转Cry1Ab/Cry2Aj和G10evo(EPSPS)基因“双抗12-5”玉米外源基因重组蛋白的消化稳定性研究

目的评价转Cry1Ab/Cry2Aj、G10evo(EPSPS)基因抗虫耐草甘膦玉米"双抗12-5"中的外源基因重组蛋白在模拟胃肠液中的消化稳定性。方法根据国家标准建立体外模拟胃肠环境消化体系,样品或对照都以5 mg/ml的浓度模拟胃液消化,2 mg/ml的浓度模拟肠液消化。分别于反应0 s、15 s、2 min、30 min和60 min时取出200μl模拟消化液,加入上样缓冲液并沸水浴5 min终止反应,再进行十二烷基磺酸钠—聚丙烯酰胺凝胶电泳(SDS-PAGE)、染色并观察结果。结果大豆胰蛋白酶抑制剂(soybean trypsin inhibitor,STI)在模拟胃、肠液中60 min仍不能被全部消化,酪蛋白(α-casein)在模拟胃、肠液中0~15 s内全部消化,Cry1Ab/Cry2Aj重组蛋白在模拟胃、肠液中0~15 s内完全消化,G10evo(EPSPS)重组蛋白在模拟胃液中0~15 s内全部消化,在模拟肠液中2~30 min内全部消化。结论转Cry1Ab/Cry2Aj、G10evo(EPSPS)基因玉米的外源基因重组蛋白在模拟胃、肠液中不具有消化稳定性,容易被降解。     

转HJC-1和G6-EPSPS基因抗虫耐草甘膦水稻表达蛋白在模拟胃肠环境中的稳定性研究

目的 研究HJC-1和G6-EPSPS基因表达的蛋白分别在模拟胃液和模拟肠液中的消化稳定性.方法 采用美国1995年药典提供的模拟胃液和模拟肠液配方,在体外建立模拟胃肠环境消化体系,测定HJC-1和G6-EPSPS基因表达的蛋白质在胃肠环境中的稳定性.蛋白质在模拟胃、肠液中的浓度分别为5.0和2.0 mg/ml.在蛋白质与模拟胃、肠液反应后的0、15、30 s,1、2、5、10、20、30和60 min准确取样,根据SDS-PAGE凝胶电泳结果,判断蛋白质在模拟胃、肠液环境中的稳定性.结果 HJC-1基因表达的蛋白质在模拟胃液和模拟肠液中均在15s内全部降解；G6-EPSPS基因表达的蛋白质在模拟胃液中30 s内全部降解,在模拟肠液中60 min内不能完全降解.结论 HJC-1基因表达的蛋白质在模拟人体胃肠环境中不稳定,易被降解.G6-EPSPS基因表达的蛋白质在模拟人体胃环境中不稳定,易被降解；在模拟人体肠环境中稳定,不易被降解.     

转Cry1Ab/Cry2Aj和G10evo(EPSPS)基因抗虫耐草甘膦玉米对大鼠的亚慢性毒性研究

目的研究喂饲转Cry1Ab/Cry2Aj和G10evo(EPSPS)基因抗虫耐草甘膦玉米对大鼠的亚慢性毒性。方法将Wistar大鼠按体重随机分为7组,即转基因玉米3个剂量组(12.5%,25.0%,50.0%),亲本玉米3个剂量组(12.5%,25.0%,50.0%)和1个常规基础饲料对照组。分别喂饲相应饲料90 d,自由进食饮水,观察大鼠体重、进食量、血液学指标、血生化指标、主要脏器系数及组织病理学检查。结果各剂量组大鼠的体重增长正常,血液学和血生化个别指标虽有统计学差异,但并无生物学意义。转基因玉米剂量组大鼠脏器系数与对应亲本组比较,差异均无统计学意义(P0.05),主要脏器组织病理学检查未发现明显异常。结论现有试验结果不能证实该转Cry1Ab/Cry2Aj和G10evo(EPSPS)基因抗虫耐草甘膦玉米对大鼠有亚慢性毒性作用。     

