弓形虫SAG1肽在烟草中的瞬时快速表达研究

发布时间：2018-05-05 17:10

  本文选题：转基因植物疫苗 + 弓形虫　； 参考：《南方医科大学》2012年硕士论文

【摘要】：转基因植物疫苗,使用简便,成本低廉,具有较好的安全性。目前已有多种优势疫苗分子在转基因植物中成功表达,且部分针对病毒和细菌的转基因植物疫苗已进入临床试验阶段,展示其具有广阔的应用前景。 弓形虫是一种寄生在人和动物体内的机会性致病原虫,可引起人兽共患弓形虫病。其对孕妇、幼儿、免疫力低下人群和畜牧业发展均构成严重威胁。因此,研制价廉易用的动物用弓形虫转基因植物疫苗,对于切断人类弓形虫病传播途径和保护畜牧业的意义重大。 本研究利用快速高效植物瞬时表达系统,采用根癌农杆菌LBA4404介导的方法侵染烟草,将弓形虫SAG1肽基因导入无土栽培的烟草中,筛选鉴定烟草叶片可溶性总蛋白(TSP),期望获得瞬时快速表达且具有免疫学活性的弓形虫SAG1肽,为新型弓形虫植物疫苗深入研发奠定基础。 一快速高效植物瞬时表达系统的实验室烟草无土栽培体系的构建 目的 科用人工气候箱在实验室建立适用于快速高效植物瞬时表达系统的烟草无土栽培体系,从而为在普通分子生物学实验室便捷实施小规模的转基因植物疫苗研发奠定基础。 方法 1.采用无土栽培的方式进行烟草培养,并且应用单因素实验观察基质松紧度、培育温度、相对湿度和光照时间等条件对烟草生长情况的影响,以当烟草达到生长强壮、整齐一致、根系发达、叶色浅绿或绿色的生长状态时的生长条件判别为适宜的培育条件。 2.基于Geminivirus快速高效植物瞬时表达系统,以绿色荧光蛋白GFP作为研究对象,采用优化的无土栽培方式进行烟草培育,并且应用单因素实验观察叶片采收时间、烟草品种、侵染工程菌浓度、侵染时机等条件对无土栽培烟草表达绿色荧光蛋白(GFP)的影响,以紫外灯检测、Western blot、ELISA等方法进行分析。 3. ELISA分析数据以SPSS13.0统计处理,两种烟草品种组间总体比较采用析因设计的方差分析,叶片采收时间组内、侵染工程菌浓度组内和侵染时机组内比较采用单向方差分析(One-way ANOVA),两种烟草品种组间每个叶片采收时间点、每个侵染工程菌浓度点、每个侵染时机点采用两样本t检验(Independent-Samples T Test)分析。P0.05表示差异具有统计学意义。 4.称取无土栽培体系培育的烟草新鲜叶片的重量,以平均每株叶片的重量作为生物量比较的评价指标。两种烟草品种组间的生物量采用两样本t检验(Independent-Samples T Test)分析,P0.05表示差异具有统计学意义。 结果 1.使用全智能人工气候植物箱能便捷、有效地实施烟草的无土栽培。当培育体系控制为：以少量基质固定烟草根系,无土栽培基质松紧度中度,即需达到握之成团、触之即散的效果；改良Hoagland营养液提供营养；光照强度为9000LX/层,光照时间为16h/d,日／夜温度为28/21℃,空气泵气石同步充气；植物箱10min/h换气；相对湿度为80%,烟草生长强壮、整齐一致、根系发达、叶色浅绿或绿色。 2.利用优化的无土栽培体系培育烟草,工程菌pBYGFPDsRed.R/LBA4404侵染的本明烟和豫烟5号均能高效表达GFP。侵染工程菌的最佳OD600为0.8；叶片采收最佳时间为侵染后4天(4dpi)；本明烟和豫烟5号最佳侵染时机分别为第6周和第5周；豫烟5号的平均生物量高于本明烟。 结论 基于人工气候箱成功构建了适用于快速高效植物瞬时表达系统的实验室烟草无土栽培体系。结合Germinivirus表达载体pBYGFPDsRed.R,在本明烟和豫烟5号中成功实现GFP的快速高效表达。首次证实豫烟5号可作为新型植物瞬时表达体系的良好受体植物。 二弓形虫SAG1肽的基因克隆以及在烟草中的瞬时快速表达 目的 选择含优势表位的弓形虫SAG1肽,以HBc呈递,优化改造合成后,构建植物瞬时高效载体,在烟草中实现其瞬时快速表达。 方法 1.选取登录号为X14080.1的弓形虫SAG1基因序列,利用www.kazusa.or.jp/codon (密码子使用频率网)查询获得烟草密码子的使用频率表。基于文献复习选取SAG1编码蛋白序列中具有T、B细胞表位的肽段,并利用BioSun软件进行表位分析。