重组人sCR1在巴斯德毕赤酵母细胞中的表达、纯化及鉴定

目的:酵母细胞SMD1168表达人sCR1,并对重组蛋白进行纯化。方法:从人外周血中提取总RNA,应用RT-PCR获得人sCR1全长cDNA,将其克隆入真核表达载体pPIC9K,构建含人sCR1的重组质粒(pPIC9K-sCR1);经测序鉴定正确后,将重组质粒转化入毕赤酵母菌细胞SMD1168中,经甲醇诱导,表达产物经SDS-PAGE分析及Western blot鉴定,并通过Ni2 -NTA agarose亲和层析纯化。结果:经甲醇诱导的含pPIC9K-sCR1的酵母细胞表达出重组人sCR1的融合蛋白,48～72hsCR1融合蛋白表达量最高。此蛋白在凝胶上表现为Mr约31000的蛋白区带,在Western blot分析中可被sCR1的CD35单克隆抗体(mAb)识别。经Ni2 -NTA agarose亲和层析纯化后得到较纯的sCR1融合蛋白。结论:人sCR1融合蛋白在酵母细胞表达系统中的高水平表达,并且有与人体天然蛋白相同的抗原活性。     

人补体受体1型SCR1-3功能域基因的克隆表达及生物活性鉴定

目的实现人补体受体1型功能域SCR1-3基因的克隆、表达及生物学活性鉴定。方法从人外周血中提取总RNA,通过逆转录获得cDNA,PCR扩增获得编码CR1-SCR1-3基因序列;克隆到毕赤酵母分泌表达质粒pPIC9K,构建重组质粒pPIC9K-CR1-SCR1-3,菌落PCR、双酶切鉴定后测序;线性化重组质粒电转化毕赤酵母KM71基因组中,经菌落PCR技术筛选在G418平板上生长的多拷贝阳性转化子;摇瓶发酵和甲醇诱导,SDS-PAGE和Western-blot鉴定目的蛋白表达;镍柱亲和层析纯化目的蛋白;用体外抑制补体溶血反应实验测定目的蛋白的生物活性。结果获得了人CR1-SCR1-3编码区序列,测序结果与与GenBank中的相应序列一致;SDS-PAGE和Western-blot表明目的基因在毕赤酵母中实现了分泌表达;体外实验证实经纯化后的CR1-SCR1-3能够明显抑制补体溶血。结论成功构建重组表达质粒pPIC9K-CR1-SCR1-3,在毕赤酵母中实现了CR1-SCR1-3的分泌表达,该蛋白具有较高的抑制补体生物活性。     

人TSLC1基因核心启动子的克隆和鉴定

目的克隆和鉴定人TSLC1基因的核心启动子区,用于开展其转录调控机制的研究。方法用PCR方法从人基因组DNA中扩增出TSLC1基因翻译起始位点上游一系列大小不等的片段,连入pGL3-Basic荧光素酶报告基因载体中。通过瞬时转染A549及NCI-H446细胞,检测细胞裂解液中的双荧光素酶活性,确定TSLC1基因的核心启动子区。结果在人TSLC1基因启动子区的分段克隆中,ATG上游-68～-329 bp片段在A549及NCI-H446细胞中均具有很强的启动活性,对TSLC1的转录起重要作用。结论人TSLC1基因翻译起始位点ATG上游-68～-329 bp区域可能为基因的核心启动子区。     

人MBL相关丝氨酸蛋白酶-1 cDNA的克隆与鉴定

目的 获得人甘露聚糖结合凝集素（ＭＢＬ）相关丝氨酸蛋白酶－１（ＭＡＳＰ－１）基因。方法 以人胎肝组织总ＲＮＡ为模板,采用ＲＴ－ＰＣＲ获取目的ｃＤＮＡ片段,克隆入ｐＧＥＭ－Ｔ载体,进行酶切图谱分析和测序鉴定。结果 以ＲＴ－ＰＣＲ方法获得了含信号顺序的全长ＭＡＳＰ－１ｃＤＮＡ,将其与ｐＧＥＭ－Ｔ载体连接,转化大肠杆菌ＴＧ１,建立了ＭＡＳＰ－１的ｃＤＮＡ克隆,酶切图谱与微机分析结果一致。序列分析表明。与     

