单克隆非特异性抑制因子-β(MNSFβ)在大肠杆菌中的表达、抗体的制备及在小鼠子宫内膜上的组织定位

为了获得足够量的单克隆非特异性抑制因子-β蛋白(monoclonal nonspecific supressor factor β,MNSFβ)及其抗体用于探讨MNSFβ在着床中的作用机理,本研究构建了表达质粒pBV220/MNSFβ-hCGβ,在大肠杆菌中表达了融合蛋白MNSFβ-hCGβ,用抗hCGβ抗体对表达产物进行鉴定,结果表明融合蛋白MNSFβ-hCGβ得到了正确表达,且分子量和理论值相近。表达产物MNSFβ-hCGβ经初步纯化后用于免疫Balb/C小鼠,制备抗体。同时,我们还构建了表达质粒pGEX-4T-2/MNSFβ,在大肠杆菌中表达了融合蛋白GST-MNSFβ。用融合蛋白GST-MNSFβ对融合前的免疫小鼠回忆刺激并进行检测,制备了抗MNSFβ多克隆抗体和单克隆抗体。应用所制备的多克隆抗体进行免疫组化研究,对MNSFβ在小鼠子宫内膜上进行了组织定位,结果显示,MNSFβ在着床日(受精后第4.5天)小鼠子宫非着床位点的表达和着床位点相比明显提高。     

肠三叶因子在大肠杆菌中的表达、纯化及其抗体制备

目的:获得肠三叶因子(ITF)的原核表达产物及抗rITF抗体,为深入研究ITF的作用机制及其受体研究奠定基础。方法:常规提取人小肠组织总RNA,用RT-PCR获得ITF编码基因片段,克隆至质粒pET32a获得原核表达栽体,双酶切和测序后转化至Origami B(DE3)用IPTG诱导表达,优化条件获得最大表达产量;用SDS-PAGE、Western blot鉴定表达产物,亲和层析纯化获得的重组蛋白rITF皮下多点注射家兔,制备多克隆抗体,并用此抗体进行大鼠肠组织免疫组化研究。结果:测序证实PCR扩增获得ITF全长基因序列与基因文库中的完全一致,将该基因片段正确插入表达载体pET32a中、优化表达条件后,重组蛋白的表达量达到50mg/L;Western blot证明重组蛋白具有良好的抗原性和特异性;通过Ni-NTA亲和层析、超滤离心后,得到90%纯度的蛋白;收集兔血清,纯化后获得特异性良好的ITF抗体,免疫组化染色肠组织显示ITF表达的部位定位于杯状细胞。结论:成功构建了表达载体pET32a-ITF,在大肠杆菌中表达并纯化获得纯度较高的rITF,并获得了生物活性较高的ITF抗体,ITF主要在肠道杯状细胞分泌表达。     

果蝇spen蛋白的抗体制备、组织特异性表达及功能分析

Spen家族蛋白参与多种生物学过程,包括神经元细胞的命运、神经元突起延伸的调节、细胞周期调控等,并且是联系Notch信号途径和生长因子受体途径的关键分子。最近的研究表明spen基因在果蝇的眼睛、翅膀和腿组织中参与Wnt信号转导。但该基因在果蝇中的功能还有很多不明确之处。文章采用基因克隆、原核表达及亲和层析等方法制备并纯化了黑腹果蝇spen的C端6×His-spen融合蛋白,以纯化的融合蛋白免疫大鼠获得了抗spen的多克隆抗体。利用制备的抗体进行免疫染色结果显示spen蛋白定位于细胞核内,并且在大脑、脂肪体、血细胞、肠和唾液腺等组织中表达量较高。分析野生型和突变体果蝇血细胞的噬菌作用,发现spen蛋白低表达的突变体吞噬外来异物明显低于野生型,结果表明spen蛋白能够调节血细胞的吞噬功能。     

原位杂交检测组织金属蛋白酶抑制因子—1 mRNA在IgA肾病肾脏中的表达

为探讨组织蛋白酶抑制因子在IgA肾病肾小球硬化中的作用。本文用原位杂交法研究组织金属蛋白酶抑制因子－1（TIMP－1）mRNA在3种类型IgA肾病（轻度系膜增生型、中度系膜增生型及局灶型）肾组织中的表达。结果表明：TIMP－1 mRNA原位杂匀的阳性信号主要分布于部分肾小管上皮细胞内。在肾小球系膜细胞也可检测到TIMP－1 mRNA阳性信号,但较肾小管弱,中度系膜增生型的TIMP－1 mRNA阳笥信号明显地较其他两型IgA肾病为强。以上结果表明：由肾小管上皮细胞及系膜细胞合成的TIMP－1通过抑制基质金属酶的活性,可导致细胞外基质（ECM）在肾小管间质及肾小球内过度沉积,从而对肾小球硬化有促进作用。     

