【作者】 吴拥军；

【导师】 赵德刚；

【作者基本信息】 贵州大学 ， 农药学， 2009， 博士

【摘要】 干扰素（IFN）作为一种动物细胞产生的细胞因子,具有广谱抗病毒活性和免疫保护作用。随着养鸡业集约化和商品化方向的发展,对病毒性疾病的有效预防与治疗,需要大量的干扰素。目前,IFN的生产主要以原核表达系统为主,后处理工艺繁琐,导致IFN活性低,生产成本高。而应用植物生物反应器生产IFN可以克服上述缺点,同时具有生产量大、可作为植物疫苗佐剂直接口服等优势。因此,研究IFN在植物中的表达特性及其生物学活性具有重要的理论价值和实践意义。研究利用GenBank数据库登录的ChIFN-γ基因序列,依据油菜密码子最高使用频率优化并设计了ChIFN-γ基因,人工合成长度614bp,包含有油菜Napin信号肽序列、Kozak序列和内质网滞留信号肽SEKDEL序列的ChIFN-γ融合基因。采用特异性引物,PCR扩增得到1158bp的Napin油菜种子特异性启动子。分析表明,Napin启动子中含有启动子基本元件及特异性元件ABREs和RY基序。应用同尾酶连接法构建了pSH-NGN、pSH-NGNL、pSH-IFNG植物表达载体用于遗传转化烟草和油菜。为了验证植物表达载体设计与构建是否正确,将含有质粒pSH-NGN、pSH-NGNL和pSH-IFNG的根癌农杆菌,以生菜为受体材料,采用真空渗透法进行瞬时表达研究。结果表明,三种植物表达载体在生菜中瞬时表达ChIFN-γ蛋白的平均含量分别为675.7pg/g、665.7pg/g及519.5pg/g。为简化蛋白提取方法,降低目的蛋白降解速度,采用不同的蛋白缓冲液提取ChIFN-γ蛋白。ELISA检测结果显示,以组成为50mM PBS,pH7.5、1%SDS、0.25MNaCl、1%β-巯基乙醇和0.05%Tween-20的缓冲液提取目的蛋白的效率最高,每克叶片最高达到1.2μg。Western blot检测表明,表达的蛋白分子量大小约30kDa。将含有质粒pSH-IFNG和pSH-NGNL的根癌农杆菌EHA105遗传转化烟草,获得24株转ChIFN-γ基因烟草。经PCR、RT-PCR、GUS组织化学染色和ELISA检测表明,ChIFN-γ基因已整合到烟草的染色体中,并能正确转录与表达。ChIFN-γ蛋白在转基因烟草植株中的表达量最高达到20μg/g,高出已报道的转基因烟草表达外源基因的通常水平（0.000017%-0.001%）。经Western blot检测,目的蛋白为30kDa和40kDa,分析为糖基化程度不同所致。对转基因烟草T₀代完整生长期的农艺性状观察发现,除虫害较少、叶片粘性增加、花苞较小、种子籽粒数少及色泽稍浅外,其它性状与野生型差异不显著。将质粒pSH-IFNG、pSH-NGNL以根癌农杆菌介导法遗传转化油菜,筛选出25株转ChIFN-γ油菜。其中Napin启动子驱动表达的ChIFN-γ蛋白最高表达量达到58μg/g,比35S启动子驱动的转化植株总体平均高出2.64倍,最高达5.17倍。表明在油菜中由Napin启动子及其信号肽组成的ChIFN-γ基因的表达盒,ChIFN-γ蛋白表达量明显高于组成型启动子35S。Napin启动子的‘渗漏’现象与其含有启动子元件有关。以转ChIFN-γ基因烟草T₀代叶片粗提总蛋白进行鸡胚成纤维细胞病变试验确定ChIFN-γ的活性效价。鸡胚成纤维细胞培养约24h呈单层时,采刚VSV(TCID₅₀=10^-5)病毒攻毒,在22h和42h时分别观察细胞形态,计算得到每毫克转基因烟草中ChIFN-γ蛋白效价为5.12x10³U。机械摩擦法接种易感型TMV病毒,6天内,转基因烟草组对病毒抑制率为100%,随着时间延长抑制率平缓下降,16天后抑制率稳定在约32%,接毒植株的生长并未表现出受到抑制的现象。对照组一周后开始出现不同程度的花叶和枯斑,感染率达到95%。说明转ChIFN-γ基因烟草植株对TMV的浸染具有显著抑制作用。采用漂浮板快速育苗,获取大量的转基因植株叶片。将转基因烟草叶片匀浆后添加至饲料中,以每克饲料添加50U ChIFN-γ蛋白和每只鸡20g饲料/天的喂养量,分组隔离喂养1-10天后,采用NDV强毒株病毒进行攻毒,20天后捕杀。结果显示,ChIFN-γ蛋白具有增加雏鸡体重的作用;血清凝集试验显示,饲喂ChIFN-γ蛋白组NDV抗体效价高于对照组;喂食300UChIFN-γ蛋白对雏鸡的保护率可达80%。表明ChIFN-γ蛋白可通过口服喂养途径显著地提高鸡体抗病毒能力。自然接种烟草蚜虫和离体利用转基因烟叶饲喂野生斜纹夜蛾幼虫及烟青虫,转ChIFN-γ基因烟叶表现出显著的抗虫效果。通过测定烟草烟碱含量、饲喂烟叶后烟青虫蛋白酶活性变化、烟草中挥发性物质和对烟草组织进行切片染色观察。发现烟碱含量与对照无显著差异;12h时,饲喂转基因烟草组的虫体蛋白酶活性较对照组低5.4%;转基因烟草叶片增厚,细胞致密,细胞壁增厚,表皮腺毛数量增加可能是导致其抗虫的直接因素之一;转基因组与野生型烟草挥发性成分存在一定的差异,转基因烟草中黑松三烯和西柏三烯二醇含量的提高与其抗虫性直接相关,ChIFN-γ基因激活的多种转录因子可能结合了腺毛发育相关基因的cis调节元件,上调了腺毛发育基因的表达,导致腺毛的数量增加,分泌的萜烯化合物增加从而具有显著地抗虫性。综上所述,植物能表达出有生物活性的ChIFN-γ蛋白,转ChIFN-γ基因植物具有一定的抗动、植病毒和抗虫作用。研究为应用植物生物反应器生产ChIFN-γ蛋白及其开发和应用奠定了基础;同时,通过直接口服饲喂转基因植物方式,对禽类疾病的预防与治疗提供了新的方向。更多还原

【Abstract】 As an animal cells cytokines, interferon （IFN） possesses broad-spectrum antiviral activity and the role of immune protection. With the intensification and commercialization of poultry industry, large amounts of interferon were needed for effective prevention against viral diseases. Conventionally, expression of recombinant IFN-γrelies mainly on microbial and mammalian cell expression systems in which the post-treatment process brings about cumbersome problem, resulting in susceptibility to proteolysis, shorter survival times and high production costs. However, plant bioreactor expression systems have demonstrated an advantage over other in vitro expression systems because it may provide an attractive and large production capacity, and inexpensive alternative to conventional production system in terms of low costs. In addition, plant-based vaccines can be directly used as direct oral advantages. Therefore, elucidation of the IFN expression characteristics and biological activity in plants may be highly important for both theoretical and practical benefits.Based on the sequence of the ChIFN-γgene from GenBank, a coding sequence for this gene was optimized by modification of codon usage of Brassica napus. The synthetic ChIFN-γgene is about 614bp along with a Napin signal peptide, Kozak sequence and an endoplasmic reticulum retention signal （SEKDEL）.With the utility of