pcDNA3/NT-3真核表达载体的构建及其表达

目的构建大鼠神经营养因子3（NT-3）基因真核表达载体并观察其在真核细胞内的表达情况。方法采用逆转录聚合酶链式反应（RT-PCR）技术从大鼠脑组织总RNA中扩增NT-3基因cDNA序列,将其克隆到真核表达载体pcDNA3中,经酶切鉴定和序列分析后,以阳离子脂质体Li-pofectamine2000介导转染L929细胞,应用免疫细胞化学和western blot鉴定NT-3在细胞内的表达。结果RT-PCR产物为822bp的特异片段,重组质粒pcDNA3/NT-3酶切后产生822bp和5.2kb的片段,DNA测序证实822bp片段的碱基序列与大鼠NT-3基因序列完全一致,成功构建了pcDNA3/NT-3重组质粒。将其转染真核细胞后,免疫细胞化学、western blot结果表明NT-3能在真核细胞中正确表达。结论成功构建了重组真核表达质粒载体pcDNA3/NT-3,为后续的研究奠定基础。     

NT-3基因过表达慢病毒载体的构建和包装及鉴定

目的构建神经营养因子-3(neurotrophin-3,NT-3)基因慢病毒载体,检测其在大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)中的表达。方法体外扩增NT-3,将NT-3全长载体GV287-GFP与扩增出的NT-3用AgeI进行酶切,将NT-3全长序列克隆入GV-287-GFP,转化大肠杆菌DHS a感受态细胞,筛选出阳性克隆进行基因测序。重组GV287-EGFP质粒、pHelper 1.0质粒和pHelper 2.0质粒三质粒共转染至包装细胞293T,培养48 h后收集细胞上清液,将病毒浓缩后在293T细胞中测定病毒滴度,并检测慢病毒载体在MSCs的转染效率。荧光显微镜观察转染是否成功,RT-PCR和Western blot检测MSCs细胞中NT-3蛋白的表达。结果测序结果和Western blot检测均证明NT-3慢病毒载体构建正确,且在细胞中正确表达。与辅助质粒共包装细胞获得慢病毒颗粒,并成功感染MSCs细胞。包装慢病毒、浓缩病毒悬液的滴度为2×109/ml慢病毒浓缩液。结论成功构建了稳定高效表达NT-3基因的慢病毒载体。     

人NT-3基因转染对雪旺细胞生物学行为的影响

目的观察神经营养素-3(NT-3)基因转染对雪旺细胞生物学行为的影响.方法采用逆转录聚合酶链反应(RT-PCR)方法,从人肝脏组织总RNA中克隆NT-3 cDNA,构建重组真核表达载体pIRES2-EGFP-NT-3;应用混合酶消化法分离、纯化雪旺细胞;采用阳性脂质体介导法将重组质粒pIRES2-EGFP-NT-3转染雪旺细胞,观察外源性NT-3对雪旺细胞生物学行为的影响.结果成功地从人肝脏组织中克隆了NT-3 cDNA,构建了重组真核表达载体pIRES2-EGFP-NT-3;分离、纯化得到了高纯度的雪旺细胞;将重组质粒转染雪旺细胞,与对照组相比,转染NT-3基因的雪旺细胞形态正常;经NT-3抗体免疫组化染色见NT-3转染组雪旺细胞胞浆呈深棕色着色,轴突着色清晰可见,与对照组相比,明显变长;空载体转染组及空白对照组胞浆及轴突着色均较淡;对各组细胞胞浆着色进行半定量分析,结果表明NT-3基因转染组灰度值显著高于对照组(P<0.01).结论 NT-3基因转染后雪旺细胞NT-3含量明显增加,轴突变长,本实验结果有望为面神经损伤后的修复提供新的途径.     

