转人源胸腺肽α₁基因蓝藻高密度培养的研究

【作者】 汪琨；

【导师】 魏文铃； 楼士林；

【作者基本信息】 厦门大学 ， 微生物学， 2002， 硕士

【摘要】 蓝藻(cyanophyte)，又称蓝细菌(cyanobacteria)，是一类光合自养型原核生物。多数蓝藻富含营养物质、无毒，常作为外源目的基因的表达系统而被广泛应用于分子生物学领域的研究。本实验室利用以蓝藻为受体的穿梭质粒表达载体和基因整合平台打靶载体两套系统，将人源胸腺素α1基因转入到蓝藻Synechococcussp．PCC7942表达载体中，筛选出高效表达人源胸腺素α1的转基因藻株，并检测到活性胸腺肽α1的表达。在成功地构建转基因藻之后，实现高密度培养和高效表达外源目的基因是转基因藻能否走向应用的关键环节之一。本研究以蓝藻无机培养基BG-11为基础，探讨接种量和各种理化因子如，温度、光照、碳源、氮源、pH值等影响转入源胸腺肽α1基因聚球藻7942及其野生藻藻细胞生长的一般规律。并在此基础上，采用正交实验和中心组合实验对高密度培养的培养基成分、培养条件和培养方式等进行优化研究，以及利用小型全自动封闭式光生物反应器进行转基因藻细胞生长规律的研究。单因子实验研究认为，选用处于对数生长期的藻细胞作为藻种，转化藻和野生藻的接种量分别大于6％和5％(v／v)(小于10％)时，可缩短藻细胞延滞期。藻细胞生长的适宜温度为28℃～32℃、培养基适宜的初始pH值为9。对于两种藻来说，全天光照可增加藻细胞生物量，光自养生长最适光照强度分别为1500Lx和2000Lx；NaHCO3比Na2CO3更有利于藻细胞生长，NaHCO3适宜浓度范围分别为10～30mg／L和20～40mg／L；KNO3是一种良好的氮源，其适宜浓度范围分别为0． 7～1． 7g/L和1． 2～2． 2g／L。正交实验研究认为，培养转化藻和野生藻的适宜培养基成分和培养条件是： NaHCO3 30mg/L和40mg/L，KNO3 1． 7g／L和2． 2g／L，初始pH值均为9． 0。从正交实验结果分析，NaHCO3 对两种藻生长影响最大，其次是初始pH值，KNO3最小。转化藻和野生藻培养收获的最大生物量(OD730) 分别达1． 461和1． 559，比生长速率(μ值)分别为0． 375 d-1和0． 382 d-1，与最初培养时的μ值(0． 182 d-1和0． 192 d-1) 相比，分别提高了106％和99％。 。P心组合实验研究认为，培养转化藻的最适宜培养基配方和培养条 件为：NaHCO。36mg／L，KNO。1．3g／L，4j］始pH值10刀，光照强度1800Lx。在卜述条件下藻细胞生长最大OD730值为1．820，收获的藻细胞干重最大达 108g／L，u值为 0．500d－‘，卜最奶培养时的卜值（0．182d－’）提高了17ti 7％，比正交实验提高了 33％。利用计算机对实验数掘进行多重回归方程的运算得到四元二次口归方程式如下： Y一68石7一 2刀85XI一 75X2一 3刀83 X 102X3一 6．632X4 ＋ 2卫IXIXZ ＋ 6．276 X 10－40lX3十0．Z183XIX4十3．757 X 10－‘XZX。＋7．973X2几＋ 3．341X 10IX3X4一 8＊23 X 10－4XIX2X3一0．2252XIX2X4一5．717 X 10－SXIX3X4一 3．794 X 103X2X3X4十 8．498 X 105XIXZX3X4一 1石38 X103X12…1086X22 一1059X10－6X32一7475X102X42 式中，Y为藻细胞生物量（OD730值X XI为碳源浓度（mg／LX XZ为氮源浓度（旮LXX3为光照强度（LXLX4为初始pH值，［。为对照，el、卜、卜、h……为各项系数。口归方程的决定系数R’达到0．88，说明方程适用于转化藻细胞密度的理论预报。 利用上述实验结果所确定的转化藻最适培养基配方和培养条件，在改装后的全自动光生物反应器中培养转人源胸腺肽a；聚球藻7942。培养3d获得的最大生物量为（OD730）为 1．sic，卜值为 0．979d‘。同摇瓶培养相比较，比生长速率比最初培养（0．182d’）提高了 437．9％，比正交实验 （0、375d－’）提高了161．lo，［L中心组合实验（0．500d－’）提高了95．8o，同时也；高于康瑞娟等在光生物反应器中的光自养培养聚球藻川02的比生长速率（0．4－0石d－）。转化藻在这种光生物反应器中培养的最大产率达0二999／（Ld人 高于刘传琳等光生物反应器培养钝顶螺旋藻m刀5012 g／ （Ld））。 最后通过 SDS－PAGE凝胶电泳和 Western blot印迹检测到了目的基固UB－TUI／转基回藻中的表达。 迎过单因子实验、正交实验和中心组合实验实现了转化藻和野生藻的高密度培养，缩短了培养时间，确定了最适宜培养基配方和培养条件，建l＿了门］预报藻细胞生物量的四元二次回归方程，为转基回藻大规模培养捉仪仙沦指导和优化的｛＿艺。更多还原

