抗人红细胞H抗原单链抗体基因克隆和表达

目的 克隆抗人红细胞H抗原单克隆抗体轻、重链可变区(VH、VL)基因并构建单链抗体(ScFv)基因及其表达载体,实现其在原核细胞中的表达.方法 从分泌抗人红细胞H抗原单克隆抗体的杂交瘤细胞株2FA中提取总RNA,采用RT-PCR法获得抗人红细胞H抗原单克隆抗体的VH、VL基因;利用重叠引物延伸法(splicing by overlap extension,SOE)将轻重链可变区基因连接,并引入连接肽(Linker)编码序列,构建VH-Linker-VL结构的单链抗体(ScFv)基因,将其克隆到原核表达载体pET-his中,转化BL21(DE3)plysS细胞,IPTG诱导表达;获得的目的蛋白经纯化后,用SDS-PAGE与Westem blotting对目的蛋白进行鉴定,间接EHISA、竞争ELISA和免疫荧光法检测目的蛋白的活性.结果 克隆的VH基因长度为351 bp,属于鼠抗体可变区重链基因家族I(B)亚群;VL基因长度为339 bp,属于鼠抗体可变区轻链基因家族I亚群;SOE法克隆的单链抗体基因为750 bp;构建的含原核表达载体的菌株经IPTG诱导后,SDS-PAGE及Western blotting法检测到Mr 32 000的目的蛋白(表达的ScFv);ScFv纯化后,经免疫荧光法、间接与竞争HLISA法检测到该ScFv蛋白具有生物结合活性.结论 成功地克隆了抗人红细胞H抗原单克隆抗体VH与VL基因和ScFv基因,构建ScFv基因的表达载体,实现了ScFv在大肠杆菌BL21(DE3)plysS细胞中的活性表达,为基于红细胞H抗原的免疫检测技术建立奠定了基础.     

超抗原葡萄球菌肠毒素与抗人大肠癌单链抗体ND-1 scFv融合基因的构建、表达及活性分析

目的:构建并表达超抗原葡萄(SEA)和鼠抗人大肠癌单链抗体ND-1scFv的融合蛋白,以提高SEA的靶向杀伤作用。方法:构建超抗原SEA和鼠抗人大肠癌单链抗ND-1scFv的融合基因ND-1 scFv/SEA的表达载体,转化到大肠杆菌E.coli M15中进行诱导表达。Ni-NTA亲和层析对表达产物进行分离、纯化。间接免疫荧光法检测融合蛋白的靶向结合活性,MTT法检测靶向杀伤效率。结果:成功构建了融合基因ND-1scFv/SEA,实现功能性表达,纯化的ND-1scFv/SEA融合蛋白与表达有ND-1相应抗原的大肠癌细胞CCL-187有高度亲和活性,通过激活外周血单核细胞,可特异性杀伤靶细胞,在4μg/mL浓度下对CCL-187的杀伤率达到91%,明显优于SEA的杀伤活性。结论:融合蛋白ND-1 scFv/SEA对大肠癌细胞CCL-187具有靶向结合和杀伤活性,为SEA用于靶向性的大肠癌治疗奠定了基础。     

人抗HER2单链抗体/精氨酸九聚体融合蛋白的基因构建、表达及鉴定

目的:构建人源性抗HER2单链抗体(scFv)/精氨酸九聚体(9R)融合蛋白基因,在大肠杆菌里表达纯化并检测该融合蛋白的活性。方法:采用PCR的方法,扩增融合基因scFv-9R,将获得的基因克隆入原核表达载体pQE30,在大肠杆菌M15中表达,表达产物经SDS-PAGE和Western blot鉴定,通过Ni-NTA螯合层析纯化,透析复性,超滤浓缩。ELISA分析scFv-9R融合蛋白抗原亲和活性,凝胶迁移实验检测scFv-9R融合蛋白与siRNA结合活性。结果:成功构建了人源性抗HER2 scFv-9R融合基因,经IPTG诱导后在M15中以包涵体形式表达。表达的目的蛋白scFv-9R能与HER2抗原结合,同时具有siRNA结合能力。结论:scFv-9R具有结合抗原与siRNA双重活性,为靶向递送siRNA的研究奠定了基础。     

