNA expression levels of Bc12 and Bax. RESULTS: Intra-tumoral injection of ricin-loaded thermosensitive hydrogel resulted in remarkable control of tumor growth. The tumor became necrotic by day 14 after administration. The histological results clearly confirmed that the tumor cells were lysed. The percentage of apoptotic cells detected by flow cytometry was higher in the ricin hydrogel group than in the other groups. Semi-quantitative RT-PCR revealed that the mRNA expression level of Bc12 was down-regulated whereas Bax was upregulated. CONCLUSIONS: Intra-tumoral injection of ricin-loaded thermosensitive hydrogel may provide an effective approach for interstitial chemotherapy in pancreatic cancer. Inducing apoptosis by downregulating Bcl2 expression and upregulating Bax expression may be a key molecular mechanism.

Protective effects of anti-ricin A-chain RNA aptamer against ricin toxicity

AIM： To investigate the therapeutic potential of an RNA ligand （aptamer） specific for the catalytic ricin A-chain （RTA）, the protective effects of a 31-nucleo- tide RNA aptamer （31RA）, which formed a high affinity complex with RTA, against ricin-induced toxicity in cell- based luciferase translation and cell cytotoxicity assays were evaluated. METHODS： To test the therapeutic potential of anti- RTA aptamers in Chinese hamster ovary （CliO） AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured us- ing luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tet- razolium compound [3-（4,5-dimethylthiazol-2-yl）- 5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H- tetrazolium]. RESULTS： Inhibition of protein synthesis by ricin in CliO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against degly- cosylated RTA （dgA） neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CliO AA8 cells. CONCLUSION： We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell- based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication.

Modification of ricin and its hepatotoxicity and activity against hepatocellular cancer in mice

AIM To observe the effects of ricin (RT) with and without chemical modification on both hepatotoxicity of mice and activity against hepatocellular cancer (HCC), and evaluate the possibility to improve RT anticancer activity via chemical modification.METHODS RT was modified with N-succinimidyl3-(2-pyridyldithio) propionate (SPDP), a heterobifunctional cross-linker, and SPDP derivative of RT (PDP-R) was obtained. The serum glutathione-s-transferase (SGST) activity, as an index of liver damage, was determined in mice intoxicated with RT and PDP-R, at various doses and time. The tissue damage of HCC in the nude mice ip injected with PDP-R was compared with that with RT at the same dose by immunohistochemical method, the relative content of both RT and PDP-R in the HCC tissues was measured by computerized image-analysis.RESULTS The SGST activities increased with doses or/and time intoxicated with both RT and PDP-R, and the increase in the value of RT group was more significant than that in the PDP-R group; the SGST activity of RT group was 2.8-fold (P＜0.01) of PDP-R group at a dose of 12.5μg/kg for 42h, showing the much lower toxicity of R-PDP than that of RT. Under an optical microscope, hemolysis and necrosis of massive cells in the HCC tissues of PDP-R group were observed and the ratio of necrosis mounted to 90.5% while the corresponding value of RT group only to 62.5%. With computerized image-analysis, the average relative content of RT and PDP-R in the HCC tissues, represented as greyness, was 140.06±3.43 and 169.10±2.74, respectively. There was significant difference between the two (P＜0.05), indicating the higher content of PDP-R in the HCC tissue than that of RT.CONCLUSION The hepatotoxicity of PDP-R to mice may be reduced by chemical modification with SPDP, but both the affinity of PDP-R to the HCC tissues and ability to kill it may be stronger than that of RT. So this might be a valuable attempt to improve the anticancer activity of RT.

Purification of Ricinus communis agglutinin and its application to research on oligosaccharides in serum of patients with lung cancer

In this report,purified Ricinus communis agglutinin (RCAl) was employed for analysis ofoligosaccharide molecules in serum of patients with lung cancer or benign diseases.By meansof DEAE-Sephadex A-50 and Sephadex G-100 chromatography,RCAl was purified from the ex-tracts of castor beans.After characterizing its bioactivity by means of red blood coagglutination andpurification by SDS-PAGE,RCAl was coupled with CNBr-activated Sepharose 4B.By RCAl af-finity chromatography,the gtycoproteins containing Galβl-4GlcNAc-linked unit at the nonreducingend of the carbohydrate chain were isolated from the gastric cancer tissue.By means of RCAl andthis glycoproteins labelled with horseradish peroxidase,a competitive binding assay was establishedand employed to detect the level of the oligosaccharides defined by RCAl in serum of pateins withlung cancer or benign diseases and of normal persons.The results suggest that expression of theseoligosaccharide molecules is higher in serum of patient with lung cancer than in serum of patientwith a benign disease of the respiratory system and healthy individuals.The positive rates were60.6%(20/33) in serum of patients with lung cancer,21.4% (6/28) with benign diseases and 4.0%(1/25) of normal persons.It is confirmed that detection of these oligosaccharide molecules in serumcan be helpful in the study and diagnosis of lung cancer.

