FAA基因治疗及其基因表达调控序列新型重组载体的构建、鉴定

目的：Y国探索新型逆转录病毒载体Lentivector及其基因表达调控序列在造血细胞中的有效表达和基因治疗中的应用,用分子克隆的方法构建Fanconis Anemis(FA)患者A组基因（FANCA基因）基因治疗新型重组载体（Lentivector)和基因表达调控序列（启动子/增强子）,进一步通过内切酶酶切位点鉴定插入的基因表达调控序列方向和目的基因的片段大小、目的原基因特异引物PCR扩增特异片段鉴定目的基因。方法：首先次质粒Friend基因表达调控序列方向和目的基因的片段大小、目的基因特异引物PCR扩增特异片段鉴定目的基因。方法：首先将质粒Friend基因表达调控序列（Fr-MuLV E/P)强启动子/增强子插入载体Lentivector pRRLsin-18中相应克隆位点,成为pRRLsin-18FrMuLV(test1);用NotI、NcoI双酶 切载体FochA-FANCA,低溶点胶回收FANCA目的基因片段;应用polylinker、adapter方法多步克隆插入新型载体Lentivector pRRLsin-18(test1)中相应特异克隆位点,获得重组载体sin-18FrMuLVE/P-FA(test2),通过酶切鉴定重组载体插入的基因表达调控序列方向和目的基因的片段大小和方向,筛选正向阳性重组质粒,用PCR法进一步证实插入的FANCA基因片段。结果：挑取的阳性克隆经多个酶酶切鉴定,有Friend质粒390bp基因表达调控序列（Fr-MuLVE/P)和4.5kbFANCA目的基因片段酶切位点,用PCR引物扩增出目的基因相应片段,证明为正向重组体。结论：已成功构建表达FANCA基因的重组逆转录病毒载体及其基因表达调控序列,为进一步应用FANCA基因的新型重组载体Lentivector经包装后转染造血干细胞及其表达情况和相应的FAA基因治疗奠定了良好的工作基础。     

肝癌抗原肽(EPVTKAEML)与人HSP70融合基因的构建及原核表达

目的:构建及原核表达肝癌抗原肽(EPVTKAEML)与人热休克蛋白70(HSP70)的融合基因.方法:采用加端PCR方法,将EPVTKAEML的基因序列融合到人HSP70基因的3′端;将融合基因克隆入原核表达载体pET-28a( )中,构建重组质粒pET-28a( )/EPVTKAEML-HSP70.经限制性内切酶BamH I、Xho I双酶切鉴定及序列测定后,转化E.coli BL21(DE3),经IPTG诱导表达融合蛋白,SDS-PAGE检测表达结果.结果:应用加端PCR方法扩增出约2.0 kb的目的片段,序列测定结果证实,EPVTKAEML的基因序列成功地融合到人HSP70基因的3′端.经BamH I、Xho I酶切鉴定证实,融合基因成功地克隆到原核表达载体pET-28a( )上;转入重组质粒的E.coli BL21(DE3)经IPTG诱导后,SDS-PAGE分析发现在相对分子质量(Mr)约72 000处有表达量明显增多的蛋白条带.结论:成功地构建并表达了肝癌抗原肽(EPVTKAEML)与人HSP70的融合基因.     

HBsAg真核表达质粒的构建和鉴定

目的构建HBsAg真核表达质粒.方法用PCR的方法从质粒pEcob6中扩增出HBsAg基因,并定向克隆到真核表达质粒pcDNA3.1(+),构建成重组质粒pcDNA3.1-S;然后用限制性内切酶消化和DNA序列测定鉴定.结果经酶切和DNA序列测定鉴定,证实重组质粒构建正确.结论真核表达质粒pcDNA3.1-S构建成功.     

