稳定高表达人ERP57基因A549细胞株的建立及其对CRT表达的影响

建立稳定高表达人ERP57蛋白的A549细胞株,并观察对CRT表达的影响。采用巢式RT-PCR从人非小细胞肺腺癌A549细胞总RNA中克隆人ERP57 cDNA,构建ERP57真核表达质粒(pcDNA3.1(+)/ERP57)脂质体转染A549细胞。经G418筛选,获得高表达人ERP57蛋白的A549细胞株。通过Western blotting检测细胞中ERP57及CRT蛋白表达情况。成功获得稳定高表达ERP57蛋白的人A549细胞株,CRT表达量无明显改变。成功获得稳定高表达ERP57蛋白的人A549细胞株,证实细胞中高表达ERP57对CRT表达量无明显影响。     

怀化医学高等专科学校医学形态学实验中心

目的:探讨抑癌基因p16对肝癌细胞生长的抑制作用及其机制。方法:将p16 cDNA亚克隆至pcDNA3.1真核表达载体上,并经脂质体介导转染至人肝癌细胞株SMMC-7721。用MTT法和Western blot分析转染细胞的生长情况。结果:成功构建重组表达质粒pcDNA3.1-p16,转染pcDNA3.1-p16的SMMC-7721细胞生长速度受到明显抑制;转染后有外源p16蛋白的表达,且伴随Bax上调,Bcl-2和cIAP2的下调。结论:重组pcDNA3.1-p16质粒能在人肝癌细胞SMMC-7721内表达,且能抑制SMMC-7721的生长,其机理与诱导肿瘤细胞凋亡相关。     

NOK过表达对肺腺癌细胞A549增殖的影响

目的:本研究通过建立稳定过表达NOK基因的A549-NOK细胞系观察其增殖变化,探讨NOK基因对A549增殖的影响。方法:运用电穿孔仪将pcDNA3.1-NOK质粒转染人肺癌A549细胞,筛选出稳定过表达NOK基因的单克隆株A549-NOK。RT-PCR检测转染前后细胞内NOK基因的表达效果。通过流式细胞术检测细胞周期,MTT法绘制细胞生长曲线。结果:RT-PCR显示A549-NOK细胞中NOK基因表达量明显增高。流式细胞术显示A549-NOK与A549、空质粒对照组细胞相比,S期细胞增多(51.7±2.2 vs 43.4±1.5,45.7±1.4,均P0.05);细胞增殖指数升高(69.4±1.1vs 58.4±1.3,57.9±1.4,均P0.05);MTT结果显示A549-NOK细胞较A549和空质粒对照组细胞生长曲线上移。结论:NOK可促进A549细胞的增殖,可能在肺癌的发生、发展中发挥重要作用。     

稳定表达乙型流感病毒血凝素蛋白细胞的构建

获得稳定表达乙型流感病毒血凝素蛋白的HEK-293-HA细胞株。采用RT-PCR方法扩增乙型流感病毒的HA基因,将其克隆至真核表达载体pcDNA3.1(+)上,构建pcDNA3.1(+)-HA重组质粒。将已鉴定正确的pcDNA3.1(+)-HA质粒与辅助质粒PLV-EF1a-EGFP(2A)Puro通过脂质体介导法共转染HEK293细胞,经嘌呤霉素筛选后得到重组细胞株HEK293-HA,通过流式细胞术法(FCM)来检测细胞中HA的表达情况。所得到的重组细胞经扩大培养后再连续培养15代,采用间接免疫荧光法(IFA)和蛋白免疫印迹法(WB)来检测HA蛋白表达的稳定性。结果表明重组质粒pcDNA3.1(+)-HA经双酶切及测序鉴定正确;共获得3株高表达阳性细胞株。细胞扩大培养后连续传代培养15代后进行Western blot和IFA检测结果表明,重组细胞的HA蛋白得到稳定的表达。已成功获得了能稳定表达乙型流感病毒血凝素蛋白的HEK-293-HA细胞,可为乙型流感病毒HA蛋白的进一步研究提供良好的基础。     