人源性抗HBc单链抗体真核表达载体的构建及其细胞内表达

目的：构建人源性抗HBc单链抗体的真核表达载体并在细胞内表达。方法： 采用DNA重组技术将特异性人源性抗HBc单链抗体基因插入真核表达载体pEGFP-c1；转染HepG2细胞，经G418筛选细胞，荧光倒置显微镜观察细胞内抗HBc单链抗体与绿色荧光蛋白融合表达情况，并用ELISA法检测HBc单链抗体基因的细胞内表达。结果： 成功地构建了人源性抗HBc单链抗体的真核表达载体。转染HepG2细胞并筛选后，经荧光倒置显微镜观察，细胞内有绿色荧光蛋白表达；ELISA检测细胞内表达的单链抗体片段具有HBcAg结合活性。结论： 人源性抗HBc单链抗体的真核表达载体的构建并在细胞内成功表达，为胞内抗HBc单链抗体的进一步研究奠定了基础。     

噬菌体表面呈现抗人红细胞血型A抗原单链抗体的研究

目的 构建表达抗人红细胞血型Ａ抗原５０Ａ杂交瘤细胞的单链抗体（ＳｃＦｖ）。方法 应用重组噬菌体抗体技术,从５０Ａ杂交瘤细胞中分离、构建单链抗体基因,并将其克隆入噬粒ｐＣＡＴＮＡＢ５Ｅ中,转化Ｅ．ｃｏｌｉ ＸＬ－Ｂｌｕｅ,辅助噬菌体援救构建５０Ａ噬菌体单链抗体库;采用完整红细胞亲和富集法淘选阳性重组噬菌体,鉴定重组噬菌体并进行序列测定分析;免疫印迹试验检测重组单链抗体的特异性抗原活性。结果 用Ｍ１３     

人源性促甲状腺激素受体单链抗体Fc 融合蛋白的制备及鉴定

目的:构建含有促甲状腺激素受体抗体(TRAb)单链抗体的重组载体pFastBac~(TM)1-ScFv-IgG1Fc,通过Bac-to-Bac表达系统实现单链抗体融合蛋白的高效表达,并检测目的蛋白表达和免疫学活性。方法:TRAb抗体的重链可变区(HV)和轻链可变区(LV)的序列以linker(G4S)连接,构建ScFv;Genbank检索人IgG1Fc序列,SnapGene软件分析上述序列和载体pFastBac~(TM)1,以基因合成方式构建重组质粒pFastBac~(TM)1-ScFv-IgG1Fc;pFastBac~(TM)1-ScFv-IgG1Fc转化感受态大肠杆菌DH10Bac生成重组杆粒,pUC/M13正向和反向引物进行PCR分析确认目的基因的序列;重组杆粒采用Cellfectin?Ⅱ试剂转染昆虫细胞Sf9生成P1病毒储液,继续感染Sf9生成高滴度的P2和P3病毒储液;P3病毒储液感染High Five~(TM)细胞,收集上清通过SPA-Sepharose CL-4B亲和层析纯化获得较纯的单链抗体融合蛋白,SDS-PAGE鉴定目的蛋白;化学发光和免疫组化检测目的蛋白抗原结合力。结果:PCR扩增得到约3 747 bp的基因序列,重组杆粒中目的基因正确插入;目的蛋白位于55 kDa附近且无杂带,纯度较高;目的蛋白浓度为248 U/L,单链抗体融合蛋白的结合力较好。结论:Bac-to-Bac表达系统成功构建并表达了TRAb单链抗体融合蛋白,抗原亲和力良好。     