小鼠水通道蛋白1基因shRNA慢病毒载体构建与鉴定

目的 应用RNA干扰技术,构建小鼠水通道蛋白1(AQP1)基因重组慢病毒载体并鉴定.方法 对小鼠AQP1 mRNA分析,设计合成的单链引物经退火形成双链寡核苷酸序列,连接入经Age Ⅰ和EcoR Ⅰ双酶切线性化的pLKO.1-TRC慢病毒质粒载体中,菌液PCR鉴定并经测序验证.测序正确后与慢病毒包装辅助质粒共转染293T细胞,收集病毒上清经高速离心浓缩后感染小鼠小胶质细胞BV-2,Western blot法分析筛选有效shRNA序列.结果 成功退火合成3对发夹shRNA序列并将其克隆到pLKO.1-TRC载体中,构建重组质粒pLKO-AQP1-SH1、2、3,经菌液PCR鉴定和测序验证载体构建成功.Real-time PCR检测发现重组质粒pLKO-AQP1-SH2感染BV-2细胞后AQP1 mRNA表达最低,Western blot结果进一步验证结果的准确性.结论 成功构建出AQP1基因shRNA慢病毒载体.     

人PSCA基因真核载体的构建与表达

目的 克隆人PSCA(prosaae stem cell antigen)分子的cDNA,构建PSCA基因的真核表达载体,并在体外进行表达和鉴定.方法 从PSCA阳性细胞系LNCaP中提取总RNA,逆转录为cDNA.根据编码人PSCA的基因序列设计引物,以cDNA为模板,采用PCR技术扩增PSCA基因,将该基因插入到pcDNA3.1(+)真核载体,构建pcDNA3.1(+)-PSCA表达质粒.经限制性酶切鉴定和DNA序列测定结果证实后,将重组质粒pcDNA3.1(+)-PSCA转染Hela细胞,检测其体外瞬时表达.结果 测序证实得到人PSCA基因序列,序列分析显示没有发生无义突变.RT-PCR和免疫细胞化学分析结果分别表明转染细胞有目的 基因和分子的表达.结论成功克隆人PSCA基因,并在真核细胞中获得表达,为进一步构建基于PSCA的肿瘤疫苗奠定了基础.     

小鼠γ-干扰素真核表达质粒的构建和鉴定

目的克隆小鼠γ-干扰素(γ-IFN)基因,构建并鉴定小鼠γ-干扰素真核表达质粒.方法从BALB/c小鼠脾脏提取总RNA,用RT-PCR方法扩增出小鼠γ-干扰素基因, 分别用EcoRⅠ、BamHⅠ双酶切扩增片段和pcDNA3.1(-),定向克隆到真核表达质粒pcDNA3.1(-),构建成真核表达质粒pcDNA3.1(-)γ-IFN.转化产物通过PCR扩增筛选,双酶切鉴定;阳性克隆进行序列分析.结果 RT-PCR产物电泳可见一约500bp大小目的片段.PCR和双酶切电泳结果均证实已插入约500bp的γ-IFN基因片段;阳性克隆测序结果表明克隆的小鼠γ-干扰素基因完全正确.结论成功构建了小鼠γ-干扰素真核表达质粒,为进一步气道内实施哮喘小鼠基因治疗打下了坚实基础.     