specific primers a specific promoter about 1,158 bp of Napin rape seeds and NOS terminator （around 220 bp） were obtained from Brassica napus genome via PCR amplification. Sequence analysis of napin gene showed that it contain promoter basic element, specific element of ABREs and RY motif. Using isocaudamer enzyme ligation method, plant expression vector pSH-NGN, pSH-NGNL and pSH-IFNG are constructed for the genetic transformation of tobacco, lettuce and rape, respectively.In order to verify the correctness of design and construction about plant expression vector, transient expression system was employed to detect lettuce （Lactuca sativa L.） vacuum-infiltrated with Agrobacterium tumefaciens containing plasmid pSH-NGN, pSH-NGNL or pSH-IFNG The ELISA detection showed that three expression levels of ChIFN-7 in lettuce were 0.6757, 0.6761 and 0.5195μg.g^-1 fresh leaf weight （FLW）, respectively. To simplify the method for extracting protein, and decrease the degradation rate of target protein, different protein extract buffers were used to extract ChIFN-γprotein. Extract solution （pH7.5） containing 50 mM PBS, 1% SDS, 0.25 M NaCl, 1%β-mercaptoethanol, and 0.05% Tween-20 demonstrated the highest extraction efficiency of ChIFN-γprotein via ELISA test, whose yield was as high as 1.2μg.g^-1 FLW. The result of western blot tests showed that the molecular weight of the protein was approximately 30 kDa.Agrobacterium tumefaciens strain EHA105 harboring pSH-IFNG and pSH-NGNL were used to carry out the genetic transformation in tobacco herein. A total of 24 transgenic ChIFN-γtobacco clones were obtained. Evidences from PCR, RT-PCR, GUS histochemical staining and ELISA quantitative tests had substantially proved that ChIFN-γgene had been integrated in the genome of tobacco, and it could transcribe and express correctly. The expression level of ChIFN-γin tobacco leaves was up to 20μg.g^-1 FLW, which is much higher than the common level （0.000017% to 0.001%）. Bands of approximately 30 kDa and 40 kDa were detected by Western analysis in transgenic tobacco leaves using an anti-chicken IFN-γantibody. The molecular weight of ChIFN-γby SDS-PAGE was higher than the predicted molecular weight, possibly duo to the different glycosylation among the lines. According to the observation of agronomic traits in the transgenic tobacco, it has been found that the character differences between the transgenes and wild type are not significant, except for the more leaves stickiness, smaller flower bud, less kernal number as well as shallower colors of the seed in the the former.Also Agrobacterium tumefaciens strain EHA105 harboring pSH-IFNG and pSH-NGNL were genetically transformed into Brassica napus. A total of 25 transgenic rape lines integrated with ChIFN-γwere obtained by screening. Plants transformed with the napin promoter targeting ChIFN-γto the nucleus, the expression content of ChIFN-γprotein could reach to 58μg.g^-1 FLW, which was around 2.46 times higher than that of transgenic plants using the constitutive cauliflower mosaic virus 35S promoter. It showed that the startup ability of in leaves of Brassica napus with the expression box containing napin promoters, and its signal peptide was obviously higher than the ones with constitutive promoters 35S. Napin promoter of ’leakage’ phenomenon is propably related to it’s promoter elements.An experiment about cytopathic effect of the chicken embryo fibroblasts had been undertaken using total crude protein extract from T₀ leaves of the transgenic ChIFN-γtobacco to determine the ChIFN-γbioactivity. Activate the passage monolayer chicken fibroblast cell when the cells culture 24 hours was infected with TCID₅₀ of vesicular stomatitis virus （VSV）. The ChIFN-γprotein titer per milligram transgenic