本文選題：轉(zhuǎn)基因植物疫苗 + 弓形蟲　； 參考：《南方醫(yī)科大學》2012年碩士論文

【摘要】：轉(zhuǎn)基因植物疫苗,使用簡便,成本低廉,具有較好的安全性。目前已有多種優(yōu)勢疫苗分子在轉(zhuǎn)基因植物中成功表達,且部分針對病毒和細菌的轉(zhuǎn)基因植物疫苗已進入臨床試驗階段,展示其具有廣闊的應(yīng)用前景。 弓形蟲是一種寄生在人和動物體內(nèi)的機會性致病原蟲,可引起人獸共患弓形蟲病。其對孕婦、幼兒、免疫力低下人群和畜牧業(yè)發(fā)展均構(gòu)成嚴重威脅。因此,研制價廉易用的動物用弓形蟲轉(zhuǎn)基因植物疫苗,對于切斷人類弓形蟲病傳播途徑和保護畜牧業(yè)的意義重大。 本研究利用快速高效植物瞬時表達系統(tǒng),采用根癌農(nóng)桿菌LBA4404介導的方法侵染煙草,將弓形蟲SAG1肽基因?qū)霟o土栽培的煙草中,篩選鑒定煙草葉片可溶性總蛋白(TSP),期望獲得瞬時快速表達且具有免疫學活性的弓形蟲SAG1肽,為新型弓形蟲植物疫苗深入研發(fā)奠定基礎(chǔ)。 一快速高效植物瞬時表達系統(tǒng)的實驗室煙草無土栽培體系的構(gòu)建 目的 科用人工氣候箱在實驗室建立適用于快速高效植物瞬時表達系統(tǒng)的煙草無土栽培體系,從而為在普通分子生物學實驗室便捷實施小規(guī)模的轉(zhuǎn)基因植物疫苗研發(fā)奠定基礎(chǔ)。 方法 1.采用無土栽培的方式進行煙草培養(yǎng),并且應(yīng)用單因素實驗觀察基質(zhì)松緊度、培育溫度、相對濕度和光照時間等條件對煙草生長情況的影響,以當煙草達到生長強壯、整齊一致、根系發(fā)達、葉色淺綠或綠色的生長狀態(tài)時的生長條件判別為適宜的培育條件。 2.基于Geminivirus快速高效植物瞬時表達系統(tǒng),以綠色熒光蛋白GFP作為研究對象,采用優(yōu)化的無土栽培方式進行煙草培育,并且應(yīng)用單因素實驗觀察葉片采收時間、煙草品種、侵染工程菌濃度、侵染時機等條件對無土栽培煙草表達綠色熒光蛋白(GFP)的影響,以紫外燈檢測、Western blot、ELISA等方法進行分析。 3. ELISA分析數(shù)據(jù)以SPSS13.0統(tǒng)計處理,兩種煙草品種組間總體比較采用析因設(shè)計的方差分析,葉片采收時間組內(nèi)、侵染工程菌濃度組內(nèi)和侵染時機組內(nèi)比較采用單向方差分析(One-way ANOVA),兩種煙草品種組間每個葉片采收時間點、每個侵染工程菌濃度點、每個侵染時機點采用兩樣本t檢驗(Independent-Samples T Test)分析。P0.05表示差異具有統(tǒng)計學意義。 4.稱取無土栽培體系培育的煙草新鮮葉片的重量,以平均每株葉片的重量作為生物量比較的評價指標。兩種煙草品種組間的生物量采用兩樣本t檢驗(Independent-Samples T Test)分析,P0.05表示差異具有統(tǒng)計學意義。 結(jié)果 1.使用全智能人工氣候植物箱能便捷、有效地實施煙草的無土栽培。當培育體系控制為：以少量基質(zhì)固定煙草根系,無土栽培基質(zhì)松緊度中度,即需達到握之成團、觸之即散的效果；改良Hoagland營養(yǎng)液提供營養(yǎng)；光照強度為9000LX/層,光照時間為16h/d,日／夜溫度為28/21℃,空氣泵氣石同步充氣；植物箱10min/h換氣；相對濕度為80%,煙草生長強壯、整齊一致、根系發(fā)達、葉色淺綠或綠色。 2.利用優(yōu)化的無土栽培體系培育煙草,工程菌pBYGFPDsRed.R/LBA4404侵染的本明煙和豫煙5號均能高效表達GFP。侵染工程菌的最佳OD600為0.8；葉片采收最佳時間為侵染后4天(4dpi)；本明煙和豫煙5號最佳侵染時機分別為第6周和第5周；豫煙5號的平均生物量高于本明煙。 結(jié)論 基于人工氣候箱成功構(gòu)建了適用于快速高效植物瞬時表達系統(tǒng)的實驗室煙草無土栽培體系。結(jié)合Germinivirus表達載體pBYGFPDsRed.R,在本明煙和豫煙5號中成功實現(xiàn)GFP的快速高效表達。首次證實豫煙5號可作為新型植物瞬時表達體系的良好受體植物。 二弓形蟲SAG1肽的基因克隆以及在煙草中的瞬時快速表達 目的 選擇含優(yōu)勢表位的弓形蟲SAG1肽,以HBc呈遞,優(yōu)化改造合成后,構(gòu)建植物瞬時高效載體,在煙草中實現(xiàn)其瞬時快速表達。 方法 1.