病毒负性调节因子在内皮细胞稳定表达细胞株的建立

建立HIV-1的调节基因Nef基因在内皮细胞稳定表达的细胞株ECV304-Nef,为研究Nef对血管内皮细胞生物学活性的影响奠定试验基础。构建真核表达载体pcDNA3.1(+)-Nef,将其质粒和pcDNA3.1(+)质粒(阴性对照)分别转染血管内皮细胞ECV304,G418筛选。通过RT-PCR检测NefmRNA在细胞中的表达;细胞免疫荧光法检测Nef蛋白的表达及定位;Western blotting检测Nef蛋白的特异性表达,获得稳定表达的细胞株。构建的重组质粒pcDNA3.1(+)-Nef经BamHI和EcoRI双酶切鉴定,得到的片段大小与理论值相符,分别为载体的5400bp和目的基因的621bp。测序结果显示碱基序列与GenBank(登录号:K03455)序列相同。转染细胞经G418筛选后获得稳定表达Nef的ECV304细胞株,RT-PCR显示转染pcD-NA3.1(+)-Nef质粒的ECV304细胞出现621bp条带,对照组无目的条带出现;荧光显微镜下观察转染pcDNA3.1(+)-Nef质粒的ECV304细胞表达的Nef蛋白主要定位于细胞质中。Western blotting结果显示,转染pcDNA3.1(+)-Nef质粒的ECV304细胞约27kD处检测到目的条带,表明pcDNA3.1(+)-Nef表达正确。     

过表达Snail增强肺癌细胞A549的体外侵袭能力

用锌指转录因子Snail诱导肺癌细胞A549发生上皮细胞-间质转化(epithelial-mesenchymal transition,EMT),检测肺癌发生EMT后细胞侵袭能力的变化,为临床筛选分子靶向药物提供依据.构建pcDNA3.1-snail载体,用pcDNA3.1-snail及空pcDNA3.1载体转染肺癌A549细胞后,进行G418筛选;光镜观察培养后细胞形态学的改变、免疫细胞化学与免疫荧光检测细胞表达E-钙黏着蛋白(E-cadherin)、波形蛋白(vimentin)的改变,Western印迹检测细胞中E-钙黏着蛋白和波形蛋白的改变,Transwell侵袭小室法进行细胞体外侵袭能力检测.采用pcDNA3.1-snail转染细胞后,A549细胞变得细长,细胞融合度降低.免疫细胞化学、免疫荧光、Western印迹结果显示,E-钙黏着蛋白表达降低,波形蛋白表达升高;transwell侵袭小室法结果显示,过表达Snail的A549细胞穿透matrigel胶的细胞数明显增多.结果提示,Snail能有效诱导肺癌发生EMT,并且能增强肺癌的体外侵袭能力.     

pcDNA3.1/myc-His-DJ-1^M26I重组载体构建及其对NIH3T3细胞增殖和凋亡研究

目的构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组表达载体,为研究DJ-1M26I突变与细胞增殖、凋亡的关系及建立转基因动物模型奠定基础。方法采用突变试剂盒将DJ-1蛋白第26位氨基酸进行突变,分别构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组表达载体,并采用脂质体介导的方法分别将其转染入NIH3T3细胞,500μg/mL G418压力筛选稳定克隆,对2种转染细胞在DNA水平、RNA水平和蛋白质水平进行鉴定,采用MTT染色方法和AnnexinV-FITC试剂盒进行转染阳性克隆细胞的细胞活力与细胞凋亡检测。结果 pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组质粒转染NIH3T3细胞经G418筛选后,PCR方法检测分别获得1个和3个阳性细胞克隆,RT-PCR及western blot方法进行DJ-1-His基因表达检测,结果均证明外源插入基因的表达,MTT实验结果初步证明转染DJ-1M26I基因的NIH3T3阳性细胞组细胞增殖速率低于正常NIH3T3细胞组（P〈0.05）,转染DJ-1基因的NIH3T3阳性细胞组细胞增殖速率与正常NIH3T3细胞相比无明显差别;细胞凋亡检测表明转染DJ-1M26I基因的NIH3T3阳性细胞组细胞凋亡率高于正常NIH3T3细胞,转染DJ-1基因的NIH3T3阳性细胞组细胞凋亡率低于正常NIH3T3细胞（P〈0.05）。结论成功构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1 M26I重组表达载体,成功筛选出稳定表达人DJ-1及DJ-1 M26I的NIH3T3细胞。DJ-1 M26I基因突变更易导致NIH3T3细胞的凋亡。     

