抗乙酰胆碱受体单链抗体基因克隆与表达

目的重建含有抗乙酰胆碱受体(AChR)单链抗体1929#(single-chain Fv antibody 1929#,scFv1929#)基因的载体并在大肠杆菌(E.coli)进行表达,提高scFv1929#的可溶性表达量。方法用聚合酶链反应法(PCR)扩增载体pHEN1中的scFv1929#结构基因,将扩增产物定向克隆至载体pET32a(+)的多克隆位点中构建新的载体pET32a(+)-scFv1929#,经EcoR v和NotⅠ双酶切后进行鉴定,并将所构建载体转化入表达宿主E.coli BL21(DE3)plysS菌株内诱导表达后集菌并裂菌,将诱导前菌体、诱导后菌体,以及裂菌后所得诱导后上清、诱导后沉淀进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,并采用Western blot(WB)法进行鉴定。结果鉴定结果显示载体pET32a(+)-scFv1929#中所插入的scFv1929#基因片段大小为730bp,与预计相符,经测序确定该基因序列无误。SDS-PAGE电泳结果显示37℃下诱导前菌体、诱导后菌体、诱导后上清、诱导后沉淀中均可在相对分子质量(Mr)接近44 000处见到蛋白条带,其中诱导后菌体及诱导后沉淀中的条带较粗大。经WB法证实融合蛋白scFv1929#具有较高特异性。结论 scFv1929#表达载体pET32a(+)-scFv1929#被成功构建,并可在大肠杆菌中高效表达,其表达产物具有较好特异性。     

抗人脑胶质瘤单克隆抗体SZ39单链抗体的构建及初步表达

目的：研究人脑胶质瘤基因工程单抗。方法：采用基因重组技术分别将抗人脑胶质瘤单抗ＳＺ３９重链和轻链可变区基因片段连接，构建表达载体，并在大肠杆菌中表达。结果：表达的ＳｃＦｖ抗体为可溶性，ＥＬＩＳＡ和ＷｅｓｔｅｒｎＢｌｏｔ检测证实能特异性地与人脑胶质瘤细胞系ＳＨＧ－４４表面抗原结合，其表达量为２２０μｇ／Ｌ。结论：成功地构建了抗人脑胶质瘤单抗ＳＺ３９ＳｃＦｖ表达质粒，并得到了与胶质瘤细胞表面抗原相结合的单链抗体。     

兔抗人免疫相关鸟苷三磷酸酶基因多克隆抗体的制备与鉴定

目的：表达人免疫相关鸟苷三磷酸酶基因（IRGM）a全长融合蛋白,制备高质量的免抗人IRGM多克隆抗体。方法：将IRGMa全长cDNA片段分别克隆到原核表达载体pGEX-4T-2和真核表达载体pCMV-myc,测序验证序列正确。在大肠埃希菌E．coliBL21中表达IRGMa／GST融合蛋白,用纯化的可溶性IRGMa／GST融合蛋白免疫新西兰大白兔,制备兔抗人IRGM抗体,采用ELISA、免疫印迹和竞争抑制等多种方法验证其敏感度和特异度。结果：自制的兔抗人IRGM多克隆抗体的特异度和敏感度均较高,效价更高于市售抗体。结论：成功制备了免抗人IRGM多克隆抗体,为进一步的功能研究打下基础。     

抗乙酰胆碱受体单链抗体原核表达载体及表达菌株的构建

目的将含有抗人乙酰胆碱受体(AChR)单链抗体基因的载体重新构建并建立表达菌株。方法采用聚合酶链反应(PCR)从质粒pHEN1中克隆单链抗体scFv1929#全长基因,将PCR产物再定向克隆至载体pET32a( )的多克隆位点中,并转入大肠杆菌BL21(D E3)plysS以构建表达菌株。结果PCR产物约730bp,与预期一致,重组阳性克隆菌株JM109 pET32a( )-scFv1929#经菌液PCR可见730 bp处有特异性条带,所提取质粒pET32a( )-scFv1929#经PCR及酶切鉴定正确,经测序后证实scFv1929#的核苷酸序列正确并正确地插入载体pET32a( )的读码框内。将新构建载体转化大肠杆菌BL21(D E3)plysS以获得表达菌株。结论已成功地重新构建含有抗AChR单链抗体基因的载体并建立大肠杆菌表达菌株BL21(D E3)plysS pET32a( )-scFv1929#,为今后表达抗AChR单链抗体scFv1929#融合蛋白打下基础。     