基因重组BDNF蛋白在大肠杆菌中的表达纯化与功能鉴定

目的构建脑源性神经营养因子（BDNF）基因的原核表达载体，并在大肠杆菌中进行表达，以获取高产量、低成本、高纯度且具有生物学活性的BDNF蛋白。方法以人的全长BDNF cDNA为模板，用PCR方法扩增成熟区BDNF的cDNA，应用基因重组技术将人BDNF cDNA克隆到质粒pET-30a（＋）中，进行限制性内切酶酶切分析和DNA测序鉴定。将重组质粒转化大肠杆菌BL21（DE3）LysS，经IPTG诱导表达后，用Ni-NTA亲和层析纯化获取蛋白，用SDS-PAGE和western blot方法鉴别，噻唑蓝（MTT）法检测重组蛋白对PC12细胞增殖的影响。结果扩增出的人BDNF cDNA片段克隆进了原核表达载体，经酶切和核酸测序鉴定，得到了正确的重组质粒pET-BDNF，并在大肠杆菌中获得了表达。纯化后的蛋白经考马氏亮蓝染色呈单一条带；用抗BDNF的抗体进行western blot分析证明目的蛋白获得了表达。基因重组BDNF蛋白能够促进PC12细胞增殖。结论本研究成功构建了表达基因重组人BDNF的原核表达载体，基因重组人BDNF蛋白在大肠杆菌中获得了表达和纯化，所获得的基因重组蛋白具有较好的生物学活性。     

真核细胞中人神经生长因子cDNA的表达

目的 构建人神经生长因子cDNA的表达载体pcDNA3．1-NGF,并在哺乳动物细胞中进行一过性表达。为以后人神经生长因子的基因治疗打下基础。方法 在本研究室已克隆人神经生长因子cDNA的基础上。构建表达载体pcDNA3．1-NGF。用DEAE-葡聚糖转染技术将pcDNA3．1-NGF导入COS-7细胞，将人神经生长因子cDNA进行一过性表达。并采用Westernblot方法检测表达情况。以鸡胚背根神经节的生长检测表达蛋白的生物活性。结果 成功构建了表达载体pcDNA3．1-NGF，Western blot方法检测出COS-7细胞表达有目的蛋白，并具有一定的生物活性。结论 用哺乳动物细胞一过性表达了人神经生长因子，并具有一定的生物活性。     

人野生型parkin基因真核表达载体的构建及其在PC12细胞中的表达

目的 克隆人野生型parkin基因并构建真核表达载体pCDNA3．1—parkin，将重组质粒转染PC12细胞获得高表达人野生型parkin基因的PC12细胞克隆。方法 从胎脑组织中提取总RNA，用RT—PCR方法获得人野生型parkin基因的全长cDNA，插入pCR2．1—TA克隆载体中进行序列测定，测序正确后将其亚克隆至表达载体pCD—NA3．1，利用脂质体将重组质粒转染PC12细胞，经G418筛选获得抗性细胞克隆，采用RT—PCR和Western Blot方法鉴定人野生型parkin基因在PC12细胞中的过表达。结果 经限制性内切酶酶切图谱分析和DNA序列测定证实目的基因已插入重组质粒，RT—PCR和Western Blot证明经G418筛选得到的转基因PC12细胞克隆中存在人野生型parkin基因的表达。结论 成功构建了人野生型parkin基因的真核表达载体，获得了稳定表达人野生型parkin基因的PC12细胞克隆，为进一步研究parkin的生物学功能以及parkin在帕金森病发病机制中的作用奠定了良好的基础。     

pcDNA3-hNGFb真核表达载体的构建及其表达

目的构建人神经生长因子β（NGF-β）基因真核表达载体并观察其在L929细胞内的表达。方法以RT-PCR从人脑组织总RNA中扩增NGF-βcDNA,将其克隆到真核表达载体pcD-NA3中,经酶切鉴定和序列分析后,以Lipofectamine2000介导转染L929细胞,应用免疫细胞化学和western blot鉴定NGF-β在细胞内的表达。结果RT-PCR产物为750bp的特异片段,重组质粒pcDNA3-hNGFb酶切后产生750bp和5.2kb的片段,DNA测序证实750bp片段的碱基序列与人NGF-βcDNA完全一致。将其转染L929细胞后,免疫细胞化学、western blot结果表明NGF-β及其前体proNGF-β能在真核细胞中正确表达。结论成功构建了重组真核表达质粒pcDNA3-hNGFb,为后续的研究奠定基础。     

腺病毒介导NT-3基因在雪旺细胞的表达

目的观察腺病毒介导的NT-3基因在培养雪旺细胞(Schwann cells, SCs)的表达.方法在293细胞中培养扩增NT-3重组腺病毒(adenovirus vector for NT-3,Ad-NT-3),用组织培养半数感染量法测定其滴度.然后用Ad-NT-3感染原代培养的SCs,逆转录酶-多聚酶链反应(RT-PCR)技术检测NT-3基因的表达.结果 Ad-NT-3扩增后获得了较高滴度的病毒.SCs经NT-3重组腺病毒感染24 h后有NT-3 mRNA的转录.结论腺病毒介导的NT-3基因可转入培养的SCs并高效表达.     