同时,将其原始密码子优化为烟草偏爱的密码子,优化时选择高／较高使用频率的密码子,且不改变其氨基酸序列,以利于在烟草中的表达。优化后的SAG1肽基因序列分别命名为P30-Ⅰ和P30-Ⅱ; P30-Ⅰ以中间插入方式与HBc连接,命名为HBc-P30-Ⅰ; P30-Ⅱ自C-端插入HBc,命名为HBc-P30-Ⅱ。对HBc-P30-Ⅰ和HBc-P30-Ⅱ进行GenScript Rare Codon Analysis Tool分析,并进一步优化。于HBc-P30-Ⅰ和P30-Ⅱ尾端加入His-Tag标签序列后,HBc-P30-Ⅰ和P30-Ⅱ由GenScript公司分别合成到麦胚乳无细胞表达载体pIVEX1.4WG和GenScript标准载体PUC57-simple中。 2. pIVEX1.4WG-HBc-P30-Ⅰ用EcoRⅠ和HindⅢ双酶切方式进行鉴定,PUC57-simple-P30-Ⅱ用NdeⅠ和SalⅠ双酶切方式进行鉴定,并进行测序验证。 3.将pIVEX1.4WG-HBc-P30-Ⅰ进行BglⅡ单酶切,大片段序列进行自身环化,构建中间载体pIVEX1.4WG-HBc;酶切鉴定后,将PUC57-simple-P30-Ⅱ的P30-Ⅱ片段通过Sal Ⅰ、 Sma Ⅰ双酶切方式克隆到pIVEX1.4WG-HBc中,构建中间载体pIVEX1.4WG-HBc-P30-Ⅱ,酶切和测序验证。 4.将pIVEX1.4WG-HBc的HBc片段通过XbaⅠ和SacⅠ双酶切方式克隆到Gemini virus载体pBYGFPDsRed.R(含有GFP和DsRed-1)中,构建快速高效植物瞬时表达载体pBYR-GFP-HBc(只含有GFP),酶切和测序验证。 5.将pIVEX1.4WG-HBc-P30-Ⅰ的HBc-P30-Ⅰ片段通过XbaⅠ和SacⅠ双酶切方式克隆到Gemini virus载体pBYGFPDsRedR (含有GFP和DsRed-1)中,构建快速高效植物瞬时表达载体pBYR-GFP-HBc-P30-Ⅰ(只含有GFP),酶切和测序验证。 6.将pIVEX1.4WG-HBc-P30-Ⅱ的HBc-P30-Ⅱ片段通过XbaⅠ和SacⅠ双酶切方式克隆到Gemini virus载体pBYGFPDsRed.R(含有GFP和DsRed-1),构建快速高效植物瞬时表达载体pBYR-GFP-HBc-P30-Ⅱ(只含有GFP),酶切和测序验证。 7.采用根癌农杆菌LBA4404介导的方法,将含有SAG1肽基因的快速高效植物瞬时表达载体侵染到无土栽培培育的本明烟和豫烟5号中,侵染后4天提取两种烟草叶片TSP,并以弓形虫anti-SAG1单抗Y3A8和His-Tag (2A8) Mouse mAb进行Western blot鉴定,以筛选获得烟草表达的具有免疫反应性的弓形虫SAG1肽。 结果 1.选取的SAG1肽编码蛋白序列经BioSun软件进行表位分析,P30-Ⅰ和P30-Ⅱ都具有多个T、B细胞表位,同时优化后的SAG1肽基因经GenScript Rare Codon Analysis Tool分析都说明HBc-P30-Ⅰ和HBc-P30-Ⅱ能够在烟草植物系统中表达。 2. GenScript公司合成的序列经酶切鉴定和测序验证,均与设计的序列相符；重组的中间载体和快速高效植物瞬时表达载体经酶切鉴定均得到预期大小片段,经序列测定确证。 3.经Western blot鉴定,在本明烟和豫烟5号中,HBc-P30-Ⅰ在预期大小区域均没有出现特异性反应条带,但是HBc-P30-Ⅱ在约46.4kDa预期大小区域均出现特异性反应条带。 结论 成功设计合成弓形虫SAG1肽基因；并构建中间载体：pIVEX1.4WG-HBc、 pIVEXl.4WG-HBc-P30-Ⅱ和快速高效植物瞬时表达载体：pBYR-GFP-HBc、 pBYR-GFP-HBc-P30-Ⅰ、pBYR-GFP-HBc-P30-Ⅱ在本明烟和豫烟5号中,成功实现以HBc呈递、含优势表位的弓形虫SAG1肽的瞬时快速表达,其为大小46.4kDa的HBc-P30-Ⅱ蛋白。
[Abstract]:The transgenic plant vaccine has the advantages of simple and convenient use , low cost and good safety .