EGFRvⅢ单链抗体治疗小鼠脑胶质瘤的研究

目的探讨表皮生长因子受体III型突变体单链抗体(EGFRvIIIscFv)对小鼠脑胶质瘤的治疗效果。方法人工合成EGFRvIIIscFv基因序列并构建到原核表达载体中,诱导表达重组蛋白,经电泳和western blotting鉴定;再通过纯化和复性,得到有功能的EGFRvIIIscFv;用酶联免疫吸附分析(ELISA)测定抗体的亲和常数后,通过立体定向仪将其注射到胶质瘤小鼠的脑内,观察荷瘤鼠的生存期,免疫荧光法检测肿瘤血管内皮生长因子(VEGF)的表达。结果双酶切鉴定和测序结果证实人工合成的EGFRv Ⅲ scFv序列与基因数据库中完全一致;经过诱导表达,电泳显示重组蛋白在大肠杆菌中以包涵体形式表达;western-blotting鉴定蛋白分子量为63kDa,与预期大小基本一致;该单链抗体亲和常数为0.203×10-8mol/L;将蛋白注射到荷瘤鼠的脑内,与对照组相比,实验组肿瘤VEGF表达量明显减少、荷瘤鼠中位生存期明显延长(P<0.01)。结论 EGFRvscFv重组蛋白可与EGFRvⅢ特异性结合;脑内注射EGFRvⅢscFv可明显降低胶质瘤血管形成,延长荷胶质瘤小鼠生存期。     

抗谷氨酸脱羧酶抗体、抗神经节苷脂抗体和抗心磷脂抗体与难治性癫痫的相关性研究

目的探讨抗谷氨酸脱羧酶抗体、抗神经节苷脂抗体和抗心磷脂抗体与成人难治性癫痫发病机制的相关性。方法本研究分为3组,即正常对照组(NC组)、可控性癫痫组(NE组)以及难治性癫痫组(IE组)各30例,其中NC组来自医院体检中心的健康体检人员;NE组及IE组来自吉林大学第一医院神经内科癫痫诊疗中心。以酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测3组的血清抗谷氨酸脱羧酶抗体、抗心磷脂抗体及抗神经节苷脂抗体数值,采用正态性检验,两组间定量资料比较。结果 IE组的血清3种抗体水平显著高于NE组及NC组,NE组和NC组的抗体浓度比较无统计学意义;3种抗体检测对于难治性癫痫的诊断具有高度一致性。结论成人难治性癫痫中谷氨酸脱羧酶抗体、抗神经节苷脂抗体和抗心磷脂抗体的检测对于难治性癫痫的诊断、治疗及预后判断具有指导作用。     

兔抗凋亡诱导因子多克隆抗体的制备、纯化与鉴定

背景：凋亡诱导因子是存在于线粒体膜间隙中的黄素蛋白,它不仅具有氧化还原和电子传递功能,还具有促细胞凋亡功能,从而在维持细胞正常生理活动中具有重要作用。 目的：制备抗凋亡诱导因子多克隆抗体并进行特性鉴定。 设计、时间及地点：单一样本观察,于2006-09/2008-08在新乡医学院免疫学研究中心完成。 材料：新西兰大白兔雌性2只,体质量1.9~2.1 kg;Jurkat E6-1细胞系购自美国ATCC公司。食管癌组织取自新乡医学院普通外科手术患者。抗原肽-KLH由上海华大天源生物科技公司合成。CNBr活化的Sepharose 4B购自美国Pharmacia公司。 方法：利用美国过敏和感染疾病研究所(NIAID)主办的抗原表位分析网站(http://beta.immuneepitope.org/home.php)的在线软件对凋亡诱导因子蛋白的氨基酸序列进行分析设计凋亡诱导因子抗原肽,制备多克隆抗体并纯化。 主要观察指标：采用间接ELISA、Western blot、免疫组织化学3种方法对获得的多克隆抗体进行特性鉴定。 结果：间接ELISA法检测显示,血清抗凋亡诱导因子抗体的效价达到1∶128 000。Western blot结果显示,存在Mr67 000条带。免疫组织化学检测结果显示,在食管癌细胞的胞质中有棕黄色的阳性颗粒存在,说明此抗体也可以用于免疫组织化学染色。 结论：实验获得了高效价的特异性的抗凋亡诱导因子抗体。     