空肠弯曲菌cdtA及其高抗原肽段的原核重组质粒的构建和表达

目的空肠弯曲菌细胞扩张毒素cdtA及其高抗原肽段蛋白人工表达。方法使用PCR方法获得cdtA、cdtA1、cdtA2编码的基因,经确证后将该基因克隆到谷胱甘肽转移酶融合表达载体(pGEX-5x-1)中,构建了pGEX-5X-1-cdtA、cdtA1、cdtA2表达质粒,在大肠杆菌BL21中获得了相应表达。结果 pGEX-cdtA、cdtA1、cdtA2重组菌株具有明显的表达带,其分子量与预期的结果相同。结论成功构建了cdtA及其高抗原肽段的原核表达重组质粒,并表达出相应蛋白为其免疫原性分析及研制疫苗奠定了基础。     

人重组NT-3基因体外转染胚胎干细胞及其表达

目的 探讨神经营养素-3(NT-3)基因体外转染小鼠胚胎干细胞(ES)的可能性及其表达情况.方法 用脂质体介导的方法,将重组真核表达载体PCDNA-3.1(+).NT-3瞬时转染ES.用细胞免疫组化及RT-PCR检测转染细胞NT-3蛋白及mRNA表达.ELISA检测细胞分泌上清液中NT-3蛋白表达.结果 细胞免疫组化结果显示,转染NT-3基因的ES细胞胞浆呈红色染色;RT-PCR得到200 bp的基因片段;ELISA检测细胞分泌上清液中NT-3蛋白表达呈阳性,与对照组有显著差别(P＜0.05).结论 重组真核表达载体PCDNA-3.1(+).NT-3可成功转染ES,获得稳定表达NT-3的ES细胞株.     

大鼠FLRT3 C端结构域蛋白的原核表达和纯化

BACKGROUND： Studies have suggested that fibronectin leucine-rich transmembrane protein 3 （FLRT3） is related to injury and regeneration of the nervous system. However, the expression and biological characteristics of these proteins remain poorly understood.
OBJECTIVE： To obtain FLRT3 C-terminal gene fragments, to effectively express and purify the target proteins. D
ESIGN, TIME AND SETTING： An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine, Central South University between October 2007 and June 2008.
MATERIALS： Three Sprague Dawley adult rats were used to extract total RNA from rat brains. The pGEX4T3 and Escherichia coil （E. coil） JM109 were purchased from Promega. E. coil BL21 was provided by Novagen.
METHODS： FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were amplified using RT-PCR technique from rat total RNA. The amplified products were cloned into the expression vector pGEX4T3. A recombinant expression vector was then constructed and introduced into E. coil BL21. IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins, followed by isolation, purification, and renaturation of inclusion bodies that comprised recombinant proteins. Finally, the purified recombinant protein was obtained.
MAIN OUTCOME MEASURES： Determination of FLRT3 C-terminal DNA sequence; expression of target proteins was assayed by SDS-PAGE electrophoresis; purified recombinant protein was identified with Western blot methods. RESULTS： FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were successfully harvested through RT-PCR amplification, and were then clones into the prokaryotic expression vector pGEX4T3. The results of the sequence were consistent with the known gene sequence. SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass of 56,600. The recombinant protein was observed in the inclusion body, and highly purified recombinant proteins were obtained through a series of methods, such as rinsing, purifying, dissolving, and renaturing.
CONCLUSION： From adult Sprague Dawley rats, FLRT3 C-terminal gene fragments were successfully cloned and shown to be effectively expressed in E. coil BL21. Moreover, highly purified GST fusion proteins were obtained.     

