转基因作物标记蛋白HPT融合表达载体构建、纯化及复性研究

目的潮霉素磷酸转移酶基因(hpt)是转基因作物中广泛应用的抗生素标记基因,本研究构建该基因的融合表达载体,以期得到足量纯化的HPT蛋白,为该外源蛋白的进一步安全性评价奠定基础。方法将hpt基因克隆到线性表达载体pET41 EK/LIC中,得到该基因的融合表达载体pET41EK/HPT,将pET41EK/HPT载体转入大肠杆菌BL21(DE3)中进行表达,沉淀中的包涵体进行了稀释复性、柱上复性和N-十二烷基肌氨酸钠(SKL)溶解稀释复性等复性研究及活性测定、标签切除、N端测序等工作。结果融合蛋白主要以无活性的包涵体形式存在,各复性方法表明SKL溶解稀释复性得到的蛋白在活性及产率方面明显高于其他两种方法,利用该方法每升培养物可获得78mg活性HPT融合蛋白,纯度约为93%,经肠激酶切割后,得到的蛋白与天然HPT蛋白N端氨基酸序列完全相同。结论利用本研究方法可得到在活性、分子量及氨基酸序列等方面与天然HPT蛋白一致的目的蛋白。     

转基因水稻中标记基因hpt表达行为的研究

目的探讨转sck基因水稻中标记基因hpt的表达行为。方法对经潮霉素抗性筛选的转基因水稻4个生长发育时期、不同组织的hpt基因进行PCR检测;提取4个生长发育时期PCR检测阳性植株的叶片总RNA,以hpt基因片断为探针进行Northernblot分析,并对不同生长时期的叶片和成熟期的茎部、根部、种子进行双夹心ELISA检测,以同种非转基因水稻M86相应时期的相应部位作阴性对照。结果转基因水稻不同生长时期、不同组织中均能扩出特异的hpt(832bp)片段;Northernblot结果显示,在4个生长发育时期的hpt基因在转录水平均有表达,但是表达水平较低,且不同生长发育时期之间无明显差别;ELISA检测结果显示HPT蛋白在4个生长时期的叶片和成熟期的茎部和根部均有表达,种子中HPT蛋白的含量不超过负对照,不能被检测。结论不同生长发育时期的叶片中hpt基因在转录水平和翻译水平表达均能稳定表达。成熟的种子中不含有可检测水平的HPT蛋白。     

转sck基因水稻种子中外源蛋白含量的检测

目的　检测转sck基因水稻T6代种子中外源蛋白CpTI及HPT的表达量。方法　对转基因水稻种子进行PCR检测;取外源基因PCR检测阳性植株的叶、茎杆、根部、种子等不同部位对CpTI和HPT蛋白表达进行Westernblot分析,并对PCR检测阳性植株的种子进行双夹心ELISA检测,以同种非转基因水稻M86种子作对照。结果　在转sck基因水稻T6代种子中扩增出sck和hpt基因;Westernblot结果显示转基因水稻的叶片、茎杆和根部的蛋白出现特异的杂交条带,种子蛋白中未出现特异杂交条带。ELISA检测结果显示,转基因水稻种子中CpTI蛋白低于双夹心ELISA的检测低限(<14 μg L) ,HPT蛋白没有被检出。结论　转基因水稻种子中CpTI蛋白含量低于检测低限,不能被定量,并且不含有可检测水平的HPT蛋白。     

转sck基因水稻中标记基因表达蛋白HPT食用安全性的初步研究

目的针对抗虫转sck基因水稻中标记基因表达蛋白HPT的食用安全性开展初步研究,主要包括该蛋白在大米中的定性、定量研究、可能的膳食摄入量估计及其潜在致敏性评估等。方法将HPT的编码序列插入带有组氨酸标签(His-tag)的原核表达载体PBV222中进行融合表达,通过脲变性和镍柱亲和层析的方法纯化融合蛋白。用纯化后的6His-HPT蛋白免疫家兔和小鼠,分别制备相应的抗HPT多克隆和单克隆抗体,进一步将这两种抗体组成双抗夹心酶联免疫检测体系,用于检测HPT在转sck基因水稻中的表达水平。在致敏性评估方面,首先将HPT序列与已知的致敏原数据库进行比较,再利用模拟的胃肠液环境进行体外消化稳定性实验。结果重组体经表达纯化后获得了高纯度(95%)的6His-HPT融合蛋白,免疫动物后获得了高效价的抗HPT多克隆及单克隆抗体,经免疫印迹检测证明所制备的抗体可与转基因水稻中及纯化出的HPT蛋白形成特异结合带。由这两种抗体组成的双抗夹心酶联免疫检测体系用于检测HPT抗原时的灵敏度为30ng/ml,可检测出转sck基因水稻叶片中HPT蛋白的表达量约为80~150ng/ml;而在相应的转基因大米中未能检出。HPT序列经与已知的致敏原数据库进行比较未见同源性,但在体外消化稳定性实验中,HPT蛋白在模拟的胃肠液环境中均迅速降解。结论就目前的检测水平来看,转sck基因大米中所含的HPT蛋白水平极低,且对消化环境不稳定,推测该标记基因表达蛋白不易引起可观察到的食用安全性问题。但利用表达的HPT蛋白直接进行动物急性毒性及致敏性实验的工作仍有待进行,有关的结果将进一步报道。     

