CAJ | 학술논문

为了克服磷酸钙转染法在真核细胞中转染效率低和不稳定性等缺点,适用于体外在哺乳细胞中高效表达外源蛋白,将绿色荧光蛋白( green fluorescent protein ,GFP)表达质粒通过常规的磷酸钙转染法导入到 HEK293T细胞中,对影响质粒转染效率的条件逐步予以优化,具体包括：在制备磷酸钙 DNA沉淀复合物时,对DNA质粒的用量和涡旋混匀时间的优化；磷酸钙 DNA沉淀形成后对转染促进剂(甘油、丁酸钠和氯喹)的浓度、作用时间及其组合进行优化和筛选.结果表明：GFP转染6孔板培养的HEK293T细胞的DNA质粒最适用量为3μg/孔,其转染效率较1和2μg/孔的平均转染效率提高了50％,较4和5μg/孔的平均转染效率提高了70％以上；涡旋混匀时间最佳为10 s ,比涡旋混匀5 s时的转染效率提高了80％；在上述优化条件基础上加入不同的转染促进剂,其中单独使用15％甘油处理细胞6.5 min时,所得转染效率较2.5、4.5、8.5和10.5 min有明显提高,15％甘油＋5 mmol/L丁酸钠联用或者单独用500μmol/L氯喹处理细胞后,转染效率较常规方法分别提高了80％和75％,且荧光表达强度有所增强.总之,该文通过对磷酸钙转染时和转染后的条件进行优化,使其转染效率较常规方法有了显著提高,相对于脂质体转染法更加安全可靠,且价格低廉,因此可适用于大规模蛋白表达质粒转染或者病毒包装,尤其适用于各种糖基化蛋白如肿瘤诊断标志物在体外的高通量表达与制备.
위료극복린산개전염법재진핵세포중전염효솔저화불은정성등결점,괄용우체외재포유세포중고효표체외원단백,장록색형광단백( green fluorescent protein ,GFP)표체질립통과상규적린산개전염법도입도 HEK293T세포중,대영향질립전염효솔적조건축보여이우화,구체포괄：재제비린산개 DNA침정복합물시,대DNA질립적용량화와선혼균시간적우화；린산개 DNA침정형성후대전염촉진제(감유、정산납화록규)적농도、작용시간급기조합진행우화화사선.결과표명：GFP전염6공판배양적HEK293T세포적DNA질립최괄용량위3μg/공,기전염효솔교1화2μg/공적평균전염효솔제고료50％,교4화5μg/공적평균전염효솔제고료70％이상；와선혼균시간최가위10 s ,비와선혼균5 s시적전염효솔제고료80％；재상술우화조건기출상가입불동적전염촉진제,기중단독사용15％감유처리세포6.5 min시,소득전염효솔교2.5、4.5、8.5화10.5 min유명현제고,15％감유＋5 mmol/L정산납련용혹자단독용500μmol/L록규처리세포후,전염효솔교상규방법분별제고료80％화75％,차형광표체강도유소증강.총지,해문통과대린산개전염시화전염후적조건진행우화,사기전염효솔교상규방법유료현저제고,상대우지질체전염법경가안전가고,차개격저렴,인차가괄용우대규모단백표체질립전염혹자병독포장,우기괄용우각충당기화단백여종류진단표지물재체외적고통량표체여제비.
Summary Transfection , involving the transfer of exogenous DNA to eukaryocyte cells , is a common technique in molecular and cellular biology , including calcium phosphate transfection , electroporation , lipofection and so on . Calcium phosphate transfection is one of the major methods for DNA transfer to eukaryocyte cells and with lower toxicity and cost than lipofection . However , the application of calcium phosphate transfection method was limited due to its low transfection efficiency and instability . <br> To establish a highly efficient and stable transfection approach to overexpress exogenous protein abundantly in HEK293T cells in vitro , green fluorescent protein ( GFP) plasmid DNA was transfected by using calcium phosphate with optimal DNA amount and vortex time of calcium phosphate‐DNA complex in absence or presence of common transfection enhancers including glycerol , sodium butyrate , and chloroquine . <br> The protocol of calcium phosphate transfection was as follows . First , HEK293T cells were seeded in a six‐well plate , incubated for 8‐24 hours at 37 ℃ in a humid incubator with 5% CO2 , and then the cells were transfected after cell confluence reached 50% 80% . Second , calcium phosphate‐DNA complex was prepared . Mixed GFP DNA plasmid with 25 μL of 2 .5 mol/L CaCl2 solution in a round‐bottom tube , added sterile water up to 250 μL ,blended 10 μL of phosphate‐solution [V(70 mmol/L Na2HPO4) ∶V(70 mmol/L NaH2PO4) =1∶1] with 250μL of 2 × Hepes buffer solution and added to the former solution , added calcium phosphate‐DNA mixture into HEK293T cells one drop at a time immediately after vortex . Finally , transfection efficiency was detected by fluorescence imaging in absence or presence of common transfection enhancers including glycerol , sodium butyrate , and chloroquine . The dosage of DNA , duration of vortex and concentrations of three common transfection enhancers were optimized during or after transfection . <br> The results showed that the optimal GFP plasmid DNA amount was 3 μg/well . Compared with GFP plasmid DNA amount of 1 or 2μg/well and 4 or 5μg/well , average transfection efficiency of optimal amount was improved by 50% and 70% , respectively . Higher transfection efficiency was also detected when the vortex time was 10 s . Under these optimal conditions , transfection efficiency of treatment with 15% glycerol alone for 6 .5 min was significantly higher than that for 2 .5 , 4 .5 , 8 .5 , or 10 .5 min . When the HEK293T cells were treated with the combination of 15% glycerol with 5 mmol/L sodium butyrate or 500 μmol/L chloroquine alone , transfection efficiency was improved by 80% and 75% , respectively . Meanwhile , GFP fluorescence intensity was enhanced . <br> Compared to lipofectamine reagents , calcium phosphate transfection with enhancers is more efficient , less toxic and less expensive . Therefore , this approach can be used to overexpress large‐scale proteins in the HEK293T cells in vitro , or to package lentiviruses/retroviruses . It is especially applicable for in vitro high flux expression and preparation for various glycosylated proteins such as cancer diagnosis markers .