FKBP12真核表达载体的构建及稳定转染A549细胞株的建立

目的:构建FK506结合蛋白12(FK506 binding proteins 12,FKBP12)真核表达载体并建立稳定转染A549细胞株。方法:RT-PCR扩增人平滑肌细胞FKBP12基因片断,构建pcDNA3.1/Hygro(+)-FKBP12真核表达载体,经琼脂糖电泳、特异性内切酶切割及测序验证其正确性。脂质体法转染真核细胞A549,HygromycinB筛选建立稳定转染的细胞株,免疫印迹法检测稳定转染的细胞株。结果:构建了FKBP12真核表达载体并建立了稳定转染的A549细胞株,成功表达FKBP12蛋白。结论:FKBP12真核表达载体成功构建及稳定转染A549细胞株的建立,为深入研究基于FKBP12靶点药物的机制奠定基础,进而为探索安全、高效的免疫抑制剂提供新的途径。     

稳定表达p120ctn的人肺腺癌A549细胞株的构建

目的:构建稳定表达p120ctn的A549细胞株,以研究p120ctn蛋白在肺癌发生和转移过程中的作用。方法:通过分子克隆,将pc DNA3.1多克隆位点插入Flag标签的编码序列,得到pc DNA.Flag表达载体。然后PCR扩增p120ctn的编码序列,插入Flag标签下游,构建pc DNA.Flag-p120ctn质粒,筛选阳性克隆并进行酶切及测序鉴定。利用脂质体Lipofectamine 2000将pc DNA.Flag-p120ctn质粒转染到肺癌细胞A549中,通过G418筛选得到稳定转染细胞株,免疫印迹法检测p120ctn的表达。结果:本文构建了融合有Flag标签的p120ctn真核表达载体并转染到A549中,免疫印迹结果表明p120ctn蛋白在A549细胞中高效的表达。结论:本文成功构建了稳定高表达p120ctn的A549细胞模型,为深入研究p120ctn在肺癌的发生和转移过程中的作用奠定了基础。     

pcDNA3.1-Canstatin-3Flag真核表达载体的构建及转染HepG2细胞中的表达

目的:构建pcDNA3.1-Canstatin-3Flag载体并稳定转染肝癌HepG2细胞,检测canstatin在mRNA水平的表达。方法:胎盘中提取总RNA,RT-PCR法获得canstatinDNA,克隆至pcDNA3.1(-)载体中,并测序,重组质粒pcDNA3.1-Canstatin-3Flag转染肝癌HepG2细胞,G418筛选出稳定转染细胞,RT-PCR检测canstatin mRNA表达。结果:1.成功构建出pcDNA3.1-Canstatin-3Flag重组质粒;2.获得稳定转染pcDNA3.1-Canstatin-3Flag的肝癌HepG2细胞;3.发现转染后的肝癌HepG2细胞canstatin在mRNA水平比未转染细胞有明显的增强。结论:获得了稳定转染pcDNA3.1-Canstatin-3Flag的肝癌HepG2细胞,为后期canstatin在肝癌中的研究提供了支持。     

维甲酸受体β基因RNAi表达质粒的构建及其对A549细胞增殖和分化的影响

目的：利用RNA干扰（RNAi）技术,构建针对维甲酸受体（RAR）β基因的小干扰RNA（siRNA）表达质粒,诱导RARβ基因沉默,并观察其对肺癌细胞A549株的细胞周期和增殖的影响。方法：依据设计siRNA的原则。针对人RARβ的mRNA序列,设计并合成编码siRNA的2条寡核苷酸序列,经退火成互补双链,再克隆到pSUPER-NEO-GFP真核表达载体中构建重组体pSUPER-RAR[β转染至A549细胞中,以空质粒和RARβ高表达质粒转染为对照,用Western印迹检测RARβ基因的表达,并采用M1Tr试验检测转染后细胞株的增殖和细胞分化情况。结果：表达人类RARβ基因的siRNA重组表达质粒构建成功;MTT试验结果表明,转染的A549-RARβ-si细胞增殖能力降低。结论：采用RNAi技术特异阻断RARB基因表达,通过转染A549细胞,可使其细胞形态发生变化,并抑制其细胞生长。     