EPSPS蛋白和PAT蛋白的模拟胃液消化稳定性分析

目的研究5-烯醇式丙酮酸莽草酸-3-磷酸合成酶(5-enolpyruvylshikimate-3-phosphate synthase, EPSPS)蛋白和膦丝菌素乙酰转移酶(phosphinothricina cetyltransferase, PAT)蛋白在模拟胃液中的消化稳定性。方法参照中华人民共和国国家标准(农业部869号公告-2-2007)模拟胃肠液外源蛋白质消化稳定性试验方法中模拟胃液的配方,在体外建立模拟胃液消化体系,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(dodecyl sulfate,sodium salt-polyacrylamide gel electrophoresis,SDS-PAGE)和蛋白印迹(Western blot),分析EPSPS蛋白和PAT蛋白在模拟胃液中不同消化时间点的降解情况。结果 EPSPS蛋白和PAT蛋白均在15 s内消化完全,SDS-PAGE和Western blot蛋白免疫印迹均未检测到蛋白残留,EPSPS蛋白和PAT蛋白在模拟胃液中极易被消化。结论 EPSPS蛋白和PAT蛋白经过胃液消化后,不具有免疫原性。     

外源蛋白在模拟胃肠环境中稳定性测定模型初探

目的　建立外源蛋白在模拟胃肠环境中稳定性的实验模型。方法　采用美国 1 995年药典提供的模拟胃液和模拟肠液配方 ,测定不同蛋白在胃肠环境中的稳定性。蛋白质在模拟胃 肠液中的浓度为 0 .5mg ml。在蛋白与模拟胃 肠液反应后的 0s、1 5s、30s、6 0s、2min、4min、8min、1 5min、30min和 6 0min准时取样 ,根据SDS PAGE凝胶电泳结果 ,判断蛋白质在模拟胃 肠液中的稳定性。结果　大豆胰蛋白酶抑制剂、牛 β 乳球蛋白A、牛 β 乳球蛋白B在模拟胃液中稳定存在 ,6 0min完全不降解 ;大豆血凝素 8min内绝大部分降解 ,但降解带 6 0min内仍有残留 ;花生血凝素 8min内降解 ;鸡卵粘蛋白、牛血清白蛋白、大豆脂肪水解酶、土豆磷酸酶均在 1 5s内降解。蛋白质在模拟肠液中的稳定性与模拟胃液不同。大豆胰蛋白酶抑制剂、牛 β 乳球蛋白A、牛β 乳球蛋白B、牛血清白蛋白、鸡卵粘蛋白、鸡卵清蛋白、大豆血凝素、花生血凝素在模拟肠液中 ,6 0min均不能完全降解 ;大豆脂肪水解酶、土豆磷酸酶及其降解带 6 0min内依然存在。结论　不同的外源蛋白在模拟胃 肠液中的稳定性不同 ,根据蛋白质稳定性差异 ,初步建立了评价外源蛋白在模拟胃 肠液中稳定性测定的模型     

重组人乳铁蛋白在体外模拟胃肠环境中稳定性研究

目的 研究重组人乳铁蛋白(recombinant human lactoferrin,rHLF)分别在体外模拟胃液和模拟肠液中的消化稳定性.方法 采用模拟胃液和模拟肠液在体外建立模拟胃肠环境消化体系,测定重组人乳铁蛋白在胃肠环境中的稳定性.重组人乳铁蛋白在模拟胃、肠液中的浓度分别为5.0和2.0 mg/ml.在重组人乳铁蛋白与模拟胃/肠液反应后的0、15、30 s和1、2、5、10、20、30、60 min取样,根据SDS-PAGE凝胶电泳结果,判断重组人乳铁蛋白在模拟胃、肠液环境中的稳定性.结果 重组人乳铁蛋白在体外模拟胃液中2 min内降解,在模拟肠液中60 min内不能完全降解.结论 重组人乳铁蛋白在体外模拟人体胃肠环境中易被降解消化.     