ANTIBODY TO ONCOGENE PROTEIN PRODUCT AND ITS CONJUGATE WITH RICIN A-CHAIN

The proteins encoded by oncogene were thought to be tumor associated antigen. The protein P110 in MGC803, a human gastric cancer cell line, was purified as immunogen. The IgY to the gastric cancer was extracted from eggs laid by immunized hen. The IgY could react immunohistochemically with gastric cancers. Positive staining rates of PAF were 80% in gastric cancers and markedly higher than in cancers of other organs and normal gastric tissue. The IgY-Ricin A was synthesized by the IgY conjugated with Ricin A- chain. TCID50 of MGC803 treated by the IgY-Ricin A was 0. 01 mg/ml and markedly lower than other cell. These results showed the IgY-Ricin A were able to react with gastric cancers selectively.

Protective effects of anti-ricin A-chain antibodies delivered intracellularly against ricin-induced cytotoxicity

AIM:To evaluate the ability of anti-ricin A-chain antibodies,delivered intracellularly,to protect against ricininduced cytotoxicity in RAW264.7 cells. METHODS:Anti-deglycosylated ricin A-chain antibody and RAC18 anti-ricin A-chain monoclonal antibody were delivered intracellularly by encapsulating in liposomes or via conjugation with the cell-penetrating MTS-transport peptide.RAW264.7 cells were incubatedwith these antibodies either before or after ricin exposure.The changes in cytotoxicity were estimated by MTT assay.Co-localization of internalized antibody and ricin was evaluated by fluorescence microscopy. RESULTS:Internalized antibodies significantly increased cell viability either before or after ricin exposure compared to the unconjugated antibodies.Fluorescence microscopy confirmed the co-localization of internalized antibodies and ricin inside the cells. CONCLUSION:Intracellular delivery of antibodies to neutralize the ricin toxin after cellular uptake supports the potential use of cell-permeable antibodies for postexposure treatment of ricin intoxication.

SELECTIVE KILLING OF GASTRIC CANCER CELLS BY MONOCLONAL ANTIBODY CONJUGATED WITH A CHAIN OF RICIN

A specific cytotoxic agent against gastric cancer was constructed by covalently coupling the ricin A chain to monoclonal artibody, MGb2. MGb2 was modified by SPDP to introduce the 3-(2-pyridylthio) propionyl radical and then treated with a reduced A chain to give a disulfide linked conjugate that retained the original binding specificity of the antibody moiety. The conjugate obtained retained the activity of the antibody and the biological activity of the A chain well.

Novel small molecule retrograde transport blocker confers post-exposure protection against ricin intoxication

Ricin is a highly toxic type 2 ribosome-inactivating protein(RIP)which is extracted from the seeds of castor beans.Ricin is considered a potential bioterror agent and no effective antidote for ricin exists so far.In this study,by structural modification of a retrograde transport blocker Retro-2cyc1,a series of novel compounds were obtained.The primary screen revealed that compound 27 has an improved antiricin activity compare to positive control.In vitro pre-exposure evaluation in Madin-Darby Canine Kidney(MDCK)cells demonstrated that 27 is a powerful anti-ricin compound with an EC50 of 41.05 nmol/L against one LC(lethal concentration,5.56 ng/mL)of ricin.Further studies surprisingly indicated that 27 confers post-exposure activity against ricin intoxication.An in vivo study showed that 1 h post-exposure administration of 27 can improve the survival rate as well as delay the death of ricin-intoxicated mice.A drug combination of 27 with monoclonal antibody mAb4 C13 rescued mice from one LD(lethal dose)ricin challenge and the survival rate of tested animals is 100%.These results represent,for the first time,indication that small molecule retrograde transport blocker confers both in vitro and in vivo post-exposure protection against ricin and therefore provides a promising candidate for the development of anti-ricin medicines.

Effect of Interstitial Chemotherapy with Ricin Temperature-Responsive Gel for Anti-Breast Cancer and Immune Regulation in Rats

Objective： To explore the effect of ricin temperature response gel on breast cancer and its regulatory effect on immune function in rats. Methods： Ricin was purified by chromatography and identified by immunoblotting. The rat subcutaneously transplanted breast cancer model was established. Forty model rats with a tumor diameter of about 3.0 cm were subjected to the study. They were randomized into four groups equally： the model group and three treated groups （blank gel, ricin, ricin-gel） were administered with blank gel, ricin, and ricin temperature response gel via percutaneous intratumor injection, respectively. The tumor was isolated 10 days later for the estimation of tumor inhibition rate （FIR） by weighing, pathologic examination, and detection of tumor apoptosis-associated genes bcl-2 and bax with semiquantitative RT-PCR. Also, peripheral blood was obtained to test T-lymphocyte subsets, the killing function of lymphocytes, and the contents of tumor necrosis factor- oL （TNF- oL ） and interleukin-2 （IL-2）. The outcomes were compared between groups. Results： The TIR in the ricin-gel group was 61.8%, with the pathologic examination showing extensive tumor tissue necrosis. Compared with the model group, after ricin temperature response gel treatment, bcl-2 expression was down- regulated, bax expression was up-regulated, CD4＋ lymphocytes and CD4＋/CD8＋ ratio in peripheral blood were increased, the killing function of lymphocytes was enhanced, and the contents of TNF- α and IL-2 were elevated （P〈0.05 or P〈0.01）. Conclusion： Intratumor injection of ricin temperature-responsive gel showed significant antitumor effect on breast cancer and could enhance the immune function in the tumor-bearing rat.