Napsin A基因对A549细胞在上皮-间质转化中增殖的影响及其机制

采用慢病毒载体质粒PLJM1将NapsinA基因转染到人肺腺癌细胞——A549细胞中,获得稳定表达Napsin A蛋白的特性并鉴定,通过转化生长因子-β1刺激A549细胞发生上皮-间质转化,体外构建上皮-间质转化模型并鉴定。MTT法检测转基因前后A549细胞在上皮-间质转化过程中生长速率的变化;流式细胞术检测其细胞周期的改变,最后予Western blot检测黏着斑激酶的表达情况,探讨Napsin A基因对A549细胞在上皮-间质转化过程中增殖的影响及其机制。结果表明转染后的A549细胞表达Napsin A蛋白明显增加(P<0.01);A549细胞发生上皮-间质转化后细胞E钙蛋白表达下调(P<0.01),Ⅰ型胶原表达上调(P<0.01);转基因细胞在体外上皮-间质转化模型中增殖速度减慢(P<0.05),且细胞周期被阻滞在G_1期(P<0.01),其表达整合素信号传导通路的基础分子——黏着斑激酶的量显著下降(P<0.01)。提示Napsin A基因可以抑制A549细胞在上皮-间质转化过程中的进一步增殖,其机制可能与抑制整合素信号传导通路有关。     

糖基化磷脂酰肌醇锚定型EGFP真核表达质粒的构建及表达

构建与增强型绿色荧光蛋白基因相连的糖基化磷脂酰肌醇(glycosyl phosphatidylinositol,GPI)序列的真核表达质粒,并检测其在A549细胞中的表达.分离人外周血淋巴细胞,提取总RNA,以RT-PCR法扩增CD24基因的243 bp GPI锚定序列,双酶切后定向克隆入pEGFP-C1质粒中,构建并鉴定pEGFP-C1-GPI质粒.经脂质体介导转染A549细胞后,在荧光显微镜下观察目的蛋白在真核细胞内的表达情况.经酶切和测序鉴定证实,所克隆的CD24 GPI序列正确,荧光显微镜观察pEGFP-C1-GPI质粒转染A549细胞可见围绕细胞膜的强绿色荧光,而对照pEGFP-C1质粒转染A549细胞仅见胞内均匀荧光.成功构建与EGFP相连的GPI真核表达质粒,且能在A549细胞膜上锚定表达EGFP-GPI融合蛋白,为构建锚定表达型肿瘤疫苗奠定基础.     

Tumor necrosis factor alpha induces spermidine/spermine N1-acetyltransferase through nuclear factor kappaB in non-small cell lung cancer cells

Tumor necrosis factor alpha (TNFalpha) is a potent pleiotropic cytokine produced by many cells in response to inflammatory stress. The molecular mechanisms responsible for the multiple biological activities of TNFalpha are due to its ability to activate multiple signal transduction pathways, including nuclear factor kappaB (NFkappaB), which plays critical roles in cell proliferation and survival. TNFalpha displays both apoptotic and antiapoptotic properties, depending on the nature of the stimulus and the activation status of certain signaling pathways. Here we show that TNFalpha can lead to the induction of NFkappaB signaling with a concomitant increase in spermidine/spermine N(1)-acetyltransferase (SSAT) expression in A549 and H157 non-small cell lung cancer cells. Induction of SSAT, a stress-inducible gene that encodes a rate-limiting polyamine catabolic enzyme, leads to lower intracellular polyamine contents and has been associated with decreased cell growth and increased apoptosis. Stable overexpression of a mutant, dominant negative IkappaBalpha protein led to the suppression of SSAT induction by TNFalpha in these cells, thereby substantiating a role of NFkappaB in the induction of SSAT by TNFalpha. SSAT promoter deletion constructs led to the identification of three potential NFkappaB response elements in the SSAT gene. Electromobility shift assays, chromatin immunoprecipitation experiments and mutational studies confirmed that two of the three NFkappaB response elements play an important role in the regulation of SSAT in response to TNFalpha. The results of these studies indicate that a common mediator of inflammation can lead to the induction of SSAT expression by activating the NFkappaB signaling pathway in non-small cell lung cancer cells.     