食物成分、食物营养评价及测定方法

玛咖（Maca）干品营养成分分析与比较；洋葱多糖的提取及其抗氧化活性研究；高剂量富硒植物硒对胃癌模型大鼠的安全性研究；姜辣素的电化学行为研究；脱色猪血红蛋白酶解产物品质及营养特性研究；转基因作物标记蛋白HPT融合表达载体构建、纯化及复性研究     

豇豆胰蛋白酶抑制剂单克隆抗体的制备及初步应用

目的　检测转豇豆胰蛋白酶抑制剂 (CpTI)基因水稻叶片中CpTI蛋白 ,来对转CpTI基因作物的检测建立初步的方法。方法　目前国际上主要通过基因检测的方法 ,而基因的检测可能存在假阳性的结果 ,而且基因的导入并不一定代表功能蛋白的表达 ,表达含量也为未可知。因此 ,通过检测转基因蛋白含量的多少是最直接和明确的方法。通过体外基因重组技术制备的CpTI蛋白来免疫动物得到单克隆抗体 ,就可通过westernblotting法检测转基因作物中的CpTI。结果　得到三株抗CpTI的单克隆抗体 ,利用这三种单克隆抗体的混合物对转CpTI蛋白的水稻叶片进行初步检测 ,得到了较为满意的结果。结论　利用抗CpTI的单克隆抗体混合物对转CpTI的水稻叶片进行westernblotting的检测方法较好     

转抗菌肽基因辣椒蛋白质检测技术研究

以转抗菌肽BD基因辣椒为材料 ,研究建立转基因食品中外源目的蛋白的检测技术 ,并建立对其忠实表达状况和安全性进行评价的模式。以柞蚕蛹免疫血淋巴作为标准 ,对照检测转抗菌肽辣椒目的蛋白表达的忠实性 ,采用针对转基因食品技术特点而预先设计的技术路线进行分析检测。研究和检测步骤为 :目的蛋白经粗提 ,达到初步纯化。再用两次CM Sepharose FF离子交换柱层析进行中度纯化。纯化产物通过测定抗菌活性、电泳及生物自显影、质谱进行鉴定。系统的检测结果表明 ,以抗菌肽目的基因构建的重组体在导入辣椒后 ,所表达的外源目的蛋白与对照标准蛋白的理化性质、抗菌活性、分子量一致 ,说明在该转基因辣椒中抗菌肽目的基因的表达是忠实的 ,与期望值相符。本研究建立了目的蛋白抗菌肽D的分离纯化及鉴定技术 ,建立了转基因辣椒外源目的蛋白的鉴定方法。方法简便快捷可行     

转基因食品中标记基因的生物安全性研究进展及对策

标记基因是帮助对转基因生物体进行筛选和鉴定的一类外源基因 ,虽然它们仅在植物筛选的早期阶段发挥作用 ,但会存留在成熟的转基因作物中 ,因此其安全性研究不容忽视。本文着重对转基因食品中标记基因的生物安全性进行综述 ,分析其可能的风险及危害 ,总结了当前已被认可安全使用的标记基因。此外 ,对转基因食品进行强化标识是加强其市场管理的重要举措 ,其基础是要开发出准确可靠的转基因成分鉴定技术。文中对最终去除转基因食品中标记基因的方法也进行了介绍 ,以彻底消除其安全性隐患     