Toxoplasma gondii is an opportunistic pathogen parasitic in human and animal , which can cause toxoplasmosis . It poses a serious threat to the development of pregnant women , young children , low immunity and animal husbandry . Therefore , it is of great significance to develop transgenic plant vaccine of Toxoplasma gondii in cheap and easy - to - use animals and to cut off the transmission path of human toxoplasmosis and protect animal husbandry .

In this study , the rapid and high efficiency plant transient expression system was used to infect tobacco by Agrobacterium tumefaciens LBA4404 . The Toxoplasma gondii SAG1 peptide gene was introduced into soilless tobacco , and the soluble total protein ( TSP ) of tobacco leaf was screened .

Construction of a rapid and efficient plant transient expression system in laboratory tobacco soilless culture system

Purpose

The invention establishes a tobacco soilless culture system suitable for the rapid and efficient plant transient expression system in a laboratory by using an artificial climate box , thereby laying a foundation for conveniently implementing small - scale transgenic plant vaccine development in the general molecular biology laboratory .

method

1 . The tobacco culture was carried out in a soilless culture manner , and the effects of the conditions on the growth of the tobacco were observed by single factor experiment , so as to judge the growth condition of the tobacco growing strong , consistent , root system developed , leaf color light green or green .

2 . Based on the rapid and efficient plant transient expression system of Geminivirus , the green fluorescent protein GFP was used as the research object , the optimized soilless culture method was adopted to cultivate tobacco , and the effects of leaf harvesting time , tobacco variety , concentration of infection engineering bacteria and time of infection on green fluorescent protein ( GFP ) were observed under the conditions of single factor experiment , UV lamp detection , Western blot and ELISA .

3 . The data of ELISA was analyzed by SPSS 13.0 . The general comparison between the two varieties of tobacco varieties was analyzed by one - way ANOVA . One - way ANOVA was used in the concentration group of infected engineering bacteria , and the concentration points of each infection engineering bacteria were compared between the two tobacco varieties .

4 . The weight of fresh leaves of tobacco cultivated by soilless culture system was weighed , and the weight of each leaf was used as the evaluation index of biomass comparison . The biomass between two tobacco varieties was analyzed by two independent - Samples T Test , and the difference was statistically significant .

Results

1 . The whole intelligent artificial climate plant box can be used for conveniently and effectively implementing soilless culture of tobacco .
improve that nutrition of Hoagland nutrient solution ;
the illumination intensity is 9000 LX / layer , the illumination time is 16 hours / day , the day / night temperature is 28 / 21 DEG C , and the air pump gas stone is synchronously inflated ;
Plant tank 10min / h ventilation ;
The relative humidity is 80 % , the growth of the tobacco is strong , neat and consistent , the root system is developed , the leaf color is light green or green .

2 . Using the optimized soilless culture system to cultivate tobacco and engineering bacteria pBYGFPDsRed . R / LBA4404 , GFP could be expressed with high efficiency .
The optimal time for blade recovery is 4 days after infection ( 4 dpi ) ;
The best time of infection was 6 weeks and 5 weeks , respectively .
The average biomass of Yuyan No . 5 was higher than that of Benjamin .

Conclusion

A laboratory tobacco soilless culture system suitable for rapid and efficient plant transient expression system was successfully constructed on the basis of artificial climate box . Combining with Germinivirus expression vector pBYGFPDsRed .

Cloning of SAG1 peptide from Toxoplasma gondii and its transient expression in tobacco

Purpose

Toxoplasma gondii SAG1 peptide with dominant epitope was selected to be presented and synthesized . After optimization and transformation , a transient high - efficient vector was constructed , and its transient expression was achieved in tobacco .

method

1 . The gene sequence of Toxoplasma gondii SAG1 with accession number X14080.1 is selected . The frequency table for obtaining the codon of tobacco is obtained by using www.kazusa . or . jp / codon ( codon usage frequency network ) .

2 . The pIVEX1 . 4WG - HB30 - 鈪，

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