本文選題：聚球藻 + 光生物反應(yīng)器��； 參考：《上海海洋大學(xué)》2016年碩士論文

【摘要】：對蝦白斑綜合癥病毒(WSSV)于1992年首次在臺灣發(fā)現(xiàn),隨后迅速蔓延至各大洲,對全世界的對蝦養(yǎng)殖業(yè)造成嚴(yán)重的病害,經(jīng)濟(jì)損失超過70億美元。國際上,尚不存在產(chǎn)業(yè)化的WSSV防治藥物。研究發(fā)現(xiàn),WSSV被膜中的特異性結(jié)構(gòu)蛋白VP28在病毒侵染細(xì)胞過程中發(fā)揮關(guān)鍵作用,該蛋白經(jīng)異源系統(tǒng)表達(dá)后,通過口服或注射接種,可提高對蝦抗WSSV的能力。我們的研究也發(fā)現(xiàn),轉(zhuǎn)vp28基因工程藍(lán)藻經(jīng)對蝦口服后攻毒,發(fā)現(xiàn)對蝦在30天內(nèi)能有效抵抗WSSV(成活率達(dá)70%-90%),表明已開發(fā)的工程聚球藻具有巨大的產(chǎn)業(yè)化前景。目前本實(shí)驗(yàn)室轉(zhuǎn)vp28基因工程聚球藻口服劑的研制已進(jìn)入實(shí)驗(yàn)準(zhǔn)備階段,其中工程聚球藻的規(guī)模培養(yǎng)及采收是其產(chǎn)業(yè)化的重要一環(huán)。我們在進(jìn)行工程聚球藻規(guī)模培養(yǎng)中,認(rèn)識到其產(chǎn)業(yè)化受3方面因素制約:1)目前規(guī)模培養(yǎng)設(shè)備及技術(shù)不成熟,生物量產(chǎn)量較低;2)重組蛋白表達(dá)率尚有提高的潛力;3)缺少安全快速高效的采收技術(shù)。為解決上述問題,本論文特從以下3方面進(jìn)行系統(tǒng)研究:1.光照強(qiáng)度對工程聚球藻生物量的調(diào)控為提高轉(zhuǎn)vp28基因工程聚球藻的生物量產(chǎn)量,光照強(qiáng)度(I)的調(diào)控依據(jù)2個因素:1)I與p H、溫度的交互作用;2)I在反應(yīng)器中的衰減作用。為此,本論文首先通過單因素實(shí)驗(yàn),確定了當(dāng)凈光合速率為極大值時,I、溫度及p H的交互范圍,分別為200-400μmol/m2s、28-48°C、6.5-8.5;并進(jìn)一步通過響應(yīng)面實(shí)驗(yàn)測定I、p H及溫度的最優(yōu)交互條件,即分別為300±10μmol/m2s、37°C、7.5,此時凈光合速率達(dá)到最大值,342μmol O2/mg Chlah。按照優(yōu)化溫度(37°C)和pH(7.5),分別于2種反應(yīng)器模型(5 L/108 W外置光源模型,100 L/120W內(nèi)置光源模型)中培養(yǎng)工程聚球藻。5 L反應(yīng)器中I在培養(yǎng)對數(shù)期可調(diào)至250-300μmol/m2s,3 d后生物量為1.5 g/L;100 L反應(yīng)器中I在培養(yǎng)對數(shù)期可調(diào)至180-250μmol/m2s,7 d后生物量為0.4 g/L。對100 L反應(yīng)器進(jìn)行改造,將燈管功率由120 W提高至300 W,同時將光源分布由3組改為6組,培養(yǎng)至對數(shù)期時,反應(yīng)器50%區(qū)域中I達(dá)到300±10μmol/m2s,并使對數(shù)期延長36 h,7 d后生物量達(dá)到0.8 g/L�？梢�,I是影響光生物反應(yīng)器最終生物量的關(guān)鍵因素,提高I的功率能夠顯著增加生物量產(chǎn)量,使I均勻分布可顯著延長生長對數(shù)期。2.光質(zhì)對工程聚球藻重組蛋白VP28表達(dá)的影響通過改變光質(zhì)可以調(diào)控工程聚球藻的生長及重組蛋白的表達(dá)。采用5 L/108 W外置光源反應(yīng)器進(jìn)行72 h培養(yǎng),通過改變?nèi)肷涔庵袉紊獾谋壤?檢測培養(yǎng)過程中生物量的變化、psb A的轉(zhuǎn)錄、vp28的轉(zhuǎn)錄及VP28蛋白的相對積累量。研究發(fā)現(xiàn):1)光質(zhì)為50%白光、33.3%藍(lán)光及16.7%紅光時,最終vp28的表達(dá)率達(dá)到2.4%,是100%白光下的3倍;重組蛋白VP28的相對積累量提高至2倍;生物量的積累達(dá)到1.22 g/L;psb AII、psb AIII的轉(zhuǎn)錄得到促進(jìn)。2)光質(zhì)為50%白光、33.3%紅光及16.7%藍(lán)光時,最終vp28的表達(dá)率達(dá)到0.5%;10μg總蛋白中VP28的相對積累量只有高比例藍(lán)光下的5%;psb AII及psb AIII基因的轉(zhuǎn)錄受到抑制,psb AI的轉(zhuǎn)錄得到促進(jìn);生物量積累達(dá)到1.68g/L�？梢�,光質(zhì)可顯著調(diào)控重組蛋白的表達(dá),并對生物量產(chǎn)生一定影響。其中藍(lán)光促進(jìn)了重組蛋白的表達(dá),抑制了生物量的積累,而紅光與藍(lán)光的作用相反。光質(zhì)對質(zhì)粒啟動子的調(diào)控機(jī)制與宿主基因psb A的調(diào)控有關(guān)。3.工程聚球藻采收技術(shù)的研究為建立快速安全高效的采收技術(shù),研究不溶性沉淀絮凝法的采收效率及安全性,主要包括3個方面:1)最適誘導(dǎo)劑及其劑量的確定;2)細(xì)胞懸浮液中胞外分泌物(AOM)的去除;3)絮凝后蛋白析出量控制。實(shí)驗(yàn)結(jié)果顯示,1)在懸浮液中增加0.25 m M鎂離子濃度,在p H 11條件下采收率達(dá)到90%以上。2)4000 rpm離心去除懸浮液中的AOM,采用新鮮培養(yǎng)液(0.3 m M鎂離子)重懸,發(fā)現(xiàn)p H調(diào)至10.5時,采收率即達(dá)到95%以上。3)發(fā)現(xiàn)光質(zhì)為50%白光、33.3%藍(lán)光及16.7%紅光時,懸浮液中AOM為2 mg/L以下。4)100%白光下,懸浮液AOM為4.5 mg/L左右。5)光質(zhì)50%白光、33.3%紅光及16.7%藍(lán)光下,懸浮液中AOM為5 mg/L以上。6)在5°C、20°C及35°C水浴條件下,分別檢測絮凝結(jié)束后上清液中的總蛋白含量,30 min后,分別為35μg/ml、24μg/ml、10μg/ml。其中,可溶性蛋白經(jīng)電泳分離后,分子量大小均在20 KDa以下,經(jīng)抗體檢測未發(fā)現(xiàn)VP28條帶。7)絮凝后及時調(diào)整下層藻細(xì)胞濃縮液至p H 7.5,上清液蛋白析出量為12μg/ml�？梢�,鎂離子的絮凝效果受到AOM的嚴(yán)重制約,高比例藍(lán)光可顯著降低AOM的產(chǎn)生,高比例紅光起相反效果。采收后藻細(xì)胞的蛋白損失受溫度及p H影響,避免高溫并及時恢復(fù)p H至藻細(xì)胞生理水平,可降低50%蛋白損失。綜上我們認(rèn)為,應(yīng)用光生物反應(yīng)器培養(yǎng)轉(zhuǎn)vp28基因工程聚球藻時,光照強(qiáng)度及光質(zhì)的合理調(diào)控能顯著提高生物量及重組蛋白產(chǎn)率;改良的不溶性沉淀絮凝法可在不損傷重組蛋白的條件下,實(shí)現(xiàn)了工程聚球藻的快速采收。
