抗人CD16单克隆抗体可变区基因的克隆和表达

目的：克隆抗人CD16单克隆抗体重，轻链可变区（VH，VL）基因并合成单链抗体（ScFv)基因。方法：从分泌抗人CD16单克隆抗体的杂交瘤细胞B88－9中提取总RNA，应用RT－PCR技术获得抗CD16单克隆抗体的VH，VL基因，用连接肽（Linker)肽VH和VL连接成具有VH－Linker-VL结构的ScFv基因，将其克隆到表达载体pcDNA3.1( )，并转杂COS－7细胞。结果：VH基因长度为354bp，属于鼠抗体可变区重链基因家族I（B）亚群，VL基因长度为333bp，属于鼠抗体可变区kappa轻链基因家族Ⅲ亚群，采用夹心ELISA方法检测到ScFv的表达。结论：抗人CD16单克隆抗体VH与VL基因的克隆和ScFv基因的构建为基于CD16的导向免疫治疗奠定了基础。     

抗人核内不均一核糖核蛋白A2/B1抗体可变区基因克隆、串联和表达

目的克隆抗人核内不均一核糖核蛋白A2/B1(heterogeneousnuclearribonucleoproteinA2/B1,HnRNPA2/B1)单克隆抗体的重链可变区(variableregionofheavychain,VH)和轻链可变区(variableregionoflightchain,VL)基因,构建抗HnRNPA2/B1单链抗体(singlechainFv,ScFv)基因,并在大肠杆菌表达。方法采用斑点ELISA、Western印迹和免疫组化检测抗HnRNPA2/B1单克隆抗体3E8的特异性,并通过逆转录-聚合酶链反应、重叠延伸PCR来克隆、串联VH和VL基因,构建抗HnRNPA2/B1ScFv基因pET28(a )表达载体,在大肠杆菌BL21中诱导表达,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳、竞争抑制ELISA检测表达产物。结果3E8抗体能特异性地与HnRNPA2/B1合成肽以及3株人肺癌细胞内HnRNPA2/B1结合;克隆的VH基因为345bp,VL基因为309bp,符合小鼠抗体可变区特性;抗HnRNPA2/B1ScFv蛋白以包涵体形式表达,分子量约28000,并具有与抗原结合活性。结论成功构建了抗HnRNPA2/B1ScFv基因克隆,并在大肠杆菌中获得了功能性表达,为阐明HnRNPA2/B1与肺癌相关性奠定了实验基础。     

Ⅱ型志贺毒素单克隆抗体可变区基因克隆及单链抗体的表达和功能鉴定

目的 从分泌抗肠出血性大肠埃希菌Ⅱ型志贺毒素中和单克隆抗体杂交瘤细胞株S2C4中克隆抗体可变区基因,构建单链抗体(ScFv),进行原核表达,并对其功能进行鉴定.方法 从杂交瘤细胞株S2C4中提取总RNA,逆转录成cDNA.在cDNA3'-OH末端添加poly-G.PCR扩增包括5'非翻译区和信号肽序列在内的抗体重、轻链可变区基因VH和VL,PCR产物装入T-A载体测序.根据测序结果,设计引物分别扩增VH和VL编码区,再通过重叠PCR,在VH和VL编码区基因之间引入连接链,构建ScFv基因,并克隆到表达载体pComb3xSS中.重组载体导入E.coli Top10F'进行表达,重组蛋白经纯化后,分别用ELISA和动物保护性实验鉴定其生物学活性.结果 VH和VL编码区基因全长分别为396 bp和378 bp,ScFv基因能在大肠埃希菌中高效表达,表达产物的分子量为34 000,用NiSO4亲和层析柱成功纯化.功能性实验表明纯化的重组蛋白可以与Stx2毒素有效结合,能保护动物抵御毒素分子的攻击.结论 成功地克隆S2C4单抗可变区基因,并构建、表达其单链抗体ScFv,为下一步进行该抗体人源化奠定实验基础.     