tobacco was 5.12×103 U as calculated by morphological observation on cells at 22h and 42 h.Using mechanical friction method, the phenotype susceptive Tobacco Mosaic Virus （TMV） was inoculated. During the initial six days, the inhibiting rate of transgenic tobacco lines to virus was 100%. Unfortunately, this rate dropped gradually, and the inhibiting rate got stable at 32% after 16 day’s treatment. Moreover, significant inhibited effect to this virus had been investigated in the transgenic lines. After one week, diverse mosaic and necrotic lesion were obtained from the control group, and the, infection rates reached 95%. All stated above had proved that the transgenic tobacco enhanced the inhibiting effects to TMV.With rapid seeding on floating plates, a large number of leaves were obtained from the transgenic plants . Basis on the data that per gram feedstuff contains 50U ChIFN-γprotein and one chick feeds on 20 g feedstuff every day, the leaf homogenate of transgenic tobacco was then added to the feedstuff. Subsequently, animal feeding experiments were proceed with five groups, in whichs 10 chickens were used for each one. The tested chickens were subjected to virulent strain Newcastle Disease Virus （NDV） for 1-10 days, and then killed after 10 days. The results showed that feeding with ChIFN-γprotein group had higher weight growth rate than that of the control （feeding-free）. Therefore, ChIFN-γprotein demonstrated the ability to add up weight of chicken. Meanwhile, the results of serum agglutination proved that NDV antibody titer of feeding ChIFN-γgroups was higher than that of the control; suggestting ChIFN-γprotein could improve the ability of antivirus in chicken. In addition, the immunoprotection ratio to virus infection reached to 80% by feeding 300U ChIFN-γprotein, thus this protein might significantly increase chicken anti-viral capacity via oral feeding mode.Natural inoculation of tobacco aphids as well as feeding wild perfect insects of Spodoptera litura or tobacco budworm with in vitro transgenic tobacco leaves sustantially verified that the, transgenic tobacco leaves showed significantly a positive effects on the resistance to insect. In tobaccos, the nicotine content, self-protease activity, volatile substances were quantified, and plant tissue was further investigated by means of slice staining. Compared with the control, no significant difference in nicotine content was obtained from the transgenic lines, and insertean protease activity was lower by 5.4% in the feeding transgenic group after 12 hours. The leaf thickness, cell density, cell wall thickening, glandular hairs increase demonstrated a positive trend toward the insect resistance in transgenic tobacco. In addition,Some differences in volatile components were investigated from the transgenic tobacco, e.g. the contents duvatriendiol and 4,8,13-Duvatriene-1,3-diol, which are closely related to the insect resistance increased directly. ChIFN-γgene activation of several transcription factors may be binding the cis-regulatory elements of trichome development-related genes, increases the expression of genes of the trichome development, leading to increase in the number of trichomes, the secretion of terpenoid, which may be linked to the main mechanisms of the transgenic ChIFN-γgene tobaccos.In conclusion, we substantially created the transgenic lines expressing active ChIFN-γprotein. Transgenic ChIFN-γgene plants may have a certain role in enhancement the resistance to animal and plant virus, as well as to insect. Our findings may provide a foundation for the application of plant bioreactor in ChIFN-γprotein production. Rather, it also demonstrates a new clue to prevent or/and recover avian disease via direct oral feeding with transgenic plants.更多还原