双功能域小分子补体受体1型衍生物的构建、表达、纯化及生物功能鉴定

目的 克隆表达出能够抑制补体活化的双功能域小分子补体受体1型(complementreceptor type 1,CR1)衍生物.方法 从外周血提取总RNA,进行RT-PCR扩增出功能性片段Ⅰ(CR1-SCR1-3);以本室构建的pET-32a-CR1-SCR15-18为模板扩增功能性片段Ⅱ(CR1-SCR15-17);利用重叠延伸PCR(SOE-PCR)法扩增出包含双功能域的融合基因.将融合基因重组于pET-32a(+)载体,转化大肠杆菌Rosetta,经酶切及DNA序列测定鉴定后,IPTG诱导表达,SDS-PAGE和Western blot对表达蛋白质进行分析鉴定.蛋白质经Ni柱亲和层析及透析复性后,采用50%补体溶血抑制试验(CH50)进行功能鉴定.结果 酶切及DNA序列测定证实成功构建出了pET-32a-CR1-SCR(1-3+15-17)重组表达载体,IPTG诱导表达后在相对分子质量(Mr)为63×103处有一明显的条带,经Westernblot鉴定为目的 蛋白,Ni柱亲和层析获得纯度约为92%的目的 蛋白,功能试验证实,在0～200 μg/ml的融合蛋白剂量范围内,随着浓度的增加,补体抑制活性增强.结论 成功构建并在大肠杆菌内表达了具有较高生物活性的重组双功能域小分子CR1衍生物,为体内保护实验研究提供实验数据.     

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。     

重组人补体受体1型SCR15-18片段对肠缺血再灌注损伤的保护作用

目的: 探讨人补体受体1型功能域SCR15-18 (CR1-SCR15-18)对大鼠肠缺血再灌注损伤的保护作用。方法: SD大鼠随机分为假手术组(SO)、缺血再灌注组(I/R)和CR1-SCR15-18保护组(CR1)。结扎肠系膜上动脉30 min,再灌注60 min,建立小肠缺血再灌注模型,再灌注前5 min注射CR1-SCR15-18蛋白(30 mg/kg)。测定肠黏膜血管的通透性、组织髓过氧化物酶(MPO)、超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;HE染色观察小肠病理改变并根据Chiu's方法评分;免疫组织化学法检测补体C3组分的沉积。结果: 与SO组相比,I/R组通透性增加,MPO活性和MDA含量显著升高,而SOD活性降低,肠黏膜病理损伤严重且有大量补体C3组分沉积。与I/R组相比,CR1组肠黏膜血管通透性显著降低,MPO活性和MDA含量显著降低,而SOD活性升高,肠黏膜损伤明显减轻,只有少量补体C3组分沉积。结论: CR1-SCR15-18蛋白抑制肠缺血再灌注时补体的活化,减轻肠组织损伤。     

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。     

人CR1-SCR1-3对补体介导的脓毒症小鼠炎症损伤的保护作用

目的观察人补体受体1型功能域SCR1-3(CR1-SCR1-3)对补体介导的脓毒症小鼠炎症损伤的保护作用。方法昆明小鼠150只,随机分为对照组(50只),脓毒症组(50只),CR1-SCR1-3保护组(50只)。采用内毒素腹腔注射致脓毒症模型,各组分别于实验前1 h腹腔注射D-氨基半乳糖600 mg/kg增敏。对照组腹腔注射等体积PBS;脓毒症腔注射LPS(50μg/kg)+等体积PBS;CR1-SCR1-3保护组注射LPS(50μg/kg)+CR1-SCR1-3(15 mg/kg)。各组留10只小鼠,观察实验后72 h生存率。其他小鼠在实验后8 h测定血清IL-1β含量,12 h后取肺组织标本检测髓过氧化物酶(myeloperoxidase,MPO)水平、免疫组织化学检测C4b沉积及观察肺病理学改变。结果脓毒症组的脓毒症反应最强烈,16 h后全部死亡,保护组的脓毒症症状减轻,16 h后生存率达40%,显著提高(P<0.05)。脓毒症组的血清促炎症介质IL-1β和肺组织MPO水平均明显升高,保护组的显著降低(P<0.001)。脓毒症组的肺组织原位C4b沉积明显增多,保护组的明显减少。病理学检查显示,保护组小鼠肺损伤较脓毒症组的明显减轻。结论补体在脓毒症的发生和发展中起重要作用,人CR1-SCR1-3对脓毒症小鼠炎症损伤具有一定的保护作用。     