基于分子对接和步进序列群的蓖麻毒素核酸适配体序列优化

目的有效结合分子对接预测和表面等离子体共振实验评价技术,获得亲和力更强、序列最短的最优适配体。方法针对前期筛选出的靶向蓖麻毒素的3条80 nt单链DNA适配体(L14、P3、L7),在明确各自二维随机区茎环序列与靶蛋白结合能力的基础上,以H-DOCK分子对接为指导,分别确定蓖麻毒素适配体随机区的最短结合单元,从而构建两端延长步进序列群,以表面等离子体共振技术测定序列群序列的亲和力和动力学参数,明确适配体的结合关键结构,从而筛选得到最优适配体。结果3条全长适配体的随机区适配体L14r、P3r、L7r均可形成一定的茎环结构,其中L14r较L14的亲和力增强9倍、L7r增强2倍、P3r基本不变。对随机区适配体和蓖麻毒素进行分子对接,结果显示,L14r、P3r、L7r的对接分数值皆优于阴性序列40T,结合关键氨基酸个数分别为11、8、9个,存在距离小于5?的预测结合位点分别为20、12、15个,具有良好的与蓖麻毒素的结合能力。进一步明确了蓖麻毒素活性口袋所容纳的适配体最短结合单元L14rm、P3rm、L7rm的序列构成,在此基础上构建出两端延长步进序列群。针对该步进群,基于结合关键氨基酸个数、结合位点个数、对接得分等参数的变化和表面等离子体共振测定结果筛选出最优适配体。所获得的最优适配体L14rm、L7rm-2亲和力继续增强了1~2倍。结论随机区适配体能有效地与蓖麻毒素结合,较之全长适配体亲和力更强,分子对接结合步进序列群设计,仅使用17条序列,便有效获得了3条最优适配体并明确其结合作用。3条结合蓖麻毒素的最优适配体——L14rm、P3r、L7rm-2的KD值分别为(64±30)、(167±19)、(120±1)nmol/L,亲和力提高到全长适配体的14、1、4倍。

蓖麻蚕对蓖麻毒素的代谢解毒作用

蓖麻蚕及蚕蛹是食用、饲用的优质蛋白质来源,但蓖麻蚕体内蓖麻毒素残留量及代谢解毒作用机制尚不明确,严重限制其食品化、饲料化开发应用。为解析蓖麻毒素在蓖麻蚕体内的代谢解毒机制,对蓖麻蚕5龄幼虫添饲或注射蓖麻毒素,分析蓖麻蚕对蓖麻毒素的吸收率、注射毒素致死中量、毒素代谢降解率,检测GST-GSH-Px解毒体系酶活性变化,并分析添饲谷胱甘肽(GSH)对蓖麻蚕存活率的影响。试验结果表明,蓖麻蚕对蓖麻毒素的吸收率约为0.12%,大部分毒素随蚕沙排出体外。蓖麻蚕全龄添饲毒素原液(2.319 mg/mL)并未死亡,但注射致死中量LD50为13.90 ng/g。添饲蓖麻毒素后,1~5龄幼虫、蚕蛹、5龄蚕血淋巴和中肠中的蓖麻毒素含量极低(0.32~0.59 ng/g);添饲毒素后幼虫对毒素的降解率达99.99%,而注射毒素幼虫对毒素的降解率为97.57%。进一步分析表明,蓖麻蚕添饲毒素后全蚕、血淋巴和中肠中GSH含量以及GR、GST、GSH-Px酶活性均显著提高;注射毒素后,全蚕和血淋巴中GSH含量先降后升,而GR、GST、GSH-Px酶活性则显著上调,中肠中GR、GST、GSH-Px酶活性呈上调趋势,但增幅比血淋巴更小,说明血淋巴和中肠是主要解毒组织,依赖GST-GSH-Px解毒防御体系完成毒素代谢;注射GSH能够延长幼虫致死中时(LT50)18.52 h,进一步确认GST-GSH-Px解毒防御体系在蓖麻毒素代谢降解中的作用。研究结果明确蓖麻蚕及蚕蛹体内蓖麻毒素含量极低,蓖麻蚕依赖血淋巴和中肠中的GST-GSH-Px解毒防御体系降解体内蓖麻毒素。上述结果为蓖麻蚕及蚕蛹的食用化、饲料化安全评价以及蓖麻蚕毒素解毒药物的研发提供了科学依据。

夹心免疫PCR方法检测蓖麻毒素

目的 建立了双抗夹心免疫PCR的检测技术,检测极微量的抗原。方法 以亲和素作为连接分子连接生物素化抗体和生物素化DNA,应用于免疫PCR中,检测极微量的抗原。结果 建立了双抗夹心兔疫PCR的检测技术,检测蓖麻毒素的灵敏度达0.1pg/mL,与夹心ELISA方法比较灵敏度高103。结论 建立的免疫PCR系统灵敏度高,可用于各种微量抗原的检测。