可溶性人类白细胞抗原—G1cDNA的克隆及序列分析

目的 建立表达可溶性HLA-G1蛋白的真核表达载体pcDNA3-sHLA-G1。方法 从癌细胞系Jeg-3细胞中提取总RNA,借助RT-PCR技术扩增可溶性HLA-G1的cDNA并把其插入真核表达载体pcDNA3,然后经酶切和测序法鉴定。结果 经酶切鉴定及测序分析。证实已成功构建pcDNA3-sHLA-G1。结论 本研究成功构建可溶性HLA-G1的真核表达载体。     

sCR1-SCR15-18蛋白减轻补体介导的大鼠脑缺血/再灌注损伤

目的: 探讨补体在大鼠大脑缺血/再灌注(ischemia-reperfusion,I/R)损伤中的作用及重组人可溶性补体受体Ⅰ型SCR15-18蛋白(sCR1-SCR15-18)的保护作用。方法: 75只雄性SD大鼠,随机分为假手术组、I/R组和sCR1 -SCR15-18保护组。采用线栓法建立大鼠大脑中动脉闭塞模型(middle cerebral artery occlusion MCAO),缺血2 h,再灌注24 h后,进行神经功能学评分,测定脑梗死体积、大脑皮质髓过氧化物酶(myeloperoxidase,MPO)活性,观察大脑皮质区补体C3b沉积和病理改变。结果: 缺血/再灌注24 h后,sCR1-SCR15-18保护组神经功能学评分,脑梗死体积及脑皮质MPO活性明显低于I/R组(P<0.05);sCR1 -SCR15-18保护组缺血脑组织补体C3b沉积明显减少,病理损伤减轻。结论: 补体在脑I/R损伤中起一定作用,sCR1-SCR15-18蛋白对大鼠I/R损伤脑具有保护作用。     

人细胞色素p450IA1基因cDNA的克隆和鉴定

在用３－甲基胆蒽诱导培养人羊膜ＦＬ细胞２４ｈ后，抽提细胞总ＲＮＡ并直接合成ｃＤＮＡ第一链。利用人工合成的一对寡核苷酸引物，采用ＰＣＲ技术特异性地扩增Ｃｙｔ ｐ５０ＩＡ１ ｃＤＮＡ。３０个循环后琼脂糖凝胶电泳显示１．５Ｋｂ大小片段，长度与预计相符。Ｓｏｕｔｈｅｒｎ杂交结果证实此片段确为Ｃｙｔ ｐ４５０ＩＡ１ ｃＤＮＡ。将此片段克隆至质粒ｐＧＥＭ－３Ｚ并进行部份序列分析。结果显示克隆片段包含Ｃｙｔ ｐ     

HIV-1gp41核心结构模拟位的筛选与鉴定

目的：筛选并鉴定HIV-1 gp4l核心表位。方法：用识别HIV-1 gp41的构象特异性单克隆抗体NC-1筛选噬菌体12肽库，通过夹心ELISA、NC-1特异性阻断实验、竞争抑制实验鉴定阳性噬菌体克隆，DNA序列分析阳性克隆。结果：经3轮筛选，随机挑取24个噬菌体克隆，ELISA鉴定表明有lO个克隆可与NC-1结合，DNA序列分析并推导氨基酸序列，共5种序列：HDVHHRWVYLLS、ITVNEWLYTSEQ、HGRSHGMFKPKR、MGPIARPHWHLN、DMYRSPRPKPDT。其中gp41N肽和C肽所形成的复合物可特异性阻断表达HDVHHRWVYLLS，VNEWLYTSEQ和MGPIARPHWHLN的克隆与NC-1的结合。结论：所得序列HDVHHRWVYLLS，VNEWLYTSEQ及MGPIARPHWHLN模拟HIV-1 gp41六螺旋束核心表位。     

抑制和激活蛋白1(Rap1)单克隆抗体的制备和鉴定

目的制备并鉴定人端粒相关蛋白抑制和激活蛋白1(Rap1)的单克隆抗体。方法用重组原核人Rap1蛋白免疫BALB/c小鼠;细胞融合后,间接ELISA筛选阳性杂交瘤,建立分泌抗人Rap1蛋白单克隆抗体(m Ab)的杂交瘤细胞株,并进行效价测定和特异性鉴定。结果制备并获得1株稳定分泌抗人Rap1蛋白m Ab的杂交瘤细胞株。ELISA测定腹水效价高达1∶10 000以上。Western blot法、免疫沉淀和免疫荧光染色证实该m Ab能特异性识别人Rap1蛋白并可用于上述不同检测。结论成功制备获得了亲和力高、特异性好的抗人端粒相关蛋白Rap1的m Ab。     