小鼠IL-17A-GST融合蛋白的克隆表达

目的: 构建含有小鼠IL-17A(mIL-17A)基因的重组原核表达载体,获得高效表达mIL-17A 的基因工程菌,以及较高产量的mIL-17A蛋白.方法:以PMA 活化后小鼠脾脏单个核细胞的总RNA 逆转录合成的cDNA为模板,PCR 法扩增mIL-17A的编码序列,并分别亚克隆至pMD18-T 载体和原核表达载体pGEX-4T-1 中,经酶切和DNA测序鉴定后,转化感受态大肠杆菌BL21(DE3),IPTG诱导表达,产物经SDS-PAGE及Western blot鉴定.结果:PCR 产物大小及其双酶切鉴定均证明所克隆的基因是mIL-17A,DNA 序列测定进一步证实与GenBank 报道的序列完全一致成功构建了重组原核表达载体pGEX-4T-1/mIL-17A,并在大肠杆菌中高效表达出相对分子质量(Mr)约40 000的具有可溶性的融合蛋白,且Western blot证实确为目的蛋白结论: 成功构建了基因重组体pGEX-4T-1/mIL-17A;并制备出可溶性IL-17A-GST融合蛋白. E3),IPTG诱导表达,产物经SDS-PAGE及Western blot鉴定.结果:PCR 产物大小及其双酶 鉴定均证明所克隆的基因是mIL-17A,DNA 序列测定进一步证实与GenBank 报道的序列完全一致成功构建了重组原核表达载体pGEX-4T-1/mIL-17A,并在大肠杆菌中高效表达出相对分子质量(Mr)约40 000的具有可溶性的融合蛋白,且Western blot证实确为目的蛋白结论: 成功构建了基因重组体pGEX-4T-1/mIL-17A;并制备出     

PML-RARα/pIRES和PML-RARα/pSecTag重组载体的构建和鉴定

目的 构建PML-RARα基因重组表达质粒,为发展PML-RARα基因疫苗提供实验依据. 方法利用RT-PCR方法从急性早幼粒细胞白血病细胞株NB4细胞中扩增出465 bp 的位于PML-RARα融合基因的框内结构的片段,克隆至真核表达载体pIRES和分泌载体pSecTag后,经酶切和序列分析鉴定重组质粒的构建情况.结果 经酶切鉴定和序列测定表明,PML-RARα目的基因已正确插入真核表达载体中,构成了目的重组载体. 结论成功构建了PML-RARα/pIRES和PML-RARα/pSecTag重组真核表达质粒,可进一步用于PML-RARα基因疫苗的实验研究.     

人瘦素基因在胎盘中的分离鉴定及在大肠杆菌中的融合表达

目的 克隆人瘦素(leptin)基因,构建融合表达载体,实现在大肠杆菌中的高效表达.方法 从人胎盘中提取RNA,利用反转录聚合链式反应(RT-PCR),克隆到人的leptin基因编码序列,对该基因片段进行T载体克隆、酶切和PCR鉴定,并且对其进行序列分析.将leptin基因插入到原核表达载体PET-28a中,转化大肠杆菌BL21(DE3)感受态细胞,利用IPTG进行诱导,对表达产物进行SDS-PAGE电泳分析,并利用脱氧胆酸钠对表达蛋白进行纯化.结果 DNA序列分析表明,从胎盘中克隆的人leptin序列与文献报道的一致,酶切鉴定证实leptin基因正确地插入到了表达载体中,经IPTG诱导和SDS-PAGE电泳分析,人leptin基因在大肠杆菌中实现了高效融合表达,表达产物的分子量为20kD,经过纯化,得到了较纯的融合表达蛋白.结论 从人胎盘中成功克隆了leptin基因,构建了原核表达载体,实现了在大肠杆菌中的高效表达,为进一步研究leptin的生物学功能奠定基础.     

Topical organization of the projections of neurons of the substantia nigra and the ventral field of the tegmentum of the midbrain to the head of the caudate nucleus of the cat

Neuroscience and Behavioral Physiology -     

Asymmetry of the internal connections of the striate cortex of the cat in the projection zone of the center of the field of vision

Studies were carried out on the organization of the internal connections of the striate cortex in cats in the projection zone of the center (0–5°) of the field of vision by microintophoretic application of horseradish peroxidase to electrophysiologically identified orientational columns. The area containing neurons showing retrograde labeling in most cases extended in the mediolateral direction. Labeled cells were located in the upper (II, III) and lower (V, VI) layers of the cortex, and the shapes and orientations of the areas containing labeled neurons in these layers coincided. Spatial asymmetry was detected in the distribution of labeled neurons relative to the orientational column studied. Labeled cells were located predominantly medial to the columns, regardless of the distance from the projection of the area centralis. Considering the visuotopical map of field 17, the asymmetry detected here provides evidence that neurons in orientational columns have more extensive connections with neurons of the peripheral part of the cortex. An asymmetrical distribution of “silent” zones around the receptive fields of neurons in orientational columns is suggested, and that these appear to receive influences from the periphery of the visual field. Laboratory of Visual Physiology and Laboratory of Central Nervous System Morphology, I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, 6 Makarov Bank, 199034 St. Petersburg, Russia. Translated from Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 82, No. 12, pp. 23–29, December, 1996.     