人TALL-1基因毕赤酵母表达载体的构建与鉴定

目的克隆人TALL-1基因全长及其胞外区片段，构建人TALL-1及其胞外区蛋白毕赤酵母分泌型表达载体。方法从人新鲜淋巴结组织中提取总RNA，利用RT-PCR技术扩增人TALL-1及其胞外区基因编码区序列，构建人TALL-1及其胞外区cDNA毕赤酵母表达载体，并进行序列测定。结果克隆获得的858bp和459bp片段与文献报道的人TALL-1全长及其胞外区基因编码区cDNA序列一致，将目的基因插入酵母表达载体pPiC-9Ka因子分泌信号肽下游。结论本实验为大量获得人TALL-1蛋白及其可溶性功能蛋白，以及探讨其生物学性能和未来在临床上的应用奠定了实验基础。     

人分泌型白细胞介素-16基因的克隆及其原核表达

目的:克隆人分泌型IL-6 cDNA,构建IL-16的原核表达质粒,并观察其在大肠杆菌中的表达.方法:采用PCR技术从健康成人外周血单个核细胞(PBMC)总cDNA文库中扩增人分泌型IL-16 DNA片段;PCR产物分离纯化后,将其克隆至pUC18-T载体中,经PCR、酶切鉴定及DNA测序确证后,将该基因定向插入原核表达载体pMAL-C2,获得重组质粒pMAL-IL-16,然后将其转化至E.coli DH5α,经IPTG诱导表达,采用SDS-PAGE及Western blot法鉴定表达产物.结果:获得了编码人分泌型IL-16的cDNA,经测序证明其长度为393 bp,编码130个氨基酸;构建了IL-16原核表达载体,并应用E.coli DH5α表达,得到1个相对分子质量(Mr)约56000的IL-16重组融合蛋白,与理论大小相符,且经SDS-PAGE及Western blot法验证.表明人分泌型IL-16原核表达载体构建并表达成功.结论:本实验成功克隆了人分泌型IL-16 cDNA,构建了IL-16原核表达载体,并在大肠杆菌中获得表达,为IL-16的纯化奠定了基础.     

人补体受体2-Fc融合蛋白真核表达载体的构建与表达

目的构建人补体受体2(CR2)-Fc融合蛋白基因表达载体,在中国仓鼠卵巢细胞(CHO)中表达人CR2-Fc蛋白。方法将人CR2片段和IgG1 Fc片段进行连接,克隆入PCI-neo表达载体。脂质体法将测序正确的重组真核表达载体转染CHO K1细胞进行蛋白表达,采用SDS-PAGE、Western blot法对蛋白表达进行检测和鉴定。结果利用双酶切及基因测序结果证实CR2-Fc基因序列正确,重组真核表达载体构建成功;SDS-PAGE检测纯化产物的相对分子质量(M r)与预期结果一致,Western blot法在同样位置检出条带。结论成功构建了CR2-Fc重组真核表达载体,获得了有活性的融合蛋白。     

人防御素-5基因的克隆和真核表达载体的构建

目的对人防御素5(HD-5)基因进行克隆,构建真核表达载体HD5-pPICZαA。方法从人cDNA文库中PCR扩增HD-5基因片段,用EcoRⅠ和XbaⅠ分别双酶切HD-5基因扩增产物和毕赤酵母表达载体pPICZαA,用T4DNA连接酶连接目的基因片段和pPICZαA,然后转化到E.coli Top10中,Zeocin筛选转化子并进行PCR、酶切和序列鉴定。结果提取阳性克隆的重组质粒,EcoR Ⅰ和Xba Ⅰ双酶切后跑电泳获得预期条带;同时重组质粒经序列测定,证实HD-5基因正确插入pPICZαA中,插入位置、方向均正确。结论成功构建人防御素5基因的真核表达载体HD5-pPICZαA,为HD-5蛋白的真核真核表达奠定基础。     