Effects of exogenous wild-type p53 on a human lung carcinoma cell line with endogenous wild-type p53

Several studies have shown that expression of exogenous wild-type p53 is detrimental to the growth of cell lines with absent or mutant p53. In this study, wild-type p53 cDNA expression plasmids were transfected into A549 lung carcinoma cells which had previously been shown by sequencing to contain wild-type p53. When a constitutively expressed wild-type p53 plasmid containing the neomycin resistance gene was transfected into these cells, no G418-resistant colonies contained the exogenous p53 cDNA even though the neomycin resistance gene was integrated. When cells were transfected with a dexamethasone-inducible wild-type p53 cDNA expression plasmid, induction of p53 expression resulted in a decreased growth rate and a decreased proportion of S-phase cells. Continuous treatment with dexamethasone resulted in continued p53 expression for 16 days, but beyond that time expression ceased and could not be reinduced. These data indicated that although the A549 cell line could proliferate in the presence of endogenous wild-type p53 there was a strong selection pressure against continued expression of additional exogenous wild-type p53.     

低氧诱导因子和角质细胞生长因子双基因重组腺病毒的构建及其在肺泡上皮细胞中的表达

目的：构建同时携带低氧诱导因子-1α（HIF-1α）和角质细胞生长因子（KGF）N腺病毒载体（pAdxsi-GFP-HIF-KGF）,观察其在防治肺损伤潜在的应用前景。方法：低氧处理A549细胞后提取总RNA并逆转录为eDNA作为模板,依据GeneBank公布的HIF-1α cDNA设计引物,并分别引入KpnI和BamHI酶切位点,PCR扩增后将目的基因HIF-1α连接到载体pShuttle-CMV-EGFP上,构建重组质粒pShuttle-GFP—HIF。然后以质粒plRES2-EGFP-KGF为模板,用引入NheI和PmeI酶切位点的引物PCR扩增KGF基因并克隆到重组质粒pShuttle-GFP-HIF上,获得穿梭质粒重组质粒pShuttle—GFP-HIF—KGF。采用细菌内重组方法将目的序列重组到pAdxsi病毒骨架栽体上构建携带HIF．10t和KGF双基因的重组腺病毒载体pAdxsi-GFP-HIF-KGF。检测重组腺病毒滴度后,转染人肺泡上皮细胞A549,检测目的基因的转染表达。结果：通过对构建质粒克隆进行测序及酶切,证实携带HIF—lot和KGF双基因的重组腺病毒载体pAdxsi-GFP-HIF-KGF构建成功,且构建的重组腺病毒纯度好、滴度高。用pAdxsi-GFP-HIF-KGF以100MOI转染A549细胞后24h后在荧光显微镜下可观察到细胞有较强的绿色荧光表达,48h时荧光更强;转染48hELISA法检测培养上清中HIF-1蛋白表达水平为（56．36±4．53）ng／mL,KGF蛋白表达水平为（60．20±2．92）ng／mL。结论：成功构建了腺病毒栽体pAdxsi-GFP-HIF-KGF,其转染效率及目的基因的蛋白表达水平较高,具有潜在的进一步在肺损伤局部应用的前景,为后期制备可以同时发挥KGF、HIF-1作用的基因治疗药物打下基础,同时为高海拔地区应激性急性肺损伤的有效防治提供实验基础。     

The 3p21.3 tumor suppressor RBM5 resensitizes cisplatin-resistant human non-small cell lung cancer cells to cisplatin

Objective: Increasing RBM5 levels inhibit tumor cell growth and promote apoptosis. In this study, we investigated the role of RBM5 in the cisplatin resistance observed in human lung non-small cell lung cancer cells and evaluated the effect of RBM5 modulation on cell growth inhibition and apoptosis induced by cisplatin in the parental non-small cell lung cancer cells A549 and their cisplatin resistant counterparts, A549/DDP cells. Methods: RBM5 mRNA and protein expression in the A549 and A549/DDP cells was analyzed by semi-quantitative RT-PCR and western blot. The A549/DDP cells were then transfected with a pcDNA3-RBM5 plasmid, and an RBM5-specific siRNA was transfected into A549 cells, prior to treatment with cisplatin. Semi-quantitative RT-PCR and western blot analyses were performed to confirm the expression of RBM5 mRNA or protein, and knockdown of RBM5 mRNA or protein, respectively. MTT assays were used to evaluate chemosensitivity to cisplatin. Apoptosis was assessed by DAPI nuclear staining and flow cytometric analysis with an Annexin-V-FITC apoptosis kit. Cytosolic cytochrome c, cleaved caspase-3 and cleaved caspase-9 were detected by western blot. Results: The expression of RBM5 mRNA and protein was significantly reduced in the A549/DDP cells compared with the A549 cells. Exogenous expression of RBM5 by the pcDNA3-RBM5 resensitized the response of A549/DDP to cisplatin, resulting in a significant increase in tumor-suppressing activity induced by cisplatin. In contrast, downregulation of RBM5 with siRNA in the A549 cells inhibited cisplatin-induced apoptosis. We also found that the RBM5-enhanced chemosensitivity was associated with the release of cytochrome c into the cytosol, activation of caspase-9 and the downstream marker caspase-3. Conclusion: Our results demonstrate that RBM5 may serve as a biomarker with the ability to predict a response to cisplatin. It may also act as a prognostic indicator in lung cancer patients. Our findings suggest that there may be clinical utility for ectopic RBM5 such as enhancing and resensitizing nonresponders to cisplatin.     