米尔比霉素肟化物ScFv噬菌体展示抗体库的构建

目的构建具16元大环内酯共性结构小分子物质的特异性抗体库。方法免疫原MILO-BSA免疫小鼠后从脾细胞中提取总RNA,RT-PCR扩增得到全套抗体重链可变区基因(VH)及轻链可变区基因(VL),SOE-PCR将VH、VL片段拼接扩增得到单链抗体可变区基因片段(ScFv)。将ScFv基因克隆到噬菌粒载体pCANTAB5E中,电转化感受态大肠杆菌,辅助噬菌体超感染得到上清液即为噬菌体抗体库。结果RT-PCR扩增出长360bp左右的抗体重链可变区基因(VH)及340bp左右的轻链可变区基因(VL),SOE-PCR扩增得到750bp左右的ScFv基因片段,成功构建了库容量为2.4×106,滴度为2.0×1012pfu/mL的噬菌体单链抗体库。结论构建的抗体库目的片段连接率较高,多样性较好,为下一步16元大环内酯类小分子物质高特异性抗体的筛选奠了基础。     

抗溶藻弧菌独特型单克隆抗体单链抗体ScFv的构建和表达

目的：克隆抗溶藻弧菌独特型单克隆抗体ScFv。方法：从分泌抗溶藻弧菌独特型单克隆抗体的杂交瘤细胞株（2F4）中提取总RNA，利用RT-PCR技术，克隆该抗体重链可变区基因（VH）和轻链可变区基因（VL），通过基因重组构建VH-VL的表达载体pTAT-ALI，转化大肠杆菌BL21后用IPTG诱导表达。结果：VH基因序列全长369碱基对，编码123个氨基酸，VL基因序列全长339碱基对，编码113个氨基酸，二者均符合小鼠免疫球蛋白可变区基因特征，含有4个框架区（FRs）、3个抗原互补决定区（CDRs）及抗体特征性的两个半胱氨酸残基。ELISA测定显示基因工程抗体ScFv与原代抗体一样，具有较高的抗原亲和力。结论：成功构建了抗溶藻弧菌独特型抗体ScFv基因并表达于细菌壁膜间隙和包涵体中，为溶藻弧菌独特型抗体单链抗体ScFv成为新一代基因工程疫苗奠定了初步基础。     

鼠抗寻常性天疱疮抗原噬菌体抗体库的构建与筛选

目的 :应用重组噬菌体抗体库技术制备抗寻常性天疱疮抗原 (PVA)的单链抗体。方法 :用PVA免疫小鼠 6w后 ,抽取小鼠脾脏细胞RNA ,逆转录为cDNA。设计针对小鼠抗体重链可变区 (VH)和轻链可变区 (VL)的特异性引物 ,PCR扩增出VH 和VL 片段。用带有VH3′端、连接肽和VL5′端序列的引物修饰VL 得VL′后 ,与VH 进行重叠延伸拼接 (splicingbyoverlapex tension ,SOE) ,连接成VH—Linker—VL(ScFv) ,并大量扩增。此ScFv经限制性内切酶Sfi1和Not1酶切后 ,以噬粒pCANTAB 5E为载体 ,构建重组质粒 ,转导TG1 感受态菌 ,建成库容为 1 5× 10 6 的噬菌体抗体库 ,用PVA筛库。结果 :构建了抗PVA的特异性单链抗体库 ,筛选出一高丰度表达单链抗PVA抗体的细胞株。结论 :从小鼠噬菌体抗体库中获得特异性的、单克隆的、单链抗PVA噬菌体抗体     

抗B型葡萄球菌肠毒素高亲和力单链抗体的制备

目的 构建针对B型葡萄球菌肠毒素(SEB)的特异性单链抗体(scFv),并进行纯化和鉴定.方法 设计抗SEB高亲和力单克隆抗体(mAb) FMU-SEB-No.2重链和轻链可变区基因片段VH和VL的引物,利用RT-PCR技术,从本室制备的抗SEBmAb FMU-SEB-No.2杂交瘤细胞株中扩增出VH和VL基因片段;然后通过在轻链引物上设计linker序列,重叠引物延伸法(SOE)将VH和VL基因拼接成scFv基因片段,并将其克隆入原核表达载体PGEX4T-1中;转化E.coli BL21(DE3)感受态细胞,IPTG诱导表达,融合蛋白经谷胱甘肽亲和层吸柱纯化.采用SDS-PAGE,Western blot,ELISA等方法对其表达水平和特异性进行分析.结果 成功从1株抗SEB mAb FMU-SEB-No.2杂交瘤细胞株中扩增出VH和VL可变区基因,并通过linker序列将其拼接成scFv片段,大小约750 bp,编码250个氨基酸.PCR、酶切鉴定和测序结果表明表达载体构建成功.转化E.coli BL21(DE3),经IPTG诱导后,以可溶形式表达相对分子质量(Mr)约54 000的融合蛋白.经谷胱甘肽亲和层析法纯化融合蛋白,可获得纯度达90％以上的scFv,ELISA和Western blot法检测结果表明,可溶性scFv与抗原SEB有较强的结合活性.结论 成功构建并表达出抗SEB的特异性单链抗体.     