食物蛋白质消化稳定性和热稳定性研究

目的建立体外模拟胃肠液消化稳定性试验和热稳定性试验方法,确定试验中的阳性和阴性参考蛋白质。方法在体外模拟胃肠液消化环境和热加工处理环境,通过SDS-PAGE凝胶电泳研究常见食物致敏原(鸡卵清蛋白OVA、牛β-乳球蛋白β-LG)、不常见食物致敏原(牛血清白蛋白BSA)和非致敏原(大豆脂肪水解酶LPE、马铃薯酸性磷酸酶PAP)在其中各观察时间点的降解情况。结果OVA在模拟胃肠液中60min不降解;β-LG在模拟胃液中60min不降解稳定存在,但在模拟肠液中30min内完全降解;BSA在模拟胃液中30min内完全降解,在模拟肠液中60min部分降解;PAP在模拟胃液中15s即完全降解,LPE在30s内完全降解;而在模拟肠液中,PAP60min完全不降解,LPE60min大部分降解。在热稳定性试验中,OVA、β-LG、BSA60min内不降解,PAP60min内完全降解。结论模拟胃液消化稳定性和热稳定性试验中设OVA为常见致敏原对照、BSA为不常见致敏原对照、PAP为非致敏原对照,模拟肠液消化稳定性试验中设OVA为常见致敏原对照、BSA为不常见致敏原对照、LPE为非致敏原对照。并据此初步建立体外消化稳定性和热稳定性试验方法。     

饲喂转Cry1Ab和epsps基因抗虫耐除草剂玉米对子三代大鼠免疫功能的影响

目的探讨转Cry1Ab和epsps基因抗虫耐除草剂玉米对F3代大鼠免疫功能的影响。方法 180只断乳F0代SD大鼠按体重随机分为AIN-93G饲料对照组、亲本玉米组和转基因玉米组。经三代繁殖产生F3代仔鼠后,对其进行抗体生成细胞检测、刀豆蛋白(concanavalin A, ConA)诱导大鼠脾淋巴细胞转化试验、自然杀伤(natural killer,NK)细胞活性检测、全血淋巴细胞亚群检测、迟发型变态反应检测及免疫器官脏器系数等检测。结果在抗体生成细胞数、ConA诱导的大鼠脾淋巴细胞增殖能力、NK细胞活性、全血淋巴细胞亚群分类及迟发型变态反应、胸腺系数等方面,转基因玉米组与亲本玉米组相比,差异均无统计学意义(P>0.05)。结论在本试验条件下,转Cry1Ab和epsps基因抗虫耐除草剂玉米饲喂SD大鼠三代后,对F3代大鼠的免疫功能未见不良影响。     

转CpTI基因大米在五指山小型猪体内消化稳定性的初步研究

目的建立五指山小型猪在体消化稳定性模型,评价转CpTI大米的胃肠消化稳定性。方法在五指山成年猪体内同时安置胃、肠瘘管,通过经口喂饲途径,以大豆作为阳性对照,测定不同时间点消化液的pH,采用蛋白凝胶电泳法分析测定不同时间点消化产物中的蛋白片段,比较转CpTI大米和亲本大米消化产物的区别。结果给样后胃液pH迅速中和至6.0左右,以后逐步降低,在4～6h后又开始回升,大豆引起胃液pH下降和回升时间均较米粉晚;13×103、17×103、34×103和50×103是大豆中几种相对消化稳定的蛋白片段,在5～6h的胃肠液中仍然能检测出高表达,其中50×103、34×103、17×103三个蛋白片段呈现胃液、肠液消化稳定性,而13kD呈现胃液消化稳定性;米粉给样0.25h后胃液中未检出任何耐受蛋白酶消化的片段或亚基,而亲本明恢86米粉和转CpTI基因米粉比较,无论是胃液或肠液中蛋白消化情况均无明显区别。结论利用五指山小型猪建立的在体消化稳定性模型是可行的,转CpTI基因米粉与亲本米粉在五指山小型猪胃液、肠液中的消化稳定性具有等同性。     