超正电荷绿荧光蛋白+36GFP作为核酸载体的细胞穿透性分析

旨在通过原核表达纯化超正电荷绿色荧光蛋白+36GFP,研究其与核酸的结合作用及作为核酸载体的细胞转导功能。将pET+36GFP-HA2质粒转化到大肠杆菌BL21(DE3)菌株中,然后表达纯化+36GFP蛋白。将得到的目的蛋白在特定浓度下分别转导293细胞、HepG2细胞、A549细胞和B16细胞,流式细胞仪检测+36GFP的转导效率;+36GFP蛋白(100 nmol/L)转导A549细胞,激光共聚焦显微镜观察结果;将+36GFP蛋白与质粒DNA按不同比例孵育,凝胶阻滞实验检测+36GFP与DNA的结合能力;激光共聚焦显微镜和流式细胞仪检测+36GFP蛋白携带质粒DNA转导细胞后报告基因的表达。结果显示,+36GFP蛋白具有较高的细胞转导效率,且随浓度升高转导效率增加,呈浓度依赖性。凝胶阻滞实验显示,+36GFP能够与质粒DNA结合,阻滞DNA在凝胶中迁移,且呈现一定的浓度依赖性。+36GFP包裹质粒转导细胞后,可高效携带质粒DNA转导进入细胞,使质粒报告基因得到表达。本研究成功表达纯化了+36GFP蛋白,证实该蛋白具有较高的细胞转导效率,可将外源核酸携带入细胞使外源基因得到表达。     

肺上皮细胞自噬体在结核分枝杆菌感染中保护作用的分子机制

目的：构建Atg5．真核表达载体并瞬时转染肺上皮细胞细胞株,探讨自噬在结核分枝杆菌感染上皮细胞中保护作用的分子机制。方法：设计针对Atg5的RNAi序列,化学合成后经过变性,退火连接到pSilencerTM3．1-H1hygro真核表达载体,经测序验证其正确性。脂质体法瞬时转染真核细胞A549,免疫印迹法检测瞬时转染的效果。用结核分枝杆菌分别感染正常和自噬表达低下的A549细胞．通过检测LDH来观察细胞的坏死情况。结果：成功的构建了pSilencerTM3．1-H1hygro真核表达载体并建瞬时转染了A549细胞株,成功抑制了细胞的自噬功能。Atg5-细胞对结核杆菌的抵抗能力下降。结论：在自噬表达低下的细胞中,细胞对结核分枝杆菌的抵抗能力有明显下降。在结核分枝杆菌感染上皮细胞的过程中,自噬是一种保护机制。     

真核表达质粒pcDNA3.1-CSRP2-HA的构建及鉴定

目的:构建并鉴定真核表达质粒PcDNA3.1-CSRP2-HA。方法:据GeneBank中人CSRP2 CDS序列设计并合成引物,提取A549细胞总RNA,并将其逆转录成cDNA作为模板,进行PCR扩增,获得CSRP2目的基因后,再与PcDNA3.1-HA载体进行连接重组,构建PcDNA3.1-CSRP2-HA真核表达质粒,经限制性内切酶消化、PCR及DNA序列测序分析等方法鉴定后,瞬时转染入A549细胞,Western blot法检验CRP2蛋白表达。结果:成功构建PcDNA3.1-CSRP2-HA真核表达质粒,Western-blot结果显示PcDNA3.1-CSRP2-HA能够在A549细胞中表达。结论:PcDNA3.1-CSRP2-HA真核表达质粒构建并鉴定成功,为后续研究CRP2在炎症诱发氧化损伤机制中的转录调控作用奠定了基础。