發(fā)布時(shí)間：2018-05-05 17:10

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

【摘要】：轉(zhuǎn)基因植物疫苗,使用簡(jiǎn)便,成本低廉,具有較好的安全性。目前已有多種優(yōu)勢(shì)疫苗分子在轉(zhuǎn)基因植物中成功表達(dá),且部分針對(duì)病毒和細(xì)菌的轉(zhuǎn)基因植物疫苗已進(jìn)入臨床試驗(yàn)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(dòng)物體內(nèi)的機(jī)會(huì)性致病原蟲,可引起人獸共患弓形蟲病。其對(duì)孕婦、幼兒、免疫力低下人群和畜牧業(yè)發(fā)展均構(gòu)成嚴(yán)重威脅。因此,研制價(jià)廉易用的動(dòng)物用弓形蟲轉(zhuǎn)基因植物疫苗,對(duì)于切斷人類弓形蟲病傳播途徑和保護(hù)畜牧業(yè)的意義重大。 本研究利用快速高效植物瞬時(shí)表達(dá)系統(tǒng),采用根癌農(nóng)桿菌LBA4404介導(dǎo)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(shí)快速表達(dá)且具有免疫學(xué)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(shí)表達(dá)系統(tǒng)的實(shí)驗(yàn)室煙草無土栽培體系的構(gòu)建 目的 科用人工氣候箱在實(shí)驗(yàn)室建立適用于快速高效植物瞬時(shí)表達(dá)系統(tǒng)的煙草無土栽培體系,從而為在普通分子生物學(xué)實(shí)驗(yàn)室便捷實(shí)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(jìn)行煙草培養(yǎng),并且應(yīng)用單因素實(shí)驗(yàn)觀察基質(zhì)松緊度、培育溫度、相對(duì)濕度和光照時(shí)間等條件對(duì)煙草生長(zhǎng)情況的影響,以當(dāng)煙草達(dá)到生長(zhǎng)強(qiáng)壯、整齊一致、根系發(fā)達(dá)、葉色淺綠或綠色的生長(zhǎng)狀態(tài)時(shí)的生長(zhǎng)條件判別為適宜的培育條件。 2.基于Geminivirus快速高效植物瞬時(shí)表達(dá)系統(tǒng),以綠色熒光蛋白GFP作為研究對(duì)象,采用優(yōu)化的無土栽培方式進(jìn)行煙草培育,并且應(yīng)用單因素實(shí)驗(yàn)觀察葉片采收時(shí)間、煙草品種、侵染工程菌濃度、侵染時(shí)機(jī)等條件對(duì)無土栽培煙草表達(dá)綠色熒光蛋白(GFP)的影響,以紫外燈檢測(cè)、Western blot、ELISA等方法進(jìn)行分析。 3. ELISA分析數(shù)據(jù)以SPSS13.0統(tǒng)計(jì)處理,兩種煙草品種組間總體比較采用析因設(shè)計(jì)的方差分析,葉片采收時(shí)間組內(nèi)、侵染工程菌濃度組內(nèi)和侵染時(shí)機(jī)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(gè)葉片采收時(shí)間點(diǎn)、每個(gè)侵染工程菌濃度點(diǎn)、每個(gè)侵染時(shí)機(jī)點(diǎn)采用兩樣本t檢驗(yàn)(Independent-Samples T Test)分析。P0.05表示差異具有統(tǒng)計(jì)學(xué)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(píng)價(jià)指標(biāo)。兩種煙草品種組間的生物量采用兩樣本t檢驗(yàn)(Independent-Samples T Test)分析,P0.05表示差異具有統(tǒng)計(jì)學(xué)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(shí)施煙草的無土栽培。當(dāng)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(dá)到握之成團(tuán)、觸之即散的效果；改良Hoagland營(yíng)養(yǎng)液提供營(yíng)養(yǎng)；光照強(qiáng)度為9000LX/層,光照時(shí)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(duì)濕度為80%,煙草生長(zhǎng)強(qiáng)壯、整齊一致、根系發(fā)達(dá)、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(hào)均能高效表達(dá)GFP。侵染工程菌的最佳OD600為0.8；葉片采收最佳時(shí)間為侵染后4天(4dpi)；本明煙和豫煙5號(hào)最佳侵染時(shí)機(jī)分別為第6周和第5周；豫煙5號(hào)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(shí)表達(dá)系統(tǒng)的實(shí)驗(yàn)室煙草無土栽培體系。