大容量噬菌体抗体库的构建及鉴定

目的　构建大容量噬菌体单链抗体库 ,从中筛选人源单链抗体 (ScFv)。方法　从正常成人外周血和新生儿脐血分离淋巴细胞 ,用RT PCR扩增轻链可变区基因 (VL)和重链可变区基因(VH) ,通过重叠PCR法将VH 和VL 拼接形成ScFv基因 ,并克隆入噬菌体表达载体PDF ,得到ScFv初级噬菌体抗体库。以高MOI超感染cre 菌株BS136 5 ,通过loxp cre定位重组系统 ,介导轻重链的组合配对 ,得到大容量抗体库 ,用多种抗原对抗体库进行生物淘筛 ,鉴定抗体库的性能。结果　获得了 6×10 10 的大容量单链噬菌体抗体库。分别用卵清蛋白、胃蛋白酶、铁蛋白、人角蛋白、人TNF α、地高辛等6种抗原进行筛选 ,均得到多样性的特异性噬菌体抗体。结论　经loxp cre定位重组系统在单细胞内重组成功地构建了大容量单链噬菌体抗体库 ,初步尝试对 6种抗原进行筛选均获成功 ,提示该抗体库可用于制备具有应用前景的人源抗体     

抗人肺癌单链抗体噬菌体库的构建及特异性单链抗体的鉴定

目的 构建人源噬菌体抗体库,并从中筛选出抗肺癌人源单链抗体。方法 提取肺癌患者癌旁淋巴结组织,通过RT-PCR扩增出重链可变区基因（VH）和轻链可变区基因（VL）,再经剪切-重叠-延伸PCR(SOE-PCR)将VH 和VL连接得到单链抗体（ScFv）。将双酶切后的ScFv基因片段克隆入噬菌体表达载体pCANTAB5E,得到初级噬菌体抗体库。以肺腺癌细胞株A549为抗原对抗体库进行4轮筛选富集,鉴定抗体库性能。将得到的阳性克隆用IPTG诱导表达并进行检测。结果 成功构建噬菌体单链抗体库。经筛选富集,噬菌体收获率得到增加,第4轮是第1轮的115倍。随机选取10个克隆,通过ELISA法检测到其中7个与A549细胞呈阳性反应,阳性率为70%。SDS-PAGE及ELISA检测证实得到人源抗肺癌单链抗体。结论 成功构建人源单链抗体噬菌体库,从中获得具有较高特异性的抗人肺癌单链抗体。     

抗人红细胞抗体和HIV抗原融合蛋白的构建及活性鉴定

目的构建及表达抗红细胞(RBC)单链抗体(ScFv)和HIV抗原的融合蛋白,并做活性测试。方法将鼠抗人红细胞单链抗体可变区重链、轻链基因与HIV抗原基因相连,重组于高效表达载体中,并在大肠埃希菌中表达。结果经ELISA检测和红细胞凝集检测,证明表达的融合蛋白具有HIV抗原和抗人红细胞的双重活性,可使含有HIV抗体的红细胞悬液凝集。结论融合蛋白构建成功,可用于HIV的快速检测。     

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.     

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.     

Counterpointing the functional role of the forebrain and of the brainstem in the control of the sleep-waking system