人sCR1转化克隆菌的快速筛选和鉴定

目的 用G418快速筛选及鉴定重组人可溶性补体受体1型（sCR1）高拷贝转化克隆菌。方法 采用的sCR1重组质粒，经电转化将其克隆入SMD1168细胞中，利用G418的抗性，快速筛选转化克隆菌，并对克隆菌株提取基因组DNA进行聚合酶链反应（PCR）鉴定；该克隆菌经甲醇诱导培养后，对其表达产物进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）及Western blot鉴定分析。结果 从G418快速筛选的酵母克隆菌株中提取的基因组DNA进行PCR鉴定，获得了高拷贝整合的重组酵母克隆菌株，经诱导培养后，对其表达产物进行SDS-PAGE及Western blot鉴定分析，该表达蛋白在SDS-PAGE上表现为相对分子质量大于30 000的蛋白区带，在Western blot分析中可被sCR1的CD35单克隆抗体（mAb）识别，成功获得高拷贝整合的重组酵母细胞株，可表达出重组人sCR1融合蛋白。结论 经高浓度G418药物快速筛选的高拷贝SMD1168细胞株，用于表达人sCR1融合蛋白。     

人血管内皮生长因子受体Flt-1胞外区2-3loop在Pichia.pastoris酵母中的表达、纯化及其生物学活性研究

目的研究人Flt-1胞外区2-3loopcDNA在Pichia.pastoris酵母中的表达,获得高效表达的具有生物学活性的重组Flt-1(2-3)。方法采用PCR扩增Flt-1(2-3)基因,经DNA序列分析后,插入含AOX1启动子和α分泌信号肽序列的Pichia.pastoris酵母表达载体中,构建重组质粒pPIC9K/Flt-1(2-3),转化酵母宿主菌GS115,筛选His     

A soluble deletion mutant of the human complement receptor type 1, which lacks the C4b binding site,is a selective inhibitor of the alternative complement pathway

The human complement receptor type 1 (CR1, CD35), is a single-chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. The SCR themselves, considered in groups of seven, form long homologous repeats (LHR) which have been designated LHR-A, -B, -C, and -D for the most common human allotype of CR1. A soluble deletion mutant of CR1 which lacks the first seven N-terminal SCR (LHR-A) as well as the transmembrane and cytoplasmic domains was produced and characterized. The resulting protein, designated sCR1[desLHR-A], lacks the C4b binding site found in LHR-A, but retains the two C3b binding sites found in LHR-B and -C, respectively. The functional activities of sCR1[desLHR-A] were quantitatively compared in vitro to those of soluble complement receptor type 1 (sCR1) which has been shown to retain all known functions of the native cell surface receptor. sCR1[desLHR-A] and sCR1 competed equally for the binding of dimeric C3b to erythrocyte CR1. sCR1[desLHR-A] and sCR1 were similar in their capacity to serve as a cofactor in the factor I-mediated degradation of the C3b and C4b α chains. sCR1[desLHR-A] and sCR1 were comparable in their capacity to inhibit erythrocyte lysis and anaphylatoxin production mediated by the alternative complement pathway. sCR1[desLHR-A], however, was significantly less effective an inhibitor of erythrocyte lysis and anaphylatoxin production than sCR1 under conditions which allow classical pathway activation. These results demonstrate sCR1[desLHR-A] to be a selective inhibitor of the alternative complement pathway in vitro.     

人血管内皮生长因子受体胞外1-3、2-3 loopcDNA在酵母中的表达及其生物学活性研究

目的 在酵姆 中实现人血管内皮生长因子（hVEGF165)受体FLT-1胞外1-3及2-3loopcDNA的高效分泌性表达,并比较两种表达产物的生物学活性。方法 构建酵母重组表达质粒pPIC9K/FLT-1(1-3)及pPIC9K/FLT-1(2-3),并分别转化酵母宿主菌CS115,表达产物经CM-SepharoseFF阳离子交换层析和SephacrylS-100分子筛层析纯化后测定其生物学活性。结果 SDS-PACE显示,表达产物以可溶性分子形式存在于上清中,诱导4d的表达量占上清总蛋白的60%以上。表达的sFLT-1（1-3）及sFLT-1(2-3)都具有结合hVEGF165的能力和抑制hVEGF165对人脐静脉内皮细胞（HUVC）的促增殖功能,其中前者的作用虽较强,但二者相差不大。结论 获得了具有生物学活性的可溶性重组TLT-1受体胞外区1-3及2-3loop小片段,为进一步的动物实验和临床应用提供有益的资料。     