結(jié)合Germinivirus表達(dá)載體pBYGFPDsRed.R,在本明煙和豫煙5號(hào)中成功實(shí)現(xiàn)GFP的快速高效表達(dá)。首次證實(shí)豫煙5號(hào)可作為新型植物瞬時(shí)表達(dá)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(shí)快速表達(dá) 目的 選擇含優(yōu)勢(shì)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(shí)高效載體,在煙草中實(shí)現(xiàn)其瞬時(shí)快速表達(dá)。 方法 1.選取登錄號(hào)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(xiàn)復(fù)習(xí)選取SAG1編碼蛋白序列中具有T、B細(xì)胞表位的肽段,并利用BioSun軟件進(jìn)行表位分析。同時(shí),將其原始密碼子優(yōu)化為煙草偏愛的密碼子,優(yōu)化時(shí)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(dá)。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(duì)HBc-P30-Ⅰ和HBc-P30-Ⅱ進(jìn)行GenScript Rare Codon Analysis Tool分析,并進(jìn)一步優(yōu)化。于HBc-P30-Ⅰ和P30-Ⅱ尾端加入His-Tag標(biāo)簽序列后,HBc-P30-Ⅰ和P30-Ⅱ由GenScript公司分別合成到麥胚乳無細(xì)胞表達(dá)載體pIVEX1.4WG和GenScript標(biāo)準(zhǔn)載體PUC57-simple中。 2. pIVEX1.4WG-HBc-P30-Ⅰ用EcoRⅠ和HindⅢ雙酶切方式進(jìn)行鑒定,PUC57-simple-P30-Ⅱ用NdeⅠ和SalⅠ雙酶切方式進(jìn)行鑒定,并進(jìn)行測(cè)序驗(yàn)證。 3.將pIVEX1.4WG-HBc-P30-Ⅰ進(jìn)行BglⅡ單酶切,大片段序列進(jìn)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(cè)序驗(yàn)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(shí)表達(dá)載體pBYR-GFP-HBc(只含有GFP),酶切和測(cè)序驗(yàn)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(shí)表達(dá)載體pBYR-GFP-HBc-P30-Ⅰ(只含有GFP),酶切和測(cè)序驗(yàn)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(shí)表達(dá)載體pBYR-GFP-HBc-P30-Ⅱ(只含有GFP),酶切和測(cè)序驗(yàn)證。 7.采用根癌農(nóng)桿菌LBA4404介導(dǎo)的方法,將含有SAG1肽基因的快速高效植物瞬時(shí)表達(dá)載體侵染到無土栽培培育的本明煙和豫煙5號(hào)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(jìn)行Western blot鑒定,以篩選獲得煙草表達(dá)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(jìn)行表位分析,P30-Ⅰ和P30-Ⅱ都具有多個(gè)T、B細(xì)胞表位,同時(shí)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(dá)。 2. GenScript公司合成的序列經(jīng)酶切鑒定和測(cè)序驗(yàn)證,均與設(shè)計(jì)的序列相符；重組的中間載體和快速高效植物瞬時(shí)表達(dá)載體經(jīng)酶切鑒定均得到預(yù)期大小片段,經(jīng)序列測(cè)定確證。 3.經(jīng)Western blot鑒定,在本明煙和豫煙5號(hào)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(jì)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(shí)表達(dá)載體：pBYR-GFP-HBc、 pBYR-GFP-HBc-P30-Ⅰ、pBYR-GFP-HBc-P30-Ⅱ在本明煙和豫煙5號(hào)中,成功實(shí)現(xiàn)以HBc呈遞、含優(yōu)勢(shì)表位的弓形蟲SAG1肽的瞬時(shí)快速表達(dá),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(hào)：1848568