转化体qXY中外源基因表达重组蛋白PA的食用安全性评价

目的 从毒性和过敏性2个方面对转化体qXY中外源基因表达重组蛋白PA进行食用安全性评价。方法 对大肠杆菌表达重组蛋白PA和转化体qXY中植物提取蛋白PA进行等同性分析后,分别采用毒性蛋白数据库和过敏原数据库对PA蛋白氨基酸序列进行毒性和过敏性生物信息学分析,对重组PA蛋白进行模拟胃/肠液消化稳定性试验,并且采用限量法对重组蛋白PA进行小鼠急性经口毒性试验。结果 大肠杆菌表达重组蛋白PA和转化体qXY中植物表达蛋白PA的表观分子量均为55 kD,且二者均可与PA蛋白特异性抗体结合,二者均无翻译后糖基化修饰,飞行质谱分析重组PA蛋白N端1-10氨基酸残基与理论序列一致,因此认为二者具有等同性。毒性生物信息学分析未发现PA蛋白与毒性蛋白数据库中已知毒蛋白具有序列相似性,CD-1小鼠急性经口毒性试验结果显示,5 000 mg/kg·BW剂量下未产生毒性效应。过敏性生物信息学发现,PA蛋白与已知过敏原高峰淀粉酶A(Asp o 21)存在较高的序列同源性,但模拟胃/肠液消化稳定性试验结果显示,重组蛋白PA在模拟胃液和肠液中0～15 s内均迅速消化,属于极易消化,不具有潜在过敏性。结论 未发现转化体...     

Effects of transgenic corn and Cry1Ab protein on the nematode, Caenorhabditis elegans

The effects of the insecticidal Cry1Ab protein from Bacillus thuringiensis (Bt) on the nematode, Caenorhabditis elegans, were studied with soil from experimental fields cultivated with transgenic Bt corn (MON810) and with trypsinized Cry1Ab protein expressed in Escherichia coli. The content of Cry1Ab protein was above the detection limit of an ELISA test in only half of the soil samples obtained from transgenic plots, ranging from 0.19 to 1.31 ng g⁻¹ dry weight. In a laboratory bioassay, C. elegans was exposed to rhizosphere and bulk soil from fields with isogenic or transgenic corn or to solutions of Cry1Ab protein (0, 24, 41, 63, 118, and 200 mg l⁻¹) over a period of 96 h, with growth and reproduction serving as the test parameters. Nematode reproduction and growth were significantly reduced in rhizosphere and bulk soil of Bt corn compared with soil from isogenic corn and were significantly correlated with concentrations of the Cry1Ab protein in the soil samples. Moreover, the toxicity of pure Cry1Ab protein to the reproduction and growth of C. elegans was concentration-dependent. As significant inhibition occurred at relatively high concentrations of the Cry1Ab protein (41 mg l⁻¹), the effects of the soil samples from Bt corn could not be assigned directly to the toxicity of the Cry1Ab protein. The results demonstrate that bioassays with the nematode, C. elegans, provide a promising tool for monitoring the potential effects of Bt toxins in aqueous medium and soils.     

Is the Cry1Ab Protein from Bacillusthuringiensis (Bt) Taken Up by Plants from Soils Previously Planted with Bt Corn and by Carrot from Hydroponic Culture?