選取登錄號為X14080.1的弓形蟲SAG1基因序列,利用www.kazusa.or.jp/codon (密碼子使用頻率網(wǎng))查詢獲得煙草密碼子的使用頻率表�；谖墨I復習選取SAG1編碼蛋白序列中具有T、B細胞表位的肽段,并利用BioSun軟件進行表位分析。同時,將其原始密碼子優(yōu)化為煙草偏愛的密碼子,優(yōu)化時選擇高／較高使用頻率的密碼子,且不改變其氨基酸序列,以利于在煙草中的表達。優(yōu)化后的SAG1肽基因序列分別命名為P30-Ⅰ和P30-Ⅱ; P30-Ⅰ以中間插入方式與HBc連接,命名為HBc-P30-Ⅰ; P30-Ⅱ自C-端插入HBc,命名為HBc-P30-Ⅱ。對HBc-P30-Ⅰ和HBc-P30-Ⅱ進行GenScript Rare Codon Analysis Tool分析,并進一步優(yōu)化。于HBc-P30-Ⅰ和P30-Ⅱ尾端加入His-Tag標簽序列后,HBc-P30-Ⅰ和P30-Ⅱ由GenScript公司分別合成到麥胚乳無細胞表達載體pIVEX1.4WG和GenScript標準載體PUC57-simple中。 2. pIVEX1.4WG-HBc-P30-Ⅰ用EcoRⅠ和HindⅢ雙酶切方式進行鑒定,PUC57-simple-P30-Ⅱ用NdeⅠ和SalⅠ雙酶切方式進行鑒定,并進行測序驗證。 3.將pIVEX1.4WG-HBc-P30-Ⅰ進行BglⅡ單酶切,大片段序列進行自身環(huán)化,構(gòu)建中間載體pIVEX1.4WG-HBc;酶切鑒定后,將PUC57-simple-P30-Ⅱ的P30-Ⅱ片段通過Sal Ⅰ、 Sma Ⅰ雙酶切方式克隆到pIVEX1.4WG-HBc中,構(gòu)建中間載體pIVEX1.4WG-HBc-P30-Ⅱ,酶切和測序驗證。 4.將pIVEX1.4WG-HBc的HBc片段通過XbaⅠ和SacⅠ雙酶切方式克隆到Gemini virus載體pBYGFPDsRed.R(含有GFP和DsRed-1)中,構(gòu)建快速高效植物瞬時表達載體pBYR-GFP-HBc(只含有GFP),酶切和測序驗證。 5.將pIVEX1.4WG-HBc-P30-Ⅰ的HBc-P30-Ⅰ片段通過XbaⅠ和SacⅠ雙酶切方式克隆到Gemini virus載體pBYGFPDsRedR (含有GFP和DsRed-1)中,構(gòu)建快速高效植物瞬時表達載體pBYR-GFP-HBc-P30-Ⅰ(只含有GFP),酶切和測序驗證。 6.將pIVEX1.4WG-HBc-P30-Ⅱ的HBc-P30-Ⅱ片段通過XbaⅠ和SacⅠ雙酶切方式克隆到Gemini virus載體pBYGFPDsRed.R(含有GFP和DsRed-1),構(gòu)建快速高效植物瞬時表達載體pBYR-GFP-HBc-P30-Ⅱ(只含有GFP),酶切和測序驗證。 7.采用根癌農(nóng)桿菌LBA4404介導的方法,將含有SAG1肽基因的快速高效植物瞬時表達載體侵染到無土栽培培育的本明煙和豫煙5號中,侵染后4天提取兩種煙草葉片TSP,并以弓形蟲anti-SAG1單抗Y3A8和His-Tag (2A8) Mouse mAb進行Western blot鑒定,以篩選獲得煙草表達的具有免疫反應(yīng)性的弓形蟲SAG1肽。 結(jié)果 1.選取的SAG1肽編碼蛋白序列經(jīng)BioSun軟件進行表位分析,P30-Ⅰ和P30-Ⅱ都具有多個T、B細胞表位,同時優(yōu)化后的SAG1肽基因經(jīng)GenScript Rare Codon Analysis Tool分析都說明HBc-P30-Ⅰ和HBc-P30-Ⅱ能夠在煙草植物系統(tǒng)中表達。 2. GenScript公司合成的序列經(jīng)酶切鑒定和測序驗證,均與設(shè)計的序列相符；重組的中間載體和快速高效植物瞬時表達載體經(jīng)酶切鑒定均得到預(yù)期大小片段,經(jīng)序列測定確證。 3.經(jīng)Western blot鑒定,在本明煙和豫煙5號中,HBc-P30-Ⅰ在預(yù)期大小區(qū)域均沒有出現(xiàn)特異性反應(yīng)條帶,但是HBc-P30-Ⅱ在約46.4kDa預(yù)期大小區(qū)域均出現(xiàn)特異性反應(yīng)條帶。 結(jié)論 成功設(shè)計合成弓形蟲SAG1肽基因；并構(gòu)建中間載體：pIVEX1.4WG-HBc、 pIVEXl.4WG-HBc-P30-Ⅱ和快速高效植物瞬時表達載體：pBYR-GFP-HBc、 pBYR-GFP-HBc-P30-Ⅰ、pBYR-GFP-HBc-P30-Ⅱ在本明煙和豫煙5號中,成功實現(xiàn)以HBc呈遞、含優(yōu)勢表位的弓形蟲SAG1肽的瞬時快速表達,其為大小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，

本文編號：1848568