重组人sCR1真核表达载体的构建、表达及其生物学活性

目的:采用酵母细胞分泌型载体pPIC9k表达人可溶性补体受体(sCR1),研究重组人sCR1融合蛋白的体外生物学活性.方法:从人外周血中提取总RNA,应用RT-PCR获得人sCR1全长cDNA,然后将其克隆入毕赤酵母细胞分泌型表达载体pPICgk中,构建含人sCR1的重组质粒(pPIC9ksCR1),经测序鉴定正确,电转化入毕赤酵母细胞SMD1168中,将经G418抗性筛选出的重组sCR1酵母细胞株进行PCR鉴定,经甲醇诱导,表达产物经SDS-PAGE分析和Westernblot鉴定,通过Ni2 -NTA agarose亲和层析纯化后进行生物学活性鉴定.结果:获得毕赤酵母细胞分泌型表达载体pPIC9k-sCRI,经G418筛选及PCR鉴定得到高拷贝整合的重组酵母细胞株,经甲醇诱导含pPIC9k-sCR1的酵母SMD1168细胞表达出重组sCR1融合蛋白.此蛋白在SDS-PAGE上表现为Mr,约31 1300的蛋白区带,在Western blot分析中可被sCR1的CD35单克隆抗体(mAb)识别.经Ni2 -NTA agarose亲和层析纯化后得到较纯的sCR1融合蛋白及较高的生物学活性.结论:人sCR1融合蛋白在酵母细胞表达系统中的高水平表达,并且有与人体天然蛋白相同的抗原性及其生物学活性.     

The anti-lipid A antibody HA-1A binds to rough gram-negative bacteria, fixes complement and facilitates binding to erythrocyte CR1 (CD35).

MoAbs to bacterial cell wall lipopolysaccharide are currently under evaluation for the treatment of Gram-negative sepsis. The mode of action of these reagents remains poorly understood. In this study we examined the ability of radiolabelled HA-1A (an IgM anti-lipid A MoAb) to bind in vitro to Salmonella minnesota (Re 595), Escherichia coli, and Streptococcus pyogenes. HA-1A was able to bind specifically to the 'rough' mutant Salm. minnesota, but not to a 'smooth' E. coli, or Strep. pyogenes. Binding to Salm. minnesota led to complement fixation which resulted in bacterial adherence to erythrocyte CR1, suggesting a possible mechanism whereby the antibody might enhance clearance of bacteria by facilitating delivery to the fixed mononuclear phagocytic system. We were not able to demonstrate the formation of immune complexes between free lipopolysaccharide and HA-1A in the presence of serum, nor the enhancement of complement-mediated binding of HA-1A:Salm. minnesota immune complexes to erythrocytes by antibiotic treatment. Binding of HA-1A to small bacterial fragments was, however, demonstrable after in vitro treatment with a beta-lactam antibiotic, which disrupts the bacterial cell wall, but not with gentamicin, an aminoglycoside antibiotic which blocks protein synthesis.     

Soluble complement receptor one (sCR1) inhibits the development and progression of rat collagen-induced arthritis

We set out to determine whether inhibition of complement using sCR1 could influence the development and progression of collagen arthritis in the Lewis rat. Collagen arthritis was successfully established in the Lewis rat, using a novel immunization schedule. In separate experiments, cobra venom factor (CVF) and sCR1 were used to achieve systemic complement inhibition. Their respective effects on disease onset and on the progression of established disease compared with saline-treated control animals was explored. Arthritis was assessed by measurement of clinical score, paw diameter and paw volume. Complement inhibition using either CVF or sCR1, prior to the onset of clinical signs of inflammation, delayed the development of disease. CVF was ineffective in the treatment of established disease, whereas sCR1 delayed the progression of disease in affected joints and prevented the recruitment of further joints while the animals were complement-depleted. In the control saline-treated groups the disease continued to progress relentlessly. We conclude that complement activation is important in the initiation and maintenance of inflammation in collagen arthritis. The potent disease-modulating effect of sCR1 provides persuasive evidence that specific complement inhibiting agents may be an effective approach to the treatment of inflammatory joint diseases     