抗阿尔茨海默病神经保护肽humanin基因的克隆与表达

目的 利川基因工程技术克隆和表达humanin基因。方法 人工合成humanin基因片段并克隆到原核表达载体pGEMEX—1和pet44a中。再将此等质粒转化至大肠杆菌，经过筛选鉴定获得高表达阳性克隆。结果 基因序列分析显示，humanin基因片段被成功地克隆到原核载体中；聚丙烯酰胺凝胶电泳(SDS-PAGE)显示，在异丙基硫代—β—D—半乳糖苷(IPTG)的诱导下，pGEMEX—1—humanin表达的融合蛋白的相对分子质量约为28000，融合蛋白含量占细菌表达蛋白总量的40％；而pet44a—humanin表达的融合蛋白的相对分子质量约为63000，融合蛋白表达量占细菌蛋白总量的20％。结论 利用基因工程技术可成功地克隆和表达humanin基因，并可获得高效表达。     

癫癎和热惊厥患儿血清抗心磷脂抗体变化及临床意义

目的 探讨癫(癎)和热惊厥患儿血清抗心磷脂抗体的变化及临床意义.方法 采用酶联免疫吸附试验(ELISA)检测39例癫(癎)患儿,45例热惊厥患儿及40例健康对照组血清中抗心磷脂抗体阳性率水平.结果 热惊厥组和癫(癎)组血清ACA-IgG阳性率均高于对照组,癫(癎)组血清ACA-IgG阳性率高于热惊厥组,差异具有统计学意义.结论 检测热惊厥和癫(癎)患儿血清中抗心磷脂抗体的变化对指导临床诊断、治疗和预防具有一定意义.     

抗NMDAR抗体脑炎研究进展

抗N-甲基-M-天冬氨酸受体(N-methyl-D-aspartate receptor,NMDA)脑炎是近年来新发现的一类副肿瘤性边缘叶脑炎(limbic encephalitis,IE),病因及机制不详。该病发病率低,常发生于伴有卵巢畸胎瘤的年轻女性,临床表现多样,以初期的上感症状发展到精神异常、意识障碍、异常运动和自主神经功能紊乱、癫发作、中枢性通气功能障碍为常见表现,脑脊液常为炎性改变。MRI在大脑皮质或小脑可表现短暂异常或颞叶内侧高信号影改变。脑电图多为非特异性改变。血及脑脊液抗NMDA受体抗体阳性可确诊。包括肿瘤切除和免疫治疗、重症监护和物理康复在内的联合治疗为最佳治疗方案。本文就抗NMDA受体脑炎的研究进展做一综述。     

Expression of secreted human single-chain fragment variable antibody against human amyloid beta peptide in Pichia pastoris*☆

BACKGROUND：Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. OBJECTIVE： Based on successful screening of a human single-chain fragment variable antibody specific to amyloidβpeptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide. DESIGN, TIME AND SETTING： A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital （Beijing, China） from January to July 2006. MATERIALS： Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library. METHODS： Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a T easy plasmid for pT-scFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvAβ was cut by EcoRI and NotI endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvAβ expression vector, which was confirmed by gene sequencing. Linearized pPIC9K-scFvAβ was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5% methanol to express human single-chain fragment variable antibody specific to amyloid β peptide. MAIN OUTCOME MEASURES： Protein electrophoresis was used to identify PCR products, gene sequencing was used to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris. RESULTS： Gene sequencing confirmed pPIC9K-scFvAβ orientation. Recombinants were obtained by linearized pPIC9K-scFvAβ transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size. CONCLUSION： The expression vector pP     

Expression of myelin proteins in the developing human spinal cord: cloning and sequencing of human proteolipid protein cDNA