抗人CD33单链抗体与CD80胞外区融合基因的构建、表达及其生物活性检测

目的构建及表达CD80胞外区与抗人CD33单链抗体(scFv)融合基因,并初步检测融合蛋白的生物活性。方法扩增CD80胞外区,利用人血清白蛋白(HAS)第三结构域亲水性的403-427位氨基酸作为链间连接肽将其与抗人CD33 scFv连接,并克隆至原核表达载体PET22b(+)中,经IPTG诱导,在大肠杆菌Rosseta(DE3)内得到融合蛋白的表达。表达产物经过溶解包涵体,镍柱亲和层析纯化和体外复性过程,获得了高纯度的融合蛋白。并用间接免疫荧光法检测融合蛋白与人CD33抗原结合活性。结果克隆出CD80胞外区序列,大小为633 bp,正确合成作为链间连接肽的人血清白蛋白(HAS)第三结构域的403~427位氨基酸。成功构建融合基因表达载体,SDS-PAGE和Western blot分析结果表明,融合蛋白在Rosseta(DE3)中得到高效表达,融合蛋白相对分子质量(Mr)约为55 000,复性后经过间接免疫荧光检测表明具有与人CD33抗原结合活性。结论成功构建PET22b(+)-CD80胞外区-CD33 scFv(ExCD80-CD33scFv)融合基因表达质粒,融合蛋白在宿主菌Rosetta(DE3)中获得了表达,并初步检测其生物学活性。     

抗阿尔茨海默病Aβ人源单链抗体基因的筛选和表达

目的 从人源噬菌体单链抗体库中筛选与阿尔茨海默病发病中起关键作用的β淀粉样多肽(Aβ)1-42特异性结合的单链可变区抗体(scFv)基因,利用原核生物大肠杆菌进行可溶性表达,以获得抗AB1-42人源抗体.方法 利用噬菌体展示技术对人源噬菌体单链抗体库进行多轮富集,以Aβ1-42为抗原经酶联免疫吸附(ELISA)检测,筛选与Aβ1-42特异性结合的阳性噬菌体克隆,再用阳性噬菌体感染E.coli HB2151进行可溶性表达,经SDS-PAGE、Western blot及ELISA法检测scFv单抗的可溶性表达水平及抗原结合活性.并对阳性scFv抗体基因进行基因测序鉴定.结果 获得了7个特异性的抗Aβ1-42 scFv阳性噬菌体克隆,其中4个克隆ELISA检测吸光度值(A值)高于阴性对照5倍以上;其中1株(A 10)获得可溶性单链抗体的成功表达,表达产物主要位于菌体中,ELISA效价显示在全菌蛋白中A值高于对照5倍以上.其相对分子质量约为33 000.DNA测序表明所获抗体的可变区基因属于scFv抗体基因,推导得到的氨基酸序列具有典型的抗体可变区结构.结论 用人源噬菌体单链抗体库筛选出抗Aβ1-42的人源抗体单链可变区基因,并成功表达了具有抗原结合活性的可溶性单链抗体,为进一步研究阿尔茨海默病的发病机制和治疗奠定了基础.     

抗HBsAg/RBC双特异噬菌体抗体的构建

目的 通过不同外壳蛋白的展示，构建抗HBsAag/RBC双特异噬菌体抗体。方法通过DNA重组技术，将抗HBsAg Fab与噬菌体基因8融合，抗RBC ScFv与噬菌体基因3融合，这2种融合基因以不同的启动子驱动，并克隆于同一噬菌体表达载体上，得到双特异噬菌体抗体表达载体，转化大肠杆菌后获取噬菌体抗体上清，用ELISA和红细胞凝集试验检测其双特异活性。结果ELISA和RBC凝集实验证明，本方法可形成双特异噬菌体抗体，可使含有HBsAg的RBC悬液产生凝集。用展示性能得到提高的蛋白8变种取代野生型蛋白8可提高双特异噬菌体抗体的活性。结论 HBsAb和RBC两种抗体分子能够通过不同噬菌体外壳蛋白同时展示于同一噬菌体表面，形成双特异噬菌体抗体。可用于人外周血HBsAg的快速血凝法检测。     

抗人HBsAg单链抗体基因的构建及其在COS-7细胞中的表达

目的：构建抗人HBsAg单链抗体基因，并分析其在COS—7细胞中的表达。方法：以从噬菌体抗体库中筛选的抗HBsAg的Fab抗体基因为模板，分别扩增其轻、重链可变区(VL、VH)基因，通过重组PCR方法将轻、重链可变区基因用连接肽(C1y4Ser)3的编码序列连接，并引入前导肽编码序列，构建具有L—VH—Linker—Vl结构的单链抗体基因。将所构建的单链抗体基因克隆入绿色荧光蛋白(GFP)融合表达载体pEGFP—N3，并转染COS—7细胞进行表达。结果：经测序表明，前导肽、连接肽、VL及Vh的序列正确。酶切鉴定证实，成功地构建了GFP基因融合表达载体。瞬时转染COS—7细胞后，通过荧光显微镜观察证实有ScFv融合蛋白的表达。转染细胞的培养上清浓缩后，进行SDS—PAGE及westem blot分析，可检出ScFv融合蛋白的分泌性表达。培养上清的间接ELISA检测证实，所表达的单链抗体具有与HBsAg结合的特异性。结论：成功地构建了抗人HBsAg单链抗体基因，并可在COS—7细胞中分泌性表达。     