[Abstract]:The shrimp white spot syndrome virus (WSSV) was first discovered in Taiwan in 1992, and then spread rapidly to various continents, causing serious diseases to the whole world's prawn breeding industry, and the economic loss was more than 7 billion dollars. Internationally, there is no industrial WSSV control drug. The study found that the specific structural protein VP28 in the WSSV membrane was infected by the virus. The cell process plays a key role in increasing the ability of the shrimp to resist WSSV by oral or injection inoculation. Our study also found that the transgenic VP28 gene engineering cyanobacteria could effectively resist WSSV (the survival rate of 70%-90%) in 30 days, indicating the developed engineering poly ball. Algae have great industrialization prospects. At present, the development of VP28 gene engineering polychlorella oral agent in our laboratory has entered the experimental preparation stage. The scale culture and collection of Engineering polychlorella is an important part of its industrialization. We recognize that the industrialization of polychlorella is restricted by 3 factors: 1) in the scale culture of polychlorella engineering. The former scale culture equipment and technology are not mature, the biomass yield is low; 2) the potential of recombinant protein expression is still improved; 3) lack of safe and rapid and efficient harvesting technology. In order to solve the above problems, this paper is to systematically study the following 3 aspects: 1. the regulation of the biomass of polychlorobacteria by 1. light intensity is to improve the transformation of VP28 gene engineering. The biomass yield of alga and the regulation of light intensity (I) are based on 2 factors: 1) the interaction between I and P H, temperature, and 2) the attenuation effect of I in the reactor. For this reason, this paper first determined the interaction range of I, temperature and P H when the net photosynthetic rate was maximum, and the interaction range of I, temperature and P H, respectively, was 200-400, 28-48 [28-48] C, and 6.5-8.5; and The optimal interaction conditions of I, P H and temperature were measured by the response surface experiment, which were 300 + 10 mol/m2s, 37, C, 7.5, at this time the net photosynthetic rate reached the maximum, 342 Mu mol O2/mg Chlah. in accordance with the optimum temperature (37 degrees C) and pH (7.5), respectively in 2 reactor models (5 L/108 W external light source model, 100 built-in light source model). In the.5 L reactor of Engineering polychlorella, the I in the logarithmic period of culture can be adjusted to 250-300 mu mol/m2s, and the biomass is 1.5 g/L after 3 D; I in the 100 L reactor can be adjusted to 180-250 micron in the logarithmic period of culture, and the biomass of 7 d is 0.4 g/L. to 100 L reactor, and the lamp power is raised from 120 to 300, and the distribution of the light source is changed from 3 to 6. When cultured to logarithmic phase, I in the 50% region of the reactor reached 300 + 10 mu mol/m2s, and the logarithmic period was extended by 36 h, and the biomass reached 0.8 g/L. after 7 d. I was the key factor affecting the final biomass of the bioreactor. The increase of the power of I could significantly increase the biomass yield, and the uniform distribution of I could significantly prolong the logarithmic phase of the growth of.2. light. The expression of polychlorella recombinant protein VP28 could regulate the growth of polychlorella and the expression of recombinant protein by changing light quality. The 5 L/108 W external light source reactor was used for 72 h culture. By changing the proportion of monochromatic light in the incident light, the change of biomass, the transcription of PSB A, the transcription of VP28 and the VP28 protein were detected. The study found that: 1) when the light quality is 50% white light, 33.3% blue light and 16.7% red light, the final expression rate of VP28 reaches 2.4%, 3 times in 100% white light; the