转基因食品安全性检验的核酸检测技术研究

以国际市场上转基因食品的主流产品转基因大豆及玉米和我国生产的转基因辣椒为材料 ,以PCR方法为基础 ,研究适用于转基因食品安全性检验的核酸检测技术。针对抗除草剂 (孟山都公司 )GM 大豆 ,转Bt 176玉米 (Novartis Ciba Geigy公司 )及转抗菌肽辣椒 (华农 )产品的插入基因及调控序列设计不同引物进行PCR检测。建立了转基因大豆、玉米、辣椒的鉴别和相关标记基因、目的基因检测的技术 ,该方法简便快速 ,检测结果与标准及申报材料相符。     

转基因豆油的检测

目的探讨转基因豆油的检测方法。方法采用PCR扩增法,对通常转基因植物共有的外源基因35s启动子和Nos终止子序列进行检测,确定其是否转基因作物。结论在转基因豆油中成功检测到35s启动子和Nos终止子的基因片段。     

Novel method for demonstrating nuclear contribution in mouse nuclear transfer

Confirmation of nuclear contribution is essential to all nuclear transfer experiments. Contribution is easily demonstrated in nuclear transfer progeny but more difficult to confirm in nuclear transfer embryos. The use of donor nuclei isolated from lacZ transgenic mice offers a clear and simple method to demonstrate contribution in nuclear transfer embryos and offspring. The unique line of transgenic mice (Zin40) used in this study displays nuclear localised lacZ expression in all cells, including embryonic blastomeres, and demonstrates distinctive blue nuclei when treated with X-gal substrate. This characteristic staining pattern provided an ideal marker for demonstrating nuclear contribution. Nuclear transfer embryos were generated following serial nuclear transfer of metaphase-arrested nuclei from transgenic and non-transgenic 4-cell embryos. Totipotency of nuclear transfer blastocysts was confirmed by the generation of live born offspring. Transgenic blastocysts and all tissue samples from fetuses and pups generated by nuclear transfer displayed distinctive blue nuclei when stained with X-gal. This staining pattern was characteristic of the transgenic mice from which the donor nuclei were isolated and clearly confirmed nuclear origin. The use of this marker will also allow the opportunity to investigate the developmental potential of nuclear transfer embryos by examining the contribution of nuclear transfer embryonic cells in chimaeric embryos.     

In vitro development of green fluorescent protein (GFP) transgenic bovine embryos after nuclear transfer using different cell cycles and passages of fetal fibroblasts

Nuclear transfer using transfected donor cells provides an efficient new strategy for the production of transgenic farm animals. The present study assessed in vitro development of nuclear transfer embryos using green fluorescent protein (GFP) gene-transfected bovine fetal fibroblasts. In experiment 1, bovine fetal fibroblasts (BFF) were transfected with linearized pEGFP-N1 by electroporation, and the enucleated oocytes were reconstructed by nuclear transfer of transfected cells (BFF-GFP). The rates of blastocyst formation did not differ significantly between BFF and BFF-GFP (18.2% v. 15.6%). In experiment 2, before nuclear transfer, the donor cell stage was synchronized by serum deprivation or forming a confluent monolayer. The rates of cleavage (67.1% v. 71.8%) and blastocyst formation (15.8% v. 15.5%) did not differ between confluent and serum-starved cells after nuclear transfer. In experiment 3, the effects of different passages of donor fibroblast cells on the development of nuclear transfer embryos were investigated. Donor cells from 'early' (at passage 8-16) showed better blastocyst development (18.9%) than those from 'late' (at passage 17-32; 10.5%). In conclusion, this study suggests that transgenic somatic cell nuclei from early passages can be reprogrammed more effectively than those from late passages. In addition, GFP, a non-invasive selection marker, can be used to select transgenic nuclear transfer embryos.     

A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants.

The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of experimental vaccines. However, an important limitation in most cases is the low concentration of the recombinant antigens in the plant tissues, which reduces the possibilities of practical applications. Because the site of insertion of the transferred DNA into the cellular chromosomal DNA is at random, different levels of foreign protein expression in independent transformants is expected. Strategies to allow the evaluation of a high number of the transgenic individuals, usually an expensive and very time consuming process, would permit the selection of those plants presenting the highest levels of recombinant protein expression. Here, we present the development of an experimental immunogen based in the expression of a highly immunogenic epitope from foot and mouth disease virus (FMDV) fused to the glucuronidase (gus A) reporter gene, which allows selection of the transgenic plants by the ss-glucuronidase (ssGUS) enzymatic activity. We produced transgenic plants of alfalfa expressing the immunogenic site between amino acid residues 135-160 of structural protein VP1 (VP135-160), fused to the ssGUS protein. Plants expressing the highest levels of the immunogenic epitope VP135-160, analyzed by Western blot, were efficiently selected based on their levels of ssGUS enzymatic activity. The FMDV epitope expressed in plants was highly immunogenic in mice which developed, after immunization, a strong anti-FMDV antibody response against a synthetic peptide representing the region VP135-160, to native virus VP1, and to purified FMDV particles. Additionally, these mice were completely protected against experimental challenge with the virulent virus. To our knowledge, this constitutes the first report of a peptide-based vaccine produced in transgenic plants that induces a protective immune response when used in experimental hosts. Also, these results demonstrated the possibility of using a novel and simple methodology for obtaining transgenic plants expressing high levels of foreign immunogenic epitopes, which could be directly applied in the development of plant-based vaccines.     