The structure,genetic polymorphisms,expression and biological functions of complement receptor type 1 (CR1/CD35)

The complement system is comprised of soluble and cell surface associated proteins that recognize exogenous, altered, or potentially harmful endogenous ligands. In recent years, the complement system—particularly component C3 and its receptors—have been demonstrated to be a key link between innate and adaptive immunity. Complement receptor type 1 (CR1), the receptor for C3b/C4bcomplement peptides, has emerged as a molecule of immense interest in gaining insight to the susceptibility, pathophysiology, diagnosis, prognosis and therapy of such diseases. In this review, we wish to briefly bring forth the structure, genetic polymorphisms, expression and biological functions of CR1.     

重组人白细胞介素10在毕赤酵母中的表达及生物学活性测定

目的在毕赤酵母中分泌表达具有生物学活性的重组人白细胞介素10(rhIL-10),用于IL-10的生理功能研究和动物试验。方法通过PCR扩增IL-10基因,插入到重组质粒α/pUC18的α因子信号序列的XhoⅠ和EcoRⅠ酶切位点处,再将融合基因αIL-10重组到表达质粒pPIC9K的BamHⅠ和EcoRⅠ之间,SalⅠ酶切线性化重组质粒IL-10/pPIC9K,电穿孔转化毕赤酵母,营养缺陷型培养基MD和G418筛选含高拷贝表达盒的酵母转化子,PCR鉴定是否含有目的基因IL-10,甲醇诱导rhIL-10的表达,SDS-PAGE和Westernblot鉴定目的蛋白,用ELISA定量测定及MC/9细胞分析IL-10生物学活性。结果成功构建了重组表达质粒IL-10/pPIC9K,获得了4个含IL-10基因的高拷贝毕赤酵母转化子,甲醇诱导后,在摇瓶水平的IL-10表达量为(0.627±0.09)mg/L,比活性为1.5×105U/mg。结论毕赤酵母分泌表达的IL-10具有良好的生物学活性。     

人白介素1受体拮抗剂的cDNA克隆和原核表达

从活化的正常人外周血单核细胞中提取总ＲＮＡ，经逆转录多聚酶链反应（ＲＴ－ＰＣＲ）获得了人白介素１受体拮抗剂（ＩＬ－１Ｒａ）ｃＤＮＡ。经过ＤＮＡ测序分析，发现该片段和国外发表的ＩＬ－１ＲａｃＤＮＡ序列一致，将该目的基因插入ｐＢＶ２２０载体，并转入大肠杆菌ＨＢ１０１中，经热诱导表达重组蛋白，ＳＤＳ－ＰＡＧＥ后发现，菌体超声裂解后，在可溶性上清中有一Ｍ１７０００的特异带，约占菌体可溶性总蛋白的８０％以上     

Increased expression of complement receptor type 1 (CR1, CD35) on human peripheral blood T lymphocytes after polyclonal activation in vitro

The receptor for C3b and C4b—complement receptor type 1 (CR1, CD35)—is present on a variety of cell types including erythrocytes, phagocytic cells, B lymphocytes and a small sub-population of T lymphocytes. The function of the receptor varies according to the different cell types, but a T lymphocytes the function is as yet not known. The present study concerns the influence of polyclonal stimulation on CR1-expressing T lymphocytes. Incubation with PHA resulted in a dose-dependent increase in the number of CR1-positive T lymphocytes. The CR1-expression T lymphocytes were found in both the CD4- and the CD8-positive subpopulation, but a significant stimulatory increase was only found in the CD4-positive population. A significant increase in the number of CR1-expressing T lymphocytes was found when monocytes were present during stimulation, indicating an importance of monocytes and/or monocyte products. However, the increase was not regulated by arachidonic acid metabolites of the cyclo-oxygenase pathway as indomethacin failed to inhibit the increase. Neither did rIL-Iα, rIL-1β, rTNFα nor rIL-6 alter the number of CR1-expressing T lymphocytes. The results of this study indicate a role for CR1 on T lymphocytes in the regulation of the immune system.