relative accumulation of recombinant protein VP28 increases to 2 times; the accumulation of biomass reaches 1.22 g/L; PSB AII, PSB AIII is transcribed to promote.2) to be 50% white light, 33.3% red light and 16.7%. In blue light, the expression rate of the final VP28 reached 0.5%; the relative accumulation of VP28 in the total protein of 10 mu g was only 5% under the high proportion of blue light; the transcription of PSB AII and PSB AIII genes was inhibited, the transcription of PSB AI was promoted; the biomass accumulation reached 1.68g/L., and the light quality could significantly regulate the expression of the recombinant protein and had a certain effect on the biomass. Blue light promotes the expression of recombinant protein and inhibits the accumulation of biomass, but the effect of red light and blue light is opposite. The study of the regulation mechanism of the plasmids promoter and the regulation of the host gene PSB A related to the.3. engineering polychlorella harvesting technology is to establish a fast and safe and efficient harvesting technology and study the extraction of insoluble precipitation flocculation method. The efficiency and safety include 3 aspects: 1) the determination of the optimum inducer and its dosage; 2) the removal of extracellular secretions (AOM) in cell suspension; 3) control of protein precipitation after flocculation. Experimental results showed that 1) increased the concentration of 0.25 m M magnesium ions in the suspension, and the recovery rate of more than 90%.2 under the condition of P H 11) and 4000 rpm centrifugation AOM in the suspension was suspended with fresh medium (0.3 m M magnesium ions), and when p H was adjusted to 10.5, the recovery rate was above 95%.3) and the light quality was 50% white, 33.3% blue light and 16.7% red light, AOM in the suspension was 2 mg/L below.4) 100% white, the suspension AOM was 4.5 mg/L.5), 50% white light, 33.3% red light and 16.7% blue light. The total protein content in the supernatant after flocculation was detected under the conditions of 5 degree C, 20 degree C and 35 degree C in the floating solution, and the total protein content in the supernatant after flocculation was detected respectively. After 30 min, the total protein content was 35 mu g/ml, 24 g/ml and 10 micron g/ml. respectively. After the soluble protein was separated by electrophoresis, the molecular weight was below 20 KDa. After the antibody detection did not detect the flocculation of the VP28 strips, it was timely after flocculation. To adjust the concentration of lower algae cell concentrate to P H 7.5, the amount of protein precipitation in the supernatant is 12 u g/ml., the flocculation effect of magnesium ions is seriously restricted by AOM, and the high proportion of blue light can significantly reduce the production of AOM. The high proportion of red light plays the opposite effect. The protein loss of the algae cells after harvesting is affected by the temperature and P H, avoiding the high temperature and recovering the H to algae in time. The level of cell physiology can reduce the loss of 50% protein. In the conclusion that the light intensity and light quality control can significantly increase the biomass and the yield of recombinant protein when using the photo bioreactor to train VP28 genetically engineered polychlorella, and the improved insoluble precipitation flocculation can achieve the engineering polymerization without damaging the recombinant protein. The fast harvest of the alga.

【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q949.2

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