Comprehensive NF1 screening on cultured Schwann cells from neurofibromas

Neurofibromatosis type 1 (NF1) is mainly characterized by the occurrence of benign peripheral nerve sheath tumors or neurofibromas. Thorough investigation of the somatic mutation spectrum has thus far been hampered by the large size of the NF1 gene and the considerable proportion of NF1 heterozygous cells within the tumors. We developed an improved somatic mutation detection strategy on cultured Schwann cells derived from neurofibromas and investigated 38 tumors from nine NF1 patients. Twenty-nine somatic NF1 lesions were detected which represents the highest NF1 somatic mutation detection rate described so far (76%). Furthermore, our data strongly suggest that the acquired second hit underlies reduced NF1 expression in Schwann cell cultures. Together, these data clearly illustrate that two inactivating NF1 mutations, in a subpopulation of the Schwann cells, are required for neurofibroma formation in NF1 tumorigenesis. The observed somatic mutation spectrum shows that intragenic NF1 mutations (26/29) are most prevalent, particularly frameshift mutations (12/29, 41%). We hypothesize that this mutation signature might reflect slightly reduced DNA repair efficiency as a trigger for NF1 somatic inactivation preceding tumorigenesis. Joint analysis of the current and previously published NF1 mutation data revealed a significant difference in the somatic mutation spectrum in patients with a NF1 microdeletion vs. non-microdeletion patients with respect to the prevalence of loss of heterozygosity events (0/15 vs. 41/81). Differences in somatic inactivation mechanism might therefore exist between NF1 microdeletion patients and the general NF1 population.     

抗RAET1G2单克隆抗体的研制和初步鉴定

目的:制备鼠源抗RAET1G2单克隆抗体,并对其特异性进行鉴定。方法:以原核表达的RAET1G2蛋白为抗原免疫BALB/c小鼠,运用B淋巴细胞杂交瘤技术制备抗RAET1G2单克隆抗体;用免疫双扩散方法鉴定Ig亚类;Western blot鉴定单克隆抗体的特异性。结果:获得了4株可分泌特异性抗RAET1G2抗体的杂交瘤细胞株7A11、9C6、10F9和12F8,Ig亚类均为IgG1。结论:制备的杂交瘤细胞株能稳定分泌特异性的抗RAET1G2抗体,并且所分泌的单克隆抗体都能识别天然的RAET1G蛋白,为进一步研究游离性的RAETlG2分子与肿瘤进展的关系以及分析各种肿瘤细胞表面的RAET1G表达情况创造了条件。     

Low expression of erythrocyte complement receptor type 1 in chronic hepatitis C patients

Primate erythrocyte complement receptor type 1 (CR1) plays an essential role in complement-associated immune complex clearance by transporting complexes to macrophages in the liver and/or spleen. Antibody-bound hepatitis C virus, which consists of immune complexes, is observed in patients with chronic hepatitis C. The aim of this study was to clarify the pathophysiological roles of erythrocyte CR1 in hepatitis C virus-infected individuals. We quantified the expression of erythrocyte CR1 with a fluorescence-activated cell sorter system in 57 chronic hepatitis C and 37 chronic hepatitis B cases and 20 normal volunteers. Complement-bound immune complexes were quantified by means of an enzyme-linked immunosorbent assay using anti-C1q and anti-C3d antibodies. Hepatitis C virus-infected patients showed lower erythrocyte CR1 and higher C3d immune complex levels than volunteers (P < 0.01 and P < 0.05, respectively). An inverse correlation was observed between the erythrocyte CR1 and C3d immune complex levels in hepatitis C virus infection (r = - 0.300, P = 0.032). The erythrocyte CR1 levels in hepatitis C virus infection were lower in patients with severe liver inflammation, cirrhosis, or hepato-cellular carcinoma than in those with mild inflammation, whereas the levels did not differ regardless of the disease stage in hepatitis B virus infection. These findings demonstrate that the expression of erythrocyte CR1 is related to immune complex quantity and the severity of liver disease in hepatitis C virus infection. © 1996 Wiley-Liss, Inc.     