【Abstract】 Cyanobacteria, or cyanophyte are photoautotrophic organisms with many special properties, making them very attractive for molecular biological studies.Funded by National High Science & Technical Program (863), our lab had succed in expressing human thymosinofi (Tai) gene in cyanobacteria Synechococcus sp.PCC7942 with the suage of shutter vector system and reconstructed plasmid vector system.This experiment began with the synthetic medium BG-11 and studied various factors, eg. temperature, Light, carbon sources, nitrogen sources & pH, etc, which influence the expression Ta?i, of in transformant 7942. Based on the results from single factor research, orthogonal experimant and central composite design(CCD) are used to optimize the medium component, calture condition and culture farm A mini-auto-closed-photolioreactor was adopted for culture.The single factor research shows, using logarithatie phase cells as inoculum, 6% (v/v) for transformant and 5% (v/v) for prototrophs, the lenghth of lag period will the be shortened. The optimized temperature is 28癈~32癈, initial pH value as 9. For both strains, lighting through the culture time will increase biomass. Optimist light strenghth is ISOOLx & 2000Lx each; NaHCOs is more geneficial than Na2CC>3, range from 10 to 30mg/L and 20 to 40mg/L each; KNCh is an essential nitrogen source, and it’s optimist scale is 0.7~1.7g/& 1.2-2.2g/L each.The orthogonal experiment shows, the optimist medium and the culture condition for the transformant is: NaHCOs, 30mg/L KNOs, 1.7g/L; initial pH value, 9.0, whilst 40mg/L, 2.2g/L & 9.0 for the prototrophs respectively. These factors give different influences on both strains growth: NaHCC>3>pH> KNC>3. The transformant tets a maxium lionass (ODy3o) of 1.461, specific growth rate (U ) of 0.375d"’, it’s 106% more than former y valae (0.182d"!). Whilst the prototroph get a maxium biomass of 1.559, n value of 0.382d"’, 99% more than former u value (0.192d~’).inThe CCD research shows that optimige medium and cultare condition for the transformant are NaHCO3, 36mg/L; KNO3, 1.3g/L; initial pH value 10.0; light strenghth, 1800 Lx. Under the condition mentioned above, the maxium biomass OD730 is 1.820, which gets a dry biomass of 1.08g/L and then value is 0.500d-1, 174.7% higher than initial value and 33% higher than that of orthogonal experiment. By with differential analyses biomass products in the Autophotobioreactor computer we got a fomula as below:Y=68.67 - 2.085X1 - 75X2 - 3.083× 10-2X3 - 6.632X4 + 2.21X,X2 + 6.276 × 10’4X1X3 + 0.2183X1X4 + 3.757 × 10’2X2X3 + 7.973X2X4 + 3.341 × 10-3X3X4 - 8.623×10-4X1X2X3 - 0.2252X1X2X4 - 5.717 × 10-5X,X3X4 -3.794× 10-3X2X3X4 +8.498 × 10-5X1X2X3X4-1.638 × 10-3X12- 1.086X22-1.059×10-6X32-7.475×10-2X42While Y-biomass (OD730), X1-concentration of carbon source(mg/L), X2-concentration of nitrogen source (g/L), X3-light strenghth (Lx), X4 -initial pH value, P0-control, P1-etc-the coefficients for the terms. The dicision coefficient R2 is 0.88, showing the fomula is fit for precipating cell density.And using the result from previous experiments, we culture 7942 in a redesigned photobioreactor. After the 3days culture, the maxim biomass (OD730) is 1.510, u value is 0.979d"’, its 437.9% higher than flask culture (0.182d-1), 161.1% higher than that of orthogonal experiment and 95.8% higher than RSM. The result is higher than calture of cyanoacteria 7002 (0.4-0.6d~’, Kang ruijuan etc.). The maxium products in this system is 0.299g/ (Ld) , higher them that of sprulinaplatensis (0.05012g/ (Ld) Liu Chuanlin etc.)Finally, the expression of UB-Ttt], is detected by SDS-PAGE and Western blot.The research work shows a high-cell density cultivation method has been reached. It will shorten the lenghth of cultare time, and derect the large-scale culture of the transformant.更多还原