人隔蛋白7的真核表达载体的构建及表达

目的 构建人隔蛋白7(SEPT7,hCDC10/SEPT7)的表达载体并对U251人脑恶性胶质瘤细胞系进行转染且检测其表达.方法 RT-PCR方法扩增SEPT7 cDNA片段,并将扩增的片段插入pCDNA3真核表达载体,构建成含SEPT7cDNA的重组载体pCDNA3/SEPT7,以脂质体介导该质粒转染人脑恶性胶质瘤细胞系U251,应用RT-PCR和免疫荧光染色鉴定转染细胞中hCDC10/SEPT7的表达.用流式细胞术对转染前后的U251细胞进行细胞周期分析.结果 成功构建含SEPT7cDNA的重组载体pCDNA3/SEPT7,并使其稳定转染U251细胞,在转染的细胞中,证实有SEPT7mRNA表达上调.细胞周期检测结果G0/G1期细胞增多,SPF降低.结论 成功构建SEPT7表达载体,并在人脑恶性胶质瘤细胞系获得表达,延迟细胞周期进展.     

重组型Caspase3对U251胶质瘤细胞促凋亡作用

目的探讨由野生型人Caspase3大小亚基颠倒构建的重组型Caspase3促U251胶质瘤细胞的调亡活性。方法运用分子克隆技术,使Caspase3基因大小亚基颠倒构建,并将重组基因克隆入绿色荧光蛋白(GFP)真核表达载体pcDNA3.1中,转染人U251胶质瘤细胞。利用透射电镜和流式细胞仪观察胶质瘤细胞凋亡的生物学特征。结果成功地获得了重组型反向Caspase3基因。经限制酶酶切分析鉴定释放330及550bp片段,PCR法鉴定反向重组成功;构建了重组型Caspase3基因的真核表达载体,转染U251胶质瘤细胞后,重组型Caspase3基因在细胞中表达,电镜显示细胞呈现凋亡的典型形态学特征。流式细胞仪可见细胞凋亡峰。结论重组型Caspase3可促进U251胶质瘤细胞的凋亡。     

人His-AWP1融合蛋白表达载体的构建及其在原核生物的表达

目的 获取了His-AWP1融合蛋白，为了阶段深入研究AWP1的结构。功能及筛取与其相互作用的蛋白打下基础。方法 应用逆转录聚合酶链反应（RT-PCR）法从人ECV304内皮细胞系中克隆AWP1cDNA，并将其重组于能表达6个组氨酸残基的原核表达质粒pET-14b中，经酶切，序列鉴定，选择正确重组克隆。将其质粒转化大肠杆菌BL21（DE3），IPTG诱导表达，用Ni^2 -NTAHis柱纯化和SDS-PAGE分离蛋白。结果 克隆到一个627bp的AWP1cDNA片段，重组质粒目的DNA测序正确，纯化出了一个分子量约为38kD的融合蛋白，结论 用基因工程方法在原核细胞表达并成功纯化出His-AWP融合蛋白。     

人14-3-3β蛋白的原核表达、抗血清制备及真核表达载体的构建

目的 纯化原核表达的14-3-3β(YWHAB)重组蛋白并制备多抗血清,构建适用于哺乳动物细胞的真核表达载体.方法 将重组蛋白表达载体pET30a(+)/YWHAB转化大肠杆菌表达菌株BL21(DE3)感受态细胞,异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达,镍-四齿螯合剂(Ni-NTA)亲和层析柱纯化重组蛋白;以纯化的重组蛋白为抗原免疫BALB/c小鼠,应用ELISA和Western blot方法分别检测抗血清的效价和特异性;应用PCR扩增添加BamH Ⅰ和EcoR Ⅰ酶切位点把YWHAB的ORF亚克隆至真核表达载体pEGFP-N1,添加BamH Ⅰ和Hind Ⅲ酶切位点把YWHAB的开放阅读框(ORF)亚克隆至真核表达载体pCDNA3.1(+),对重组载体进行酶切和PCR鉴定.结果 YwHAB重组蛋白以可溶性形式表达,分子量为32 000,与预期分子量一致;纯化后的重组蛋白纯度达90%以上,ELISA结果显示其抗血清的效价为1:50 000,Western blot结果表明抗血清的特异性较好;酶切和PCR鉴定结果表明真核表达载体pEGFP-N1/YWHAB和pCDNA3.1(+)YWHAB构建成功.结论 通过亲和层析纯化获得人14-3-3β重组蛋白,进而免疫BALB/c小鼠制备多抗血清,为进一步研究人14-3-3β的功能成功构建了其真核表达载体.     

