【作者】 单志；

【导师】 杨婉身；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 质粒DNA (plasmid DNA)是基因工程的重要载体,从细胞内提取质粒已成为实验室中经常性的基础工作,构建快捷、简单、经济、易于自动化和环境友好型的供体细胞富集和质粒DNA纯化方法具有重大意义。建立在磁性固相载体基础上的纯化方法可使细胞或DNA直接从液体粗原料中钓出,免去了离心、沉淀等纷繁复杂的中间步骤,因而自二十世纪90年代以来得到重大发展,但已报道的磁性载体对核酸的纯化能力普遍偏低,使其应用受到限制。对于细胞富集,免疫磁性分离法虽行之有效但却受制于样品制备、生产成本和储藏寿命,不适于批量菌体分离,而基于非特异性吸附反应富集细胞的磁性分离手段却鲜见文献报道。同时,已报道的磁性分离法从大肠杆菌中提取质粒DNA时仍需两次以上离心操作：一次是从液体培养基中回收细胞,另一次是去除变性的基因组DNA／蛋白质复合体。而离心是个耗时、劳动密集型操作,对生物大分子的剪切力较大,离心介入后不易实现高通量样品分析的自动化、微型化和集成化。针对以上问题,本研究提出了制备高吸附容量、pH敏感型、多功能羧基功能化磁性纳米粒子(carboxyl-functionalized magnetic nanoparticles, CMNPs)的技术；利用其构建集细胞捕获、变性基因组DNA／蛋白质复合体去除和质粒DNA捕获于一体的技术路线；提出基于共絮凝行为富集微生物和核酸的思路；建立无RNase介入分离质粒DNA的方法,主要工作及结果如下：1.以FeCl3, FeSO4, NaOH为原料,采用化学共沉法制备了Fe304磁性纳米粒子。分别以Bis[2-(methacryloyloxy) ethyl] phosphate为偶联剂、甲基丙烯酸为功能单体、过硫酸钾为引发剂,控制反应温度,通过超声辐照原位聚合技术制备了羧基功能化磁性纳米粒子。结果表明超声空化作用下铁氧体可以成为聚合反应的活性中心,超声功率越大则产物的粒径越小,在400、600和800W超声功率下产物的平均粒径分别为96.87、55.97749.61nm; TEM和XRD表明产物为尖晶石铁氧体,粒径约7nm; FTIR分析证明聚甲基丙烯酸对铁氧体纳米粒子的成功包覆；TGA-DTG分析表明包覆层含量为7-12%；VSM测试表明包覆后饱和磁化强度为47emu／g,具有较强磁响应性；对pH敏感,在中性环境中5min内可悉数回收,而在酸性条件下达到相同的回收率则小于2.以“捕获率”和捕获后“菌体/CMNPs复合体在管壁上吸附的牢固程度”为指标,对有潜力使CMNPs与大肠杆菌结合的20种化学试剂进行了筛选鉴定,发现酸、钙盐和锌盐是有效的“偶联剂”。对表现最好的盐酸和氯化钙辅助CMNPs捕获大肠杆菌的条件进行了优化。结果表明,大肠杆菌的捕获效率随pH降低而急剧升高,在pH 5左右达到较平稳的值。细胞浓度越高,CMNPs用量越多,磁分离时间越长,则相同条件下捕获率也越高。在较优的条件组合下(细胞OD600大于0.2,粒子用量为0.85mg,磁分离时间3min, pH值3.5-1.5)可以捕获90%以上的大肠杆菌。以氯化钙作为“结合液”时,钙离子与菌体浓度的比值决定了大肠杆菌的捕获效率,二者之间关系可用Logistic模型函数拟合,并受CMNPs用量和磁分离时间的影响。在捕获行为中,钙离子可能起到离子桥作用,通过与纳米粒子的羧基和菌体表面的活性基团发生配位,形成菌体/CMNPs复合体。3.以CMNPs为固相载体,基于SPRI(solid phase reversible immobilization, SPRI)法对大肠杆菌粗裂解液中的质粒DNA进行了纯化研究。结果表明：质粒DNA回收量与结合液浓度之间存在临界关系,当PEG8000>0.15g/mL, NaCl浓度>0.5mol／L时才能获得稳定的质粒DNA收率。质粒DNA回收量随着CMNPs用量的增加而增加,超过最佳值后因不可逆吸附或妨碍洗脱收率反而下降。质粒DNA在纳米粒子表面的吸附与解吸附是快速过程,所需平衡时间分别为150s和40s。0-70℃之间,温度升高对质粒DNA在CMNPs表面吸附影响不大而RNA吸附量急剧下降。利用温度“调谐”差示吸附DNA和RNA的现象,建立了不需要RNase预处理而提取较纯质粒DNA的方法。4.建立了基于CMNPs快速、小量制备质粒DNA的方案。该法整合大肠杆菌捕获、裂解液去杂和质粒DNA富集于一体,操作简单,无需离心,对仪器的依赖性较低且不涉及有毒试剂；质粒DNA质量高,主要以超螺旋构象存在,A260/A280比值大于1.75,可满足酶切等一般的分子生物学实验要求。产量和质量同QIAGEN公司的标准提取试剂盒法相当但成本只有其1／5,整个过程用时不到20min,短于试剂盒法和醇沉淀法。本法特别适用于高通量、自动化、微型化和集成化样品提取和分析平台的构建。5.现已报道的磁性载体核酸分离方法,对常规盐离子含量较低(折合NaCl浓度<0.5mol／L)的样品(如PCR产物),要么用到高浓度高离液序列盐(chaotropic)创造吸附或洗脱条件,对核酸具有损伤作用并造成体积放大；要么用高系黏度结合液,降低了磁性粒子的可控性,且生产成本高、环境不友好。针对以上问题,本研究提出了以生物相容性极强的精胺和亚精胺作为CMNPs/核酸“结合液”,以脱盐质粒DNA／RNA为核酸供体,构建了一种新的核酸富集方法。结果表明：精胺和亚精胺作为结合液时,CMNPs对质粒DNA和RNA选择吸附性不显著。以亚精胺为结合液时,质粒DNA在1～2mmol／L亚精胺浓度内在CMNPs表面吸附；而RNA在0.5-5.5mmol／L之间具有普遍的吸附能力,但最大吸附同样在1～2mmol／L浓度范围。以精胺为“结合液”,CMNPs对质粒DNA的吸附率在1～7mmol／L浓度内达100%；而对RNA吸附率在1.5～5mmol/L浓度之间接近90%,约10%小分子RNA不能被吸附。延长时间可提高核酸的回收率,孵育5min之内可达到100%吸附。凝聚试剂被充分剔除后核酸的解吸附率可达100%。精胺辅助CMNPs富集核酸的能力远强于亚精胺,以其作为结合液时CMNPs对核酸饱和吸附量可达2pg核酸／μg载体。6.对于低盐浓度核酸溶液,降低pH也可驱使其在CMNPs表面高效吸附。在碱性溶液中(pH>8.0), CMNPs几乎不吸附核酸；而在酸性溶液中,CMNPs对核酸的吸附率可达100%。可见,DNA/RNA在羧基纳米粒子表面的吸附与否,可通过pH调控迅速开启或关闭。更多还原

【Abstract】 Because plasmid DNA (pDNA) is routinely used as an important genetic engineering vector, the development of a rapid, simple, cost-effective and environmental-friendly bacteria enrichment and pDNA extraction method amenable for automation is of considerable advantage. Conventional pDNA extraction techniques requiring centrifugation, precipitation are not easily adapted to automated systems. In contrast, magnetic nanoparticle extraction methods demonstrate remarkable simplicity owing to the nature of the particles, which can fish bioentities directly out of biological matrices, and received considerable attention since 1990’. However, the present magnetic adsorbents and related protocols