Prolonged waking reduces human immunodeficiency virus glycoprotein 120- or tumor necrosis factor alpha-induced apoptosis in the cerebral cortex of rats

The human immunodeficiency virus (HIV) induces neuronal death, presumably by apoptosis. This effect may be triggered by the glycoprotein 120 (HIVgp120) released by HIV when infecting a cell, and mediated by tumor necrosis factor alpha (TNF), a pro-inflammatory cytokine. Both molecules, HIVgp120 and TNF, increase sleep when administered acutely in the brain. On the other hand, sleep deprivation increases the levels of several growth factors. In this context, we challenged rats with HIVgp120 or TNF simultaneously with sleep deprivation. Our results indicate that both HIVgp120 and TNF increase neuronal death in the rat cerebral cortex, but not hippocampus, and that this effect is completely prevented by total deprivation of sleep. These results suggest that acute total deprivation of sleep protects against the HIVgp120 and TNF deleterious effects.     

Recombinant fusion proteins for haemagglutination-based rapid detection of antibodies to HIV in whole blood

Recombinant fusion proteins, consisting of a monovalent anti-human RBC monoclonal antibody B6, and conserved immunodominant peptide of HIV-1 envelope glycoprotein gp41 or HIV-2 envelope glycoprotein gp36, have been designed and purified after over-expression in E. coli. These fusion proteins are Fab-based and were obtained by assembling the light chain with Fd (variable domain and the first constant domain of the heavy chain) or Fd fusions containing HIV-derived peptide, and following a protocol of in vitro denaturation of inclusion bodies and subsequent renaturation to assemble functional Fab. Using a multistep column chromatographic procedure, monomeric Fab and Fab fusion proteins containing HIV-derived peptide were purified to high degree, free of aggregates. The yield of various proteins on the laboratory scale (1-2 l of shake flask culture) was in the range of tens of milligram. Purified anti-human RBC Fab fusion proteins containing sequences derived from HIV-1 gp41 and HIV-2 gp36 were highly specific for detection of antibodies to HIV-1 and HIV-2, respectively. The described design, expression and purification protocols will make it possible to produce specific recombinant reagents in large quantities for agglutination-based rapid detection of antibodies to HIV in whole blood.     

Nicotine prevents HIVgp120-caused electrophysiological and motor disturbances in rats

Human immunodeficiency virus (HIV)-associated dementia (HAD) is a frequent complication in HIV+ subjects. Several electrophysiological markers and motor control are altered in HIV+ subjects, including event-related potentials (N2-P3 changes). These are electrophysiological indicators of cognitive processing. The mechanisms by which HIV induces neurophysiological abnormality is still under research. However, several neurotransmitters have been implicated. For example, glutamate and the vasoactive intestinal neuropeptide (VIP). In this study, we support further this notion indicating that HIVgp120, a glycoprotein derived from HIV, is involved in the pathogenesis of neuropsychiatric abnormalities. We also have observations suggesting that one HIVgp120 mechanism of action is to interfere with cholinergic neurotransmission. Our results indicate that event-related potentials (ERP) were affected by HIVgp120, in particular N2 and P3. In addition, motor coordination was severely affected. Both parameters were maintained near normality when rats were simultaneously treated with nicotine. These results support further an HIVgp120-caused alteration of cholinergic neurotransmission that might be part of the etiology of neuropsychiatric disturbances.     