The use of transgenic Plasmodium berghei expressing the Plasmodium vivax antigen P25 to determine the transmission-blocking activity of sera from malaria vaccine trials

P25 is a major surface protein of Plasmodium ookinetes. Antibodies against P25 prevent the formation of oocysts in the mosquito and thereby block transmission of the parasite through an endemic population. Plasmodium vivax transmission-blocking vaccines based on Pv25 have undergone human trials and inhibit transmission significantly. The current assay to determine transmission-blocking activity (TBA) of these sera, the 'standard membrane feeding assay', is complex and can be performed by few groups worldwide that require both mosquito breeding facilities and access to volunteers naturally infected with P.vivax--a costly, and uncontrolled source of parasites. Here we report the development of novel assays to determine TBA using two clones (Pv25DR and Pv25DR3) of transgenic rodent parasites (Plasmodium berghei) expressing Pv25. We show that oocyst development of the transgenic parasites is inhibited by monoclonal antibody against Pv25 with the same kinetics exhibited by wild type parasites when exposed to mouse monoclonal antibodies targeted to a paralogous protein P28. Human transmission-blocking sera from a clinical vaccine trial of Pv25 inhibited oocyst development of Pv25DR and Pv25DR3, whereas non-blocking sera did not. We further show transmission-blocking activity can be determined in a simple assays of ookinete development in vitro, assays that obviate the need for mosquito colonies. These results demonstrate that transgenic rodent malarias expressing proteins from human Plasmodium species can be cheap, safe, and simple tools for testing TBA from sera. To this end the cloned lines have been deposited with, and are freely available from, MR4.     

A dietary mixture containing fish oil, resveratrol, lycopene, catechins, and vitamins E and C reduces atherosclerosis in transgenic mice

Chronic inflammation and proatherogenic lipids are important risk factors of cardiovascular disease (CVD). Specific dietary constituents such as polyphenols and fish oils may improve cardiovascular risk factors and may have a beneficial effect on disease outcomes. We hypothesized that the intake of an antiinflammatory dietary mixture (AIDM) containing resveratrol, lycopene, catechin, vitamins E and C, and fish oil would reduce inflammatory risk factors, proatherogenic lipids, and endpoint atherosclerosis. AIDM was evaluated in an inflammation model, male human C-reactive protein (CRP) transgenic mice, and an atherosclerosis model, female ApoE*3Leiden transgenic mice. Two groups of male human-CRP transgenic mice were fed AIDM [0.567% (wt:wt) powder and 0.933% (wt:wt oil)] or placebo for 6 wk. The effects of AIDM on basal and IL-1β-stimulated CRP expression were investigated. AIDM reduced cytokine-induced human CRP and fibrinogen expression in human-CRP transgenic mice. In the atherosclerosis study, 2 groups of female ApoE*3Leiden transgenic mice were fed an atherogenic diet supplemented with AIDM [0.567% (wt:wt) powder and 0.933% (wt:wt oil)] or placebo for 16 wk. AIDM strongly reduced plasma cholesterol, TG, and serum amyloid A concentrations compared with placebo. Importantly, long-term treatment of ApoE*3Leiden mice with AIDM markedly reduced the development of atherosclerosis by 96% compared with placebo. The effect on atherosclerosis was paralleled by a reduced expression of the vascular inflammation markers and adhesion molecules inter-cellular adhesion molecule-1 and E-selectin. Dietary supplementation of AIDM improves lipid and inflammatory risk factors of CVD and strongly reduces atherosclerotic lesion development in female transgenic mice.     