The uptake of the insecticidal Cry1Ab protein from Bacillus thuringiensis (Bt) by various crops from soils on which Bt corn had previously grown was determined. In 2005, the Cry1Ab protein was detected by Western blot in tissues (leaves plus stems) of basil, carrot, kale, lettuce, okra, parsnip, radish, snap bean, and soybean but not in tissues of beet and spinach and was estimated by enzyme-linked immunosorbent assay (ELISA) to be 0.05 ± 0.003 ng g⁻¹ of fresh plant tissue in basil, 0.02 ± 0.014 ng g⁻¹ in okra, and 0.34 ± 0.176 ng g⁻¹ in snap bean. However, the protein was not detected by ELISA in carrot, kale, lettuce, parsnip, radish, and soybean or in the soils by Western blot. In 2006, the Cry1Ab protein was detected by Western blot in tissues of basil, carrot, kale, radish, snap bean, and soybean from soils on which Bt corn had been grown the previous year and was estimated by ELISA to be 0.02 ± 0.014 ng g⁻¹ of fresh plant tissue in basil, 0.19 ± 0.060 ng g⁻¹ in carrot, 0.05 ± 0.018 ng g⁻¹ in kale, 0.04 ± 0.022 ng g⁻¹ in radish, 0.53 ± 0.170 ng g⁻¹ in snap bean, and 0.15 ± 0.071 ng g⁻¹ in soybean. The Cry1Ab protein was also detected by Western blot in tissues of basil, carrot, kale, radish, and snap bean but not of soybean grown in soil on which Bt corn had not been grown since 2002; the concentration was estimated by ELISA to be 0.03 ± 0.021 ng g⁻¹ in basil, 0.02 ± 0.008 ng g⁻¹ in carrot, 0.04 ± 0.017 ng g⁻¹ in kale, 0.02 ± 0.012 ng g⁻¹ in radish, 0.05 ± 0.004 ng g⁻¹ in snap bean, and 0.09 ± 0.015 ng g⁻¹ in soybean. The protein was detected by Western blot in 2006 in most soils on which Bt corn had or had not been grown since 2002. The Cry1Ab protein was detected by Western blot in leaves plus stems and in roots of carrot after 56 days of growth in sterile hydroponic culture to which purified Cry1Ab protein had been added and was estimated by ELISA to be 0.08 ± 0.021 and 0.60 ± 0.148 ng g⁻¹ of fresh leaves plus stems and roots, respectively. No Cry1Ab protein was detected in the tissues of carrot grown in hydroponic culture to which no Cry1Ab protein had been added. Because of the different results obtained with different commercial Western blot (i.e., from Envirologix and Agdia) and ELISA kits (i.e., from Envirologix, Agdia, and Abraxis), it is not clear whether the presence of the Cry1Ab protein in the tissues of some plants under field condition and in carrot in sterile hydroponic culture was the result of the uptake of the protein by the plants or of the accuracy and sensitivity of the different commercial kits used. More detailed studies with additional techniques are obviously needed to confirm the uptake of Cry proteins from soil by plants subsequently planted after a Bt crop.     

Effects of Activated Bt Transgene Products (Cry1Ab, Cry3Bb) on Immature Stages of the Ladybird Adalia bipunctata in Laboratory Ecotoxicity Testing

Insect-active Bacillus thuringiensis (Bt) proteins are expressed by several transgenic crop plants to control certain pests, but nontarget organisms such as ladybirds also can be exposed to these proteins in the field. We developed an improved ecotoxicity testing protocol and conducted feeding trials in a laboratory setting to test for possible adverse effects of different concentrations of microbially produced trypsin-activated Cry1Ab and Cry3Bb toxins on the coccinellid Adalia bipunctata. Larval/pupal mortality, development time, and overall body mass accumulation were recorded. Even at the lowest concentration (5 μg/ml), A. bipunctata larvae fed with the lepidopteran-active Cry1Ab toxin exhibited significantly higher mortality than the control group. In experiments with the coleopteran-active Cry3Bb, only a concentration of 25 μg/ml resulted in a marginally significantly higher mortality compared to the control. Both experiments revealed a slight decline in mortality at the highest concentration of 50 μg/ml, though this was statistically significant only in the Cry1Ab treatment. No differences were detected for development time and body mass of newly emerged adults. Dilutions of the expression vector pBD10—used as a control to exclude effects of the toxin production method—at concentrations between 10 and 100 μg/ml revealed no significant effects on either of the studied parameters. This suggests that the increased mortality of larvae in the toxin feeding trials was caused directly by the activated Bt toxins and raises questions regarding their commonly postulated specificity and their mode of action in A. bipunctata. Implications of the reported results for ladybird populations and their biological pest control functions in transgenic crop ecosystems are discussed.     