Establishment of an HIV cell-cell fusion assay by using two genetically modified HeLa cell lines and reporter gene

Infection of human cells with the human immunodeficiency virus type I (HIV-1) can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 together with the appropriate human chemokine receptors. In this study, a T-tropic HIV cell-cell fusion assay was established that utilized CD4, human CXCR4 and HIV NL4-3 gp160 as fusion components and a T7 polymerase-activated luciferase as a reporter system. The HeLa T4 cells used, expressed CD4 and CXCR4, and the applied HeLa KS386 cells expressed HIV NL4-3 gp160. By combining HeLa T4 cells with HeLa KS386 cells, an approximately about 100- to 300-fold increase in luciferase activity could be elicited relative to the control. The addition of anti-CD4 monoclonal antibody (Mab) (RPA-T4) or anti-CXCR4 Mab (12G5) in the assay significantly inhibited the fusion event; in contrast, an anti-CCR5 Mab (2D7) had no effect, indicating that the fusion assay was CD4 and CXCR4 dependent. In this report, fusion events could be monitored by both the luciferase reporter system and syncytia formation. Fusion events were monitored and compared using these two approaches. The luciferase reporter system was found to be more sensitive than syncytia formation. Moreover, compared with previous HIV fusion models, such as using recombinant vaccinia viruses, this system has several advantages, including simplicity and sensitivity. Finally, the system provides a powerful tool to study fusion mechanisms mediated by T-tropic HIV gp160, as well as to screen for fusion-blocking antibodies and antiviral agents.     

抗HBsAg和抗红细胞双特异Diabody的构建及表达

目的构建及表达抗乙肝病毒表面抗原（ＨＢｓＡｇ）和抗红细胞（ＲＢＣ）双特异Ｄｉａｂｏｄｙ，用于血清中ＨＢｓＡｇ的快速检测。方法将抗ＨＢｓＡｇ的人抗体轻重链可变区基因与抗红细胞的鼠抗体重轻链可变区基因交叉配对构建成两个杂合Ｄｉａｂｏｄｙ基因，并将两个配对基因组建在一个表达载体中，成为双特异Ｄｉａｂｏｄｙ表达载体，并在大肠杆菌中分泌表达。结果经ＥＬＩＳＡ检测和红细胞凝集实验测定，证明所表达的双特异性Ｄｉａｂｏｄｙ具有抗ＨＢｓＡｇ和抗ＲＢＣ双重功能，并可使含有ＨＢｓＡｇ的ＲＢＣ悬液产生血球凝集。结论所表达的双特异性Ｄｉａｂｏｄｙ具有双重抗体活性，可用于血清中ＨＢｓＡｇ的快速检测     

RANTES, MDC and SDF-1alpha, prevent the HIVgp120-induced food and water intake decrease in rats

Human immunodeficiency virus (HIV)-wasting syndrome might be facilitated by the HIVgp120 affecting the immunological system. We studied the effect (subchronic administration: 5 days) of HIVgp120, and a few immune-response mediators: regulated upon activation normal T-cell expressed and presumably secreted (RANTES), stromal derived factor-1alpha (SDF-1alpha), macrophage-derived chemokine (MDC), and their combination, on food and water intake in rats, motor control and pain perception. Eighty male adult Wistar rats received an intracerebroventricular (icv) administration of: vehicle 5 microl/day or 0.92 nmol daily of HIVgp120IIIB, RANTES, SDF-1alpha, or MDC, and the combination of RANTES+HIVgp120IIIB, SDF-1alpha+HIVgp120IIIB, or MDC+HIVgp120IIIB. Food and water intake was measured every day during administration, and 24 and 48 h after the last administration. Rats were also weighed the first and the last day of experiment in order to detect the impact of these treatments in the body weight. HIVgp120IIIB significantly decreased food and water intake. These rats gain less weight than the control (vehicle) and chemokines-treated subjects with exception of those treated with SDF-1alpha that also gain less weight. In addition, HIVgp120 deteriorated motor control. HIVgp120IIIB effects on food and water intake, and motor control were prevented by these chemokines. HIVgp120+RANTES, HIVgp120+SDF-1alpha, and SDF-1alpha alone induced hyperalgesia. Results suggest an interaction between HIVgp120 and the chemokine system to generate the HIV-wasting syndrome, the motor abnormalities and changes in pain perception.     