cDNA cloning, prokaryotic expression and purification of rat α-synudein

Objective To clone the cDNA of rat α-Syn gene, investigate its prokaryotic expression and produce purified recombinant rat α-Syn protein. Methods Rat α-Syn cDNA was amplified from the rat brain total RNA by RT-PCR and was cloned into pGEX-4T-1, a prokaryotic expressing vector. The recombinant plasmid containing rat α-Syn gene was transformed into E. Coli BL21 to express a fusion protein with rat α-Syn protein tagged by glutathione-S-transferase （GST）. The fusion protein was then cleaved by thrombin during passing through the GST-agarose 4B column to release the recombinant rat α-Syn protein. The recombinant rat a-Syn protein was further purified using Superdex S200 gel filtration. Results DNA sequencing confirmed that the cloned cDNA contained 420 base pairs encoding 140 amino acids, which was identical to the reported amino acid sequence of rat α-Syn. After transformation, the recombinant plasmid pGEX-ra-Syn expressed a soluble protein that was inducible by IPTG. The purified recombinant protein was shown to be single band on SDS-PAGE, with a molecular size of around 18000, which was identical to the reported molecular size of rat α-Syn. Western blot analysis demonstrated that the recombinant protein was recognized by specific antibody against α-Syn. Conclusion The rat α-Syn gene was successfully expressed in prokaryotic expression system and highly purified rat α-Syn recombinant protein was produced.     

pEGFPN1-GDNF真核表达载体的构建及其表达

目的 构建人胶质细胞源性神经营养因子(GDNF)基因真核表达载体并观察其在COS-7细胞内的表达.方法 应用RT-PCR从U251细胞总RNA中扩增GDNF cDNA,将其克隆至真核表达载体pEGFPN1,经酶切鉴定及序列分析后,以Fugene HD介导转染COS-7细胞,应用免疫细胞化学和Western blot鉴定其在细胞内的表达.结果 RT-PCR产物为650bp的特异片段,重组质粒pEGFPN1-GDNF经双酶切产生650bp和4.7kb的片段,测序分析结果与文献报道结果完全一致.将其转染COS-7细胞后,免疫细胞化学、Western blot结果表明GDNF蛋白能在COS-7细胞中正确表达.结论 成功构建了pEGFPN1-GDNF真核表达载体,为进一步开展帕金森病的基因治疗研究奠定了基础.     

The gene expression of coagulation factor VIII in mammalian cell lines.

Both the full-length and B domain-deleted cDNA of factor VIII were constructed in plasmid pcDNA3, respectively, and successfully expressed in Cos-7 cells. The yield of recombinant factor VIII-deltaB (0.4 U/mL/10(6) cells/day) was approximately four times higher than that of the recombinant factor VIII. In addition, it was indicated that the gene expression of factor VIII is specific for cells from different tissues. The highest expression level was found in the hepatocellular carcinoma line SMMC-7721, followed by kidney, ovary, and lung cell lines. To compare the efficiency of gene expression of recombinant factor VIII, the factor VIII-deltaB gene was further reconstructed in different forms in the expression plasmid pCMV-dhfr for transient gene expression in Chinese hamster ovary cells. The redundant 5'- and 3'-untranslated sequences of factor VIII-deltaB were deleted. The cDNA encoding the heavy and light chains of factor VIII were constructed, respectively. Among them the high yield of the recombinant factor VIII was found in the coexpression of the heavy and light chain cDNA fragments of factor VIII. The deletion of the redundant 5'-untranslated sequence of factor VIII-deltaB was also beneficial for gene expression. As expected, the gene coexpression of factor VIII-deltaB and von Willibrand Factor cloned by the long-polymerase chain reaction method was also helpful for enhancing the expression level of recombinant factor VIII. A monoclonal antibody raised against factor VIII was prepared and used for the specific assay of recombinant factor VIII by the competitive ELISA method, the assay results were consistent with those determined by the one-stage bioassay.