A full-length clone for the human proteolipid protein (PLP) was isolated from a cDNA library constructed from poly(A)+ RNA isolated from fetal spinal cords obtained at 15-24 weeks of conceptional age. The sequence of the human PLP cDNA was determined, and the deduced amino acid sequence was found to be identical with that of rat PLP. Comparison of human and rat PLP cDNA clones indicated that the coding regions retained 97% homology and that there were also other areas of conserved sequence. The human 5'-untranslated region was 93% homologous to that of the rat. The 3'-untranslated region was, overall, 73% homologous to that of the rat with areas containing greater than 84% homology in the first 400 and last 200 nucleotides. The most variability within the 3'-untranslated region occurred between nucleotides 2,000-2,500, where homology with the rat cDNA dropped to 55%. Expression of PLP in the human spinal cord between 11 and 23 weeks after conception was examined and compared with the expression of the myelin basic protein (MBP). RNA was isolated from pooled human spinal cords obtained at three periods of development: 11-14 weeks, 17-19 weeks, and 21-23 weeks. Northern blot analysis revealed a 3.2-kilobase (kb) PLP mRNA that was present at higher abundance in the 21-23-week spinal cord RNA than in the 17-19-week or the 11-14-week samples. The 17-19-week RNA sample also contained a PLP-hybridizing band at 2.2 kb which may possibly have arisen by utilization of alternative polyadenylation signals. Messenger RNA for MBP was detectable at 11-14 weeks but was readily evident in both the 17-19- and 21-23-week age groups. Immunoblot analysis of whole spinal cord homogenates indicated that polypeptides for MBP preceded the appearance polypeptides for PLP by 3-4 weeks.     

cDNA cloning and mRNA expression analysis of the human neuronatin

The authors cloned the nearly complete cDNA of human neuronatin with the aid of an expressed sequence tag (EST) database, and analyzed its expression in various human tissues by Northern blot analysis. The nucleotide and deduced amino acid sequences of the human neuronatin showed a high similarity to those of rodents. The Northern blot analysis revealed that the human neuronatin message was expressed predominantly in the fetal brain in the brain-specific manner, but only faintly in the adult brain. Among the various adult human tissues examined, the anterior pituitary gland was shown to be the only place where the neuronatin mRNA was strongly expressed. Intense neuronatin expression was also observed in several human pituitary adenomas, including ACTH-producing, GH-producing, and nonfunctioning adenomas, but hardly detected in other brain tumors.     

重组人生长激素基因的转染和基因表达产物的定量分析

目的：定量分析基因表达产物人生长激素（ｈＧＨ），并确定基因表达期限。方法：我们利用磷酸钙介导的方法将重组ｈＧＨ基因导入培养的人胚皮肤成纤维细胞内，之后用Ｎｏｒｔｈｅｒｎ杂交证实转染细胞能够表达ｈＧＨ基因。在此基础上又采用放免法对ｈＧＨ进行了定量测定。结果：从细胞转染的第１２天算起，ｈＧＨ水平逐渐提高，到２３天进入一稳定表达阶段，到９０天仍在进行稳定表达。此时每毫升培养液中的细胞数为１．３×１０６个，ｈＧＨ含量为７９．３２±３．２６６３ｎｇ。结论：转染细胞至少能持续表达ｈＧＨ９０天。     

黑色素瘤相关性抗原E1基因的克隆与测序

目的 克隆表达人黑色素瘤相关性抗原MAGE-E1片段，以便研究其对神经系统恶性肿瘤的生物学作用。方法 从人胶质瘤细胞系BT-325中提取mRNA，用RT-PCR法从中扩增出MAGE—E1片段，采用DNA重组技术将其克隆于pGEM—T Easy载体中并测序。结果 RNA体外扩增得到的片段为400bp，PGEM—MAGE-El经酶切鉴定正确，测序结果与Genebank比较完全相同。结论 该片段可用于真核及原核表达。     

大鼠GLUT3基因重组腺病毒载体的构建及鉴定

目的构建携带大鼠葡萄糖转运体3（GLUT3）基因的复制缺陷型重组腺病毒载体，为研究GLUT3的生物学功能提供工具。方法采用逆转录-聚合酶链反应（RT-PCR）的方法获取大鼠GLUT3基因全长cDNA片段，将其克隆至穿梭质粒pShuttle中构建穿梭质粒pShuttle-GLUT3，经酶切后与线性化的腺病毒骨架质粒pAdeno-X体外连接并转化大肠杆菌DH5α，构建重组腺病毒质粒pAd-GLUT3，酶切线性化重组腺病毒质粒后转染293细胞包装成重组病毒颗粒，重组腺病毒在293细胞中反复扩增数代后，分别用聚合酶链反应（PCR）及免疫印记法（western blot）的方法从基因和蛋白表达水平鉴定重组的腺病毒。结果经DNA测序和PCR分析显示GLUT3 cDNA序列正确。成功筛选出重组腺病毒质粒pAd-GLUT3后在HEK293细胞中成功包装出重组病毒，包装后冻融细胞行PCR及western blot检测表明重组腺病毒包装成功。结论本研究成功构建了携带大鼠GLUT3基因的复制缺陷型重组腺病毒载体。     