Whole-blood agglutination assay for on-site detection of human immunodeficiency virus infection

Simple and rapid diagnostic tests are needed to curtail human immunodeficiency virus (HIV) infection, especially in the developing and underdeveloped nations of the world. The visible-agglutination assay for the detection of HIV with the naked eye (NEVA HIV, which represents naked eye visible-agglutination assay for HIV) is a hemagglutination-based test for the detection of antibodies to HIV in whole blood. The NEVA HIV reagent is a cocktail of highly stable recombinant bifunctional antibody fusion proteins with HIV antigens which can be produced in large quantities with a high degree of purity. The test procedure involves mixing of one drop of the NEVA HIV reagent with one drop of blood sample on a glass slide. The presence of anti-HIV antibodies in the blood sample leads to clumping of erythrocytes (agglutination) that can be seen with the naked eye. Evaluation with commercially available panels of sera and clinical samples has shown that the performance of NEVA HIV is comparable to those of U.S. and European Food and Drug Administration-approved rapid as well as enzyme-linked immunosorbent assay kits. The test detects antibodies to both HIV type 1 (HIV-1) and HIV-2 in a single spot and gives results in less than 5 min. The test was developed by keeping in mind the practical constraints of testing in less developed countries and thus is completely instrument-free, requiring no infrastructure or even electricity. Because the test is extremely rapid, requires no sample preparation, and is simple enough to be performed by a semiskilled technician in any remote area, NEVA HIV is a test for the hard-to-reach populations of the world.     

A quantitative GFP-based bioassay for the detection of HIV-1 Tat transactivation inhibitors.

The Tat function of the human immunodeficiency virus (HIV) represents an important target for the development of new anti-HIV drugs. A rapid, sensitive and simple bioassay was developed for the detection of HIV transactivation inhibitors. A reporter plasmid based on the expression of the green fluorescent protein (GFP) under control of the HIV-1 long terminal repeat (LTR) was constructed. This reporter gene can be quantified by simply measuring the fluorescence irradiated by GFP-producing cells, without the need of extraction procedures or enzymatic assays. Cells, stably expressing HIV-1 Tat protein, were transfected with this plasmid and the inhibitory effect of anti-Tat drugs was assessed by measuring the inhibition of fluorescence. Using this assay system the anti-transactivation activity of several known compounds was confirmed. This is the first HIV transactivation assay using GFP reporter gene in microtiter plates. The assay can be used for the detection and quantification of HIV transactivation, and for the high throughput evaluation of anti-transactivation drugs in different cellular backgrounds.     

分泌型ICOS-mIg重组融合蛋白定量检测方法的建立和应用

目的:建立双抗体夹心ELISA法,定量检测重组可诱导共刺激分子(ICOS)-mIg融合蛋白的分泌型表达,并对其灵敏度、特异性及线性检测范围进行评价。方法:用分子生物学技术制备重组ICOS-mIg融合蛋白。以羊抗小鼠IgG为包被抗体,HRP标记的马抗小鼠IgG为检测抗体,通过配对试验、方阵滴定试验及绘制mIgG浓度与A450值的标准曲线,建立定量检测重组ICOS-mIg融合蛋白的双抗体夹心ELISA法。结果:建立了双抗体夹心ELISA法,检测的线性范围为7.8~500μg/L,标准曲线的回归方程为:y=-0.7864 1.1635log(x),R2=0.9911,P<0.0001。应用该方法可快速检测哺乳动物细胞表达的重组ICOS-mIg融合蛋白的分泌量。结论:建立了一种可快速定量检测重组ICOS-mIg融合蛋白分泌表达的双抗体夹心ELISA法。该法灵敏、准确、快速、实用性好,对优化ICOS-mIg融合蛋白表达细胞的筛选和大规模制备具有重要价值。     

Biological and pharmacokinetic properties of a novel immunoglobulin-CD4 fusion protein

Summary We report a CD4-immunoglobulin fusion protein in which the first two extracellular domains of human CD4 replace the Fc region of the immunoglobulin. When co-expressed with a gene encoding an immunoglobulin light chain, the protein was covalently assembled into a form having an Mr consistent with that expected for two fusion heavy chains and two immunoglobulin light chains. The antigen specificity of the antibody was retained, however, binding to HIV gp120 was lost. Pharmacokinetic analysis revealed the in vivo half-life of the fusion protein to be 2.4 h in mice.