Characteristics of the topographic interrelation of the head of the pancreas and the duodenum and pathology of the organs of the pancreatoduodenal area

Seventy pancreatoduodenal complexes of 55 patients with chronic pancreatitis and tumours of this zone and 15 patients died from other diseases are studies histotopographically . The pieces of the pancreatic head tissue in the medial wall of the duodenum were found in 12 cases of the first group and in 4 control cases. The pancreatic tissue consisted either of all elements of this organ or cystically dilated ducts and seemed to infiltrate different layers of the duodenum wall. Three variants of the pancreatic head structure are suggested on the basis of anatomo-topographical interrelationships of the pancreatic head and duodenum. In 12 out of 14 cases chronic pancreatitis and carcinoma of organs of this zone were combined with the variants of the pancreatic head structure, in 2 cases there was a true heterotopy . Pathogenetic significance of these variants for the development of chronic pancreatitis is discussed.     

Influence of the level of the transmembrane potential on the dynamics of the extinction of the amplitude of the synaptic reactions of the snail under voltage clamping conditions

The influence of the level of the transmembrane potential on the dynamics of the extinction of the amplitudes of summary excitatory postsynaptic currents arising in identified giant parietal neurons in response to rhythmic stimulation of the intestinal nerve was investigated in a preparation of the isolated CNS of the common snail in order to identify the possibility of the participation of the postsynaptic element in synaptic plasticity. It was demonstrated that, at a greater value of the transmembrane potential, the decrease in the amplitudes of the postsynaptic currents which have been induced by rhythmic stimulation takes place more rapidly. It was also demonstrated that at a higher frequency of stimulation of the nerve, the effect of the influence of the membrane potential level on the dynamics of the synaptic reactions is more prominent. The data obtained may be regarded as an argument in favor of the possibility of the participation of the post-synapse in plasticity. This study was partially supported by the Russian Basic Research Fund (project No. 94-04-12209). Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I. P. Pavlova, Vol. 45, No. 1, pp. 171–179, January–February, 1995.     

Effects of the lesion of the postcommissural part of the septum on the behavior of the rat.

The effects of the lesion of the postcommissural part of the septum on behavior of the rat has been studied. Results may be summarized as follows. An increase in the exploratory behavior in the open field which decreases rapidly; a decrease in the number of defecations in this test and a decrease in time leaving a dark environment for exploration. In the shuttle box test, no facilitation of the acquisition, but a permanent and quite significant increase in the intertrial activity has been found. We conclude that the lesions tend to decrease the emotivity of the subjects. An interpretation on the basis of the species -- specific defensive reactions explains the transitory and permanent effects of the lesions on the spontaneous activity.     

Effect of the conditions of adaptation of the optic analyzer to the function of the thermoreceptors of the human skin

Summary A discussion is presented of the effect produced by light stimulation applied to the optic analyzer alone on the function of the temperature analyzer. The cutaneous-temperature analyzer was kept on constant illumination and temperature. The light effects from the retinal photoreceptors on the cutaneous thermoceptor system led to reflex changes of the functional rate of the latter. Conditions of the optic analyzer of light adaptation caused reflex adjustment of cold receptors as to heating. Dark adaptation of the eyes changed the functional state of the cold receptors, corresponding to their adjustment to low temperatures.(Presented by Active Member AMN SSSR V. V. Parin) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 57, No. 3, pp. 3–6, March, 1964