Comparative analysis of intrathecal antibody synthesis and DNA amplification for the diagnosis of cytomegalovirus infection of the central nervous system in AIDS patients

We evaluated 49 paired cerebrospinal fluid (CSF) and serum samples of 35 patients infected with the human immunodeficiency virus type 1 (HIV-1) for laboratory evidence of cytomegalovirus (CMV) infection. The patients were grouped according to clinical criteria as probable CMV encephalitis/polyradiculomyelitis, CMV retinitis, cerebral toxoplasmosis, progressive multifocal leukoencephalopathy, HIV-1-related cognitive/motor complex, HIV-1-associated myelopathy, and other neurological diseases. Paired CSF and serum samples were analysed for CMV deoxyribonucleic acid (DNA) by polymerase chain reaction (PCR), quantitative intrathecal synthesis of immunoglobulin G (IgG) antibodies specific for recombinant phosphoprotein 150 (pp150) of CMV and CMV-specific serum IgM. Intrathecal synthesis of pp150-specific IgG was detected in 26% of patients (9/35), serum IgM was found in 23% of patients (8/35), and PCR of CSF was positive in 11% of patients (4/35). Detection of CMV-specific DNA in CSF preceded the intrathecal antibody synthesis in three patients for whom serial samples were available. PCR results of the CSF became negative in one patient with CMV polyradiculomyelitis after successful therapy with 9-[2-hydroxy-l(hydroxymethyl) ethoxymethyl] guanine (DHPG). PCR has a higher diagnostic specificity in the acute phase of CMV infection than intrathecal antibody synthesis. The serum IgM response to CMV cannot be used to monitor a compartmentalized immune response in the central nervous system while an intrathecal immune response seems to be associated with recovery either spontaneously or as a result of treatment.     

Generation and characterization of a novel single-chain antibody fragment specific against human fibrin clots from phage display antibody library

A novel single-chain fragment variable (scFv) antibody was developed directed against human fibrin clots by using the human single fold scFv libraries I+J (Tomlinson I+J). Three positively binding scFvs were evaluated by scFv-phage enzyme-linked immunosorbent assay (ELISA) and DNA sequencing. Then the positive scFv was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography (IMAC) with a yield of about 1 mg/l, the expression of soluble scFv was verified by Western blot analysis. The purified scFv could specifically recognize human fibrin clots and indicate no binding ability with human fibrinogen shown by ELISA. Furthermore, we will amplify the gene of the positive scFv by polymerase chain reaction (PCR) for future study of its role in diagnosis and therapy of thrombus-correlated diseases.     

重组大鼠质粒pEGFP-GDNF的构建及真核细胞转染

目的　构建携带大鼠胶质细胞源性神经营养因子(GDNF)基因的真核细胞表达载体,为应用GDNF进行如帕金森综合征之类的神经元退化性疾病的基因治疗打基础。方法　采用RT- PCR方法从大鼠胎脑组织总RNA中扩增出该基因的c DNA序列,并克隆到增强型绿色荧光蛋白(EGFP)报告基因的真核表达载体p EGFP- C1中,对重组质粒p EGFP- GDNF进一步鉴定。采用电转及阳离子脂质体将重组质粒p EGFP- GDNF转染至SH- SY5 Y细胞。结果　大鼠GDNF c DNA已正确地克隆到真核表达载体p EGFP- C1中,而构建成重组大鼠质粒p EGFP-GDNF。GDNF基因可稳定表达在细胞中。结论　真核细胞表达载体p EGFP- GDNF以及表达GDNF工程细胞SH-SY5 Y的成功构建,为进一步开展GDNF基因治疗PD等中枢神经系统疾病奠定了基础。