Corrosion behavior of Cu and the Cu-Zn-Al shape memory alloy in simulated uterine fluid

Chemical immersion tests, electrochemical methods and atomic absorption spectrometry were employed to investigate the corrosion behavior of Cu and the Cu-Zn-Al shape memory alloy (SMA) in simulated uterine fluid. The effect of pH on corrosion rate and corrosion potential was also investigated. The results indicated that in the static state in simulated uterine fluid, dealuminumification of the Cu-Zn-Al alloy occurred with Cl- combining with aluminum ions to form hydroxyl aluminum chloride. The hydroxyl aluminum chloride hydrolyzed readily and facilitated further dealuminumification corrosion. The corrosion process of Cu and Cu-Zn-Al SMA in simulated uterine fluid was controlled by cathodic reduction of oxygen. Because the tendency for surface ionization is greater for aluminum than for zinc, a compact protective aluminum layer was formed, which inhibited the cathodic reduction of oxygen. Hence, the corrosion rate of Cu-Zn-Al SMA was smaller than that of Cu in simulated uterine fluid. With increasing pH, the corrosion rate of Cu and Cu-Zn-Al SMA in simulated uterine fluid decreased and the open-circuit potential moved in a positive direction.     

肝癌癌组织及癌旁组织中生物钟基因Cry1蛋白的表达及意义

目的探讨肝癌癌组织及癌旁组织中生物钟基因Cry1蛋白的表达及意义。方法采用免疫组织化学染色检测80例肝癌患者癌组织和癌旁组织及30例正常肝组织中生物钟基因Cry1蛋白的表达,探讨Cry1蛋白表达与患者临床病理特征之间的关系。结果 Cry1蛋白的阳性染色位于细胞核和细胞质中,主要位于细胞核;正常肝组织、癌旁组织和肝癌组织中阳性表达率分别为93.33%、52.50%和28.75%,阳性表达评分分别为(6.27±1.25)分、(4.19±0.89)分和(2.24±0.46)分,差异均有统计学意义(P<0.05)。Cry1蛋白表达水平与患者肿瘤直径大小、包膜是否完整、是否淋巴结转移以及Edmonfson分级有关(P<0.05),与性别无关(P>0.05)。结论 Cry1在机体中主要起维持生物节律的作用,其在肝癌组织及癌旁组织中表达水平显著低于正常肝组织,可能对肿瘤浸润生长及转移具有一定抑制作用。     

The release of cupric ion in simulated uterine: New material nano-Cu/low-density polyethylene used for intrauterine devices

With the development of IUDs, a number of copper-bearing devices are now commercially available, including the copper-T, the Multiload and the copper-T in various other forms, so-called "the second-generation" IUDs. In this article, we report on nano-Cu/low-density polyethylene composite as a potential copper carrier in IUD. Two issues for the new material are addressed: the effectiveness of polymers in reducing the initial burst in cupric ion release and the amount and pattern of continuing release. The aim of this study was to investigate copper ion release from this composite as a basis for considering its used in an IUD.     

Investigation of the release behavior of cupric ion for three types of Cu-IUDs and indomethacin for medicated Cu-IUD in simulated uterine fluid

BACKGROUND: A number of copper-bearing intrauterine devices (Cu-IUDs) are now commercially available in China. The release behavior of cupric ion from Cu-IUDs is essential to the success of contraception, and the release behavior of indomethacin from medicated Cu-IUD is related to its therapeutic effect. STUDY DESIGN: In this study, analytical methods were established to investigate the release behavior of cupric ion of three existing types of Cu-IUDs and indomethacin of one medicated Cu-IUD (Yuangong 365 Cu-IUD). Cu-IUDs were incubated in simulated uterine fluid (SUF). The concentrations of cupric ion and indomethacin were analyzed by flame atomic absorption spectrometer (FAAS) for 250 days and UV 752 spectrophotometer for 300 days, respectively. RESULTS: The release behavior of cupric ion for three types of Cu-IUDs was biphasic, which consisted of the initial burst release and then slow and constant release. The release of cupric ion from the medicated Cu-IUD Yuangong 365 showed a zero-order process. In vitro release experiment confirmed a sustained release of indomethacin from Yuangong 365 and the release was in accordance with the Weibull equation. CONCLUSION: The cupric ion release appears to be more constant in the medicated Cu-IUD than in the others. In view of the feature of indomethacin release from medicated Cu-IUD, it is conjectured that the design of this device could be useful to avoid the adverse effects caused by all Cu-IUDs.