Plant-derived measles virus hemagglutinin protein induces neutralizing antibodies in mice

Measles remains a significant problem in both the developed and developing world, and new measles vaccination strategies need to be developed. This paper examines the strategy of utilizing transgenic plants expressing a measles antigen for the development of an oral sub-unit measles vaccine. A 1.8 kb fragment encompassing the coding region of the measles virus hemagglutinin (H) protein was cloned into a plant expression cassette. Three different expression constructs were tested: pBinH (H gene alone), pBinH/KDEL (addition of a C-terminal endoplasmic reticulum-retention sequence SEKDEL) and pBinSP/H/KDEL (further addition of an authentic N-terminal plant signal peptide). The highest levels of recombinant H protein production were observed in plants transformed with pBinH/KDEL. Mice inoculated intraperitoneally with transgenic plant derived recombinant H protein produced serum anti-H protein antibodies that neutralized the measles virus (MV) in vitro. Mice gavaged with transgenic tobacco leaf extracts also developed serum H protein-specific antibodies with neutralizing activity against MV in vitro. These results indicate that the plant-derived measles H protein is immunogenic when administered orally and that, with further development, oral vaccination utilizing transgenic plants may become a viable approach to measles vaccine development.     

HBcrAg的研究进展

乙型肝炎是一种严重危害人类健康的传染病。患者血中的HBeAg、HBcAb以及血清HBVDNA等含量常用来反映病情的发展及预后。近年来,HBcrAg作为一种新的血清学标志在患者病情的发展评价中被寄予厚望。本文就HBcrAg分析方法的发展、在临床上诊断中的优势以及临床应用等方面作了概述。     

骨钙素与儿童生长发育

骨钙素是成骨细胞分泌的骨蛋白 ,骨钙素水平与成骨细胞活性成正相关 ,是一项很好的反映骨形成与骨转化的生化指标。小儿骨生长发育有自身特点 ,骨钙素水平的改变类似儿童身高生长曲线 ,可以很好地应用于评价儿童生长发育及监测生长障碍患儿对治疗的反应     

Immunogenicity study of plant-made oral subunit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV)

Currently, killed-virus and modified-live PRRSV vaccines are used to control porcine reproductive and respiratory syndrome disease (PRRS). However, very limited efficacy of killed-virus vaccines and serious safety concerns for modified-live virus vaccines demand the development of novel PRRSV vaccines. In this report, we investigated the possibility of using transgenic plants as a cost-effective and scalable system for production and delivery of a viral protein as an oral subunit vaccine against PRRSV. Corn calli were genetically engineered to produce PRRSV viral envelope-associated M protein. Both serum and intestine mucosal antigen-specific antibodies were induced by oral administration of the transgenic plant tissues to mice. In addition, serum and mucosal antibodies showed virus neutralization activity. The neutralization antibody titers after the final boost reached 6.7 in serum and 3.7 in fecal extracts, respectively. A PRRSV-specific IFN-γ response was also detected in splenocytes of vaccinated animals. These results demonstrate that transgenic corn plants are an efficient subunit vaccine production and oral delivery system for generation of both systemic and mucosal immune responses against PRRSV.     

Hazardous effects of effluent from the chrome plating industry: 70 kDa heat shock protein expression as a marker of cellular damage in transgenic Drosophila melanogaster (hsp70-lacZ)

Hazardous effects of an effluent from the chrome plating industry were examined by exposing transgenic Drosophila melanogaster (hsp70-lacZ) to various concentrations (0.05, 0.1, 1.0, 10.0, and 100.0 micro L/mL) of the effluent through diet. The emergence pattern of adult flies was affected, along with impaired reproductive performance at the higher dietary concentrations of the effluent. Interestingly, the effect of the effluent was more pronounced in male than in female flies. The effect of the effluent on development of adult flies was concurrent with the expression pattern of the heat shock protein 70 gene (hsp70), both in larval tissues and in the reproductive organs of adult flies. We observed a dose- and time-dependent expression of hsp70 in third instar larvae exposed for different time intervals. Absence of hsp70 expression in larvae exposed to 0.1 micro L/mL of the effluent indicated that this is the highest nontoxic concentration for Drosophila. The stress gene assay in the reproductive organs of adult flies revealed hsp70 expression in the testis of male flies only. However, trypan blue dye exclusion tests in these tissues indicate tissue damage in the male accessory gland of adult flies, which was further confirmed by ultrastructural observations. In the present study we demonstrate the utility of transgenic Drosophila as an alternative animal model for evaluating hazardous effects of the effluent from the chrome plating industry and further reveal the cytoprotective role of hsp70 and its expression as an early marker in environmental risk assessment.