are limited in terms of binding capacity. Although considered as an important cell enrichment method, the immunomagnetic separation of cells are limited in terms of sample preparation, cost and shelf life, and especially not suitable for large-scale cell separation purpose. Nevertheless, there are few successful cell enrichment methods based on nonspecific adsorption between cells and magnetic particles. Second, most magnetic solid phase procedures involve two centrifugation steps:the first to harvest cells from liquid culture and the second to pellet denatured genomic DNA/protein complexes after cell disruption and neutralization. These centrifugation procedures are both time-and labor-intensive; moreover, this step was not amenable to the miniaturization and automation required of high-throughput biological sample preparation. Furthermore, the potential for shearing damage to biomacromolecules during extensive centrifugation is unavoidable.In view of above-mentioned reasons, the present study prepared multifunctional carboxylated magnetic nanoparticles (CMNPs) with high binding capacities. With the help of the nanoparticles, a facile method was constructed for plasmid DNA extraction by integration of bacteria capture, lysate clearance and DNA purification. A new idea for enrichment of microorganisms and nucleic acid based on co-agglomeration with magnetic nanoparticles was also proposed. Even without digestion of RNA primarily by RNase, plasmid can be separated selectively from DNA/RNA mixture by carful tune of temperature.1. Iron oxide nanocrystals were prepared by coprecipitating di and trivalent Fe ions in alkaline solution. The as-prepared iron oxide was then coated by ultrasound assisted in situ polymerization method by using Bis[2-(methacryloyloxy) ethyl] phosphate as coupling agent, methacrylic acid as functional monomer and potassium persulfate as initiator. The results indicated iron oxide could act as polymerization center. When the ultrasound power increased from 400w to 600w and then 800W, the related hydrodynamic size was 96.87,55.97 and 49.61 nm, respectively. The spinel crystalline with size 7 nm was indicated by transmission electron microscopy (TEM) and X-ray diffraction (XRD) characterization. The fourier transform infrared spectroscopy (FTIR) study and thermogravimetric analyses (TGA-DTG) confirmed the successful functionalization of carboxyl groups on the surface of magnetic nanocrystals with organic content 7-12%. The saturation magnetization value for coated particles obtained by vibrating sample magnetometer (VSM) measuring was 47 emu/g. The coated particles were sensitive to environmental pH. When flocculated on addition of acid, the particles are easily separated within 30 s, while it generally needs 5 min to reach a recovery of nearly 100%under neutral condition.2. Using "cell capture efficiency" and the "firmness of immobilized cell/CMNPs on the wall" after magnetic separation as index, we found acid, calcium salt and zinc salt were effective CMNPs-E.coli cell coupling agents, of which HCI and CaCI2 were the best choice. The performance of HCI and CaCl2 was studied sufficiently to optimize best conditions. The results indicated that capture of cells with CMNPs increased sharply when pH was below 5.0, then achieving steady value of upward 90%capture. The cells capture efficiency increase with elevating of cell density, more addition of CMNPs amount and elongated separation time. Under optimized condition (0.85 mg of CMNPs, pH 3.5-1.5), typically, more than 90%of E.coli cells could be recovered within 3 min from 1.5 ml of overnight culture with OD6oo higher than 0.2. When CaCl2 was used as binding agent, the cell capture efficiency was controlled by ratio of [ca2+] vs. OD600, and also affected by CMNPs amount and separation time, which fit Logistic model equation perfectly. In this event, Ca2+ might act as salt bridge between CMNPs and E.coli cells.3. A rapid protocol for extraction and purification of high quality plasmid DNA from bacteria with CMNPs was reported using solid phase reversible immobilization method (SPRI). In this case, a more expressive DNA adsorption was achieved at a PEG800>0.15 g/mL and an NaCl concentration>0.5 mol/L. The recoveries of plasmid DNA increase with more addition of CMNPs, while decrease when used too much. The adsorption and elution of plasmid DNA was rapid, with equilibrium time of 150 and 40 s, respectively. Below 70℃, plasmid DNA adsorption was not affected while RNA recoveries decrease dramatically with increase of temperature. Hence, an RNase-free method will work for purification of pDNA by carful modulation of temperature.4. By using pH sensitive multifunctional magnetic nanoparticles, a facile, centrifugation-free and small-scale plasmid DNA extraction method was established by integration of bacteria capture, lysate clearance and DNA purification. This method was simple without involvement of expensive apparatus and hazard agents; the recovered pDNA was mainly composed of supercoli form with A260/A280>1.75 generally. The biological quality of pDNA was validated by restriction enzyme digestion. The quality and quantity of pDNA were comparable with that extracted by QIAGEN Kit, yet, the cost associated with the current method was only 1/5 of the commercial one. The entire procedure took less than 20 min, whereas the other method took at least 30 min.5. To generate adsorption or elution conditions, the present magnetic-particle-based purification method usually employ high concentration chaotropic salts which are damage to nucleic acid, or vicious agents which make particles difficult to disperse or recover. Besides, these agents are expensive and not environmental-friendly, and large volume consumption of these agents inevitably increased the handle volume. For samples with low salt concentration (e.g. PCR product), the present methods suffer one or more of above mentioned problems. In view of these considerations, a new method was constructed for DNA/RNA enrichment by using high biocompatible spermine or spermidine as binding buffer. The results showed neither of the two compaction agent could generate a selectivity for pDNA or RNA. Only when spermidine concentration was 1-2 mmol/L can pDNA adsorb to CMNPs’surface, and that for RNA was 0.5-5.5 mmol/L. When spermine is 1-7 mmol/L,100%of pDNA can be recovered, while RNA was around 90%when spermine was 1.5-5 mmol/L, leaving 10%of small RNA in supernatant. DNA and RNA could completely elute when compaction agents were scripted. The saturation binding capacity was 2μg DNA/μg particle when spermine was used as binding agent, which was higher than other methods ever reported.6. Low pH could also produce driving fore for the binding of nucleic acid to the surface of CMNPs. In solutions with pH higher than 8.0, the adsorption was nearly zero, while in acidic solutions,100%DNA and RNA could be fished out of solution with slight degradation of DNA.更多还原