The aminoglycoside G418 suppresses muscarinic receptor-activated calcium release in stably transfected murine N1E-115 neuroblastoma cells

The aminoglycoside G418 inhibited the release of calcium (Ca²⁺) from internal stores coupled to muscarinic receptors in murine N1E-115 neuroblastoma cells carrying the aminoglycoside resistance gene neomycin phosphotransferase (NPT). No significant effect was observed on responses coupled to histamine or bradykinin receptors. Cells were transfected using the eukaryotic expression vector pHβAPr-1-neo and selected using G418. Two groups were differentiated either in the continued presence of G418 or in the absence of G418. Carbachol (1 mM), histamine (200 μM) and bradykinin (100 nM) were administered to cells for thirty seconds and changes in [Ca²⁺]_i were measured with fluorescence video microscopy of single cells loaded with the Ca²⁺ indicator fura-2. The effects of G418 on carbachol evoked Ca²⁺ release included a 73% reduction in the number of cells responding, a two fold increase in the time to reach half-maximal response, a 35% reduction of the peak [Ca²⁺]_i in response to agonist and an elevation of resting [Ca²⁺]_i from 99± 14 nM (mean ± S.E.M.) to 155 ± 27 nM. Acute application (20 min) of G418 to transfected cells differentiated without G418 also reduced the percentage of cells responding to carbachol. This effect was less pronounced in non-transfected parent cells. Thus, the mechanism might involve a metabolite of G418 produced in cells expressing NPT. These results indicate that G418 attenuates Ca²⁺ release coupled to muscarinic receptors.     

ataxin-3相互作用蛋白的筛选及鉴定

目的 筛选ataxin-3的相互作用蛋白并进行互作结构域分析，探讨ataxin-3的功能和脊髓小脑型共济失调Ⅲ型／马查多-约瑟夫病的发病机理。方法应用酵母双杂交系统3，从成人脑eDNA文库中筛选和鉴定突变型ataxin-3的互作蛋白，构建ataxin-3蛋白羧基端Bait质粒，进行互作结构域分析。应用激光共聚焦显微镜观察ataxin-3与所筛到的互作蛋白在哺乳动物细胞中的共定位情况。结果分离获得5个新的ataxin-3互作蛋白，视紫红质-二磷酸鸟苷解离抑制因子α、苏素-1、氨氯吡嗪脒敏感性神经元阳离子通道2和2个未知新序列。结构域分析显示除1个未知蛋白与ataxin-3蛋白羧基端互作外，其余4个均与氨基端互作。在SH-SY5Y细胞的细胞核内，野生型ataxin-3与苏素-1共定位，突变型ataxin-3所形成的核内蛋白聚合体也与苏素-1共定位。结论 发现1个未知蛋白可能与ataxin-3蛋白羧基端互作，苏素1可能与ataxin-3蛋白氨基端互作，苏素化可能参与了ataxin-3蛋白的翻译后修饰和脊髓小脑型共济失调Ⅲ型/马查多-约瑟夫病的发病过程。     

重组双功能域补体受体Ⅰ型分子在Vero细胞中的稳定表达及其抑制补体活化的初步研究

构建包含人重组双功能域人补体受体Ⅰ与绿色荧光蛋白(GFP)的重组质粒,观察融合蛋白在非洲绿猴肾细胞(Vero)内表达并检测其抑制补体活化的能力。PCR方法扩增出重组双功能域CR1分子,限制性内切酶XhoⅠ和SalⅠ将重组分子连入真核表达载体pEGFP-N2中,构建出重组质粒pEGFP-N2/CR1-2D,脂质体转染Vero细胞中。新霉素G418筛选出稳定表达细胞克隆,荧光显微镜下观察绿色荧光融合蛋白在细胞内的表达。用Vero细胞和免疫小鼠获得的抗Vero细胞多克隆抗体激活补体后,通过检测乳酸脱氢酶的释放来分析重组蛋白抑制补体活化的功能。结果显示pEGFP-N2/CR1-2D质粒经酶切及测序分析证实载体构建正确。转染细胞后,荧光显微镜下观察到重组质粒pEGFP-N2/CR1-2D在Vero细胞中能够大量表达,G418筛选出了稳定表达细胞克隆,乳酸脱氢酶活性检测显示,与对照组相比CR1-2D能够显著的抑制补体的活化(P0.05),初步证实了其能够抑制补体的活化。