This paper reviews the lifetime contributions of the author to the field of sleep-wakefulness (S-W), reinterprets results of the early studies, and suggests new conclusions and perspectives. Long-term cats with mesencephalic transection show behavioral/polygraphic rapid eye movement sleep (REMS), including the typical oculo-pupillary behavior, even when the section is performed in kittens prior to S-W maturation. REMS can be induced as a reflex. Typical non-rapid eye movement S (NREMS) is absent and full W/arousal is present only after a precollicular section. The isolated forebrain (IF) rostral to the transection exhibits all features of W/arousal and NREMS [with electroencephalographic (EEG) spindles and delta waves], arousal to olfactory stimuli, and including the appropriate oculo-pupillary behaviors. These features also mature normally after neonatal transection. REMS is absent from the IF. After deprivation there is NREMS pressure and rebound in the IF, but the decerebrate cat only shows pressure for REMS. Most IF reactions to pharmacologic agents are within expectations, except for the tolerance/withdrawal effects of long-term morphine use which are absent. In contrast, these effects are supported by the brainstem (i.e. seen in the decerebrate cat). In cats with ablation of the telencephalon, or diencephalic cats, delta waves are absent in the thalamus. EEG thalamic spindle waves are seen triggering S for only 4-5 days after ablation. Therefore, true NREMS is absent in chronic diencephalic cats although pre- and postsomniac behaviors persist. These animals are hyperactive and show a pronounced, permanent insomnia; however, a low dose of barbiturate triggers a dramatic REMS/atypical NREMS rebound. Cats without the thalamus (athalamic cats), initially show a dissociation between behavioral hyperactivity/insomnia and the neocortical EEG, which for 15-20 days exhibits only delta and slower oscillations. Fast, low-voltage W rhythms appear later on, first during REMS, but spindle waves and S postures are absent from the start, such that these cats also display only atypical NREMS. Athalamic cats also show barbiturate-sensitive insomnia. Cats with ablation of the frontal cortices or the caudate nuclei remain permanently hyperactive. They also show a mild, but significant hyposomnia, which is permanent in afrontal cats, but lasts for about a month in acaudates. The polygraphic/behavioral features of their S-W states remain normal. We conclude and propose that: (a) the control of the S-W system is highly complex and distributed, but is organized hierarchically in a well-defined rostro-caudal manner; the rostral-most or highest level (telencephalon), is the most functionally complex/adaptative and regulates the lower levels; the diencephalic/basal forebrain, or middle level, has a pivotal role in inducing switching between S and W and in coordinating the lowest (brainstem) and highest levels; (b) W can occur independently in both the forebrain and brainstem, but true NREMS- and REMS-generating mechanisms exist exclusively in the forebrain and brainstem, respectively; (c) forebrain and brainstem S-W processes can operate independently from each other and are preprogrammed at birth; this helps understanding normal and abnormal polygraphic/behavioral dissociations in humans and normal dissociations/splitting in aquatic mammals; (d) NREMS homeostasis is present in the IF, but only REMS pressure after deprivation persists in the decerebrate cat; (e) the thalamus engages in both NREMS and W; (f) insomnia in diencephalic cats is the result of an imbalance between antagonistic W- and S-promoting cellular groups in the ventral brain (normally modulated by the telencephalon); (g) the EEG waves, which are signature for each S-W state, appear to truly drive the concomitant behaviors, e.g. a hypothetical human IF could alternate between behavioral NREMS and W/arousal/awareness; (h) a role for REMS is to keep the individual sleeping at the end of the self-limiting NREMS periods. The need for accelerating research on telencephaling NREMS periods. The need for accelerating research on telencephalic S-W processes and downstream control of the lower S-W system levels is emphasized.     

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 -     

Ultrastructure of the endothelium of the drainage system of the eye

The endothelium of the ocular drainage system (Schlemm’s canal, collector tubules, and aqueous veins) in primary juvenile glaucoma undergoes degenerative dystrophic changes with compensatory hypertrophy and proliferation at the initial stages of the glaucomatous process and atrophy and desquamation at advanced and terminal stages. Progressive decrease in the pinocytous function of endotheliocytes, reduction of the protein-synthesizing and mitochondrial compartments of the cytoplasm, and formation of autophagosomes reflect the process of endotheliocyte degeneration in general. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 5, pp. 574–577, May, 2008     

The development of the ligament of the head of the femur

The hip joints of 30 human male and female fetuses and stillborns between 20 mm and 350 mm crown-rump length were studied by light microscopy. The ligament of the head of the femur developed in situ as a condensation of mesenchyme at the end of the second month of intra-uterine life (IUL), and was vascularized by branches of acetabular vessels early in the fourth month. In the majority of fetuses older than 5.5 months IUL, vessels in the ligament passed a short way into the femoral head within cartilage canals, to supply a small region around the fovea capitis. The remainder of the head was supplied by vessels in canals from around the upper part of the neck. The ligament changed from predominantly cellular to fibrous during the last 4 months of IUL. This increase in strength suggested significant mechanical functions in utero: limitation of adduction-flexion and opposition to postero-superior dislocation were the most likely.     

电磁辐射生物效应的机理研究述评

电磁辐射好似一束带刺儿的玫瑰,她所奉献的物质文明绚丽多彩,日新月异;但人类在享用的同时,也必须提防被刺儿扎着。随着社会的发展,环境中的电磁辐射剂量（能量密度）会呈几何级数式增长。有人把电磁污染称为除空气、水、噪声污染以外的第四类污染。而且是看不见、听...