破骨细胞形成抑制因子TNFR结构区在大肠杆菌中表达、抗体制备及活性测定

破骨细胞形成抑制因子(OPG)对骨的重建与再吸收有重要调节作用,其TNFR结构区行使抑制破骨细胞形成与活性的功能。通过PCR将该区基因片段克隆出来,插入表达载体PET-28a质粒多克隆位点,重组质粒转入大肠杆菌BL21中进行表达,表达产物以包涵体形式存在,包涵体经变性复性后,亲合层析获得重组蛋白。纯化的产物作为抗原免疫兔,得到较高特异性的兔源多克隆抗体。利用小鼠降血钙实验检测变性复性后产物的活性,结果表明该重组蛋白有一定的生物活性。     

基质金属蛋白酶-2、-9及其组织抑制因子（TIMPs）在动情周期大鼠子宫中的表达

基质金属蛋白酶（MMPs)家族的作用是降解所有细胞外基质,其活性受其特异性组织抑制因子（TIMPs)的抑制。细胞外基质成分的降解与重组在动物生殖生长过程中起重要作用,其变化可以通过MMPs和TIMPs两者表达水平的变化进行监测。大鼠虽然没有月经形成,但是在其子宫内膜也出现类似灵长类的生殖生物学变化。本文从MMPs和TIMPs两者的表达水平,对大鼠子宫内膜的这些变化进行了研究。于大鼠动情周期的不同时期,将算处死,取子宫制备酶粗提液和组织切片,采用酶谱法(zymography)和原位杂交方法研究动情周期大鼠子宫中MMP-2和-9的活性变化以反MMP-2、-9和TIMP-1、-2、-3mRNA的表达。并通过光密度扫描方法对酶谱结果进行半定量分析。所用杂交探针见Table 1．酶谱结果显示：在动情周期大鼠子宫中只检测到67kDa的MMP-2活性,而没有检测到MMP-9的活性(Fig.1)。MMP-2的活性在动情前期最高,动情期和动情后期次之,同情期最低(Flg．2)。原位杂交结果显示：MMP-2、-9、TIMP-1、-2、-3m RNA主要在子宫内膜基底部的基质细胞中表达。MMP-2和-9 mRNA在动情前期、动情期和动情后期子宫内膜基底部的基质细胞和动情期子宫环行肌层中表达均较强,在间情期表达较弱。MMP-9 mRNA在动情期腔上皮和腺上皮细胞中也有较弱的表达(Fig．3)。TIMP-1 mRNA在动情期和动情后期子宫内膜基底部的基质细胞中以及动情期腔上皮和腺上皮细胞中表选较强,在动情前期和间情期表达较弱(Fig．4)。TIMP-2 mRNA在动情期和间情期子宫内膜基底部的基质细胞中以度在动情期上皮细胞中表达较强,在动情前期和动情后期表逮较弱(Fig.4)。TINP-3 mRNA在动情期子宫内膜基底部的基质细胞中表达最强,在动情后期和间情期次之,动情前期表达最弱。TIMP-3 mRNA在动情期子宫环行肌层中也有较强的表达(Fig.5)。由此可见,MMP-2的活性和MMIP-2的基固表达基本一致,MMP-2和-9及其组织抑制因子TIMPs在时间和空间上的表达也基本一致。提示：MMP-2,-9及TIMP-1、-2、-3共同参与了动情周期大鼠子宫内膜的周期性变化。     

银鲫HIRA多克隆抗体的制备及组织特异性表达分析

Hir/Hira基因家族的成员广泛存在于多种生物体中,但有关其在生物体发育过程中的具体功能还不甚清楚.对果蝇的研究表明,dHira基因产物可能在受精时精核的解凝过程和雄性原核的正常形成过程中起重要作用.本研究组前期已经分别克隆出雌核发育银鲫和两性生殖彩鲫的Hira基因(cagHira 和caHira),本实验在银鲫cagHira基因的特异区域,设计一对引物,以银鲫成熟卵母细胞总RNA逆转录出的cDNA为模板,扩增出cagHira的特异片段.再将该片段克隆到原核表达载体pET-32a上,转化BL21(DE3)菌株,经诱导后表达出融合蛋白.分析表明,该融合蛋白主要以包涵体形式表达.以纯化的融合蛋白作为抗原去免疫小鼠,制备多克隆抗血清,经蛋白质印迹分析检测,该抗血清(稀释到1:2000)与包涵体蛋白识别反应良好,确定获得了具有高效价的特异性银鲫CAGHIRA多克隆抗体,为进一步研究HIRA在鱼类发育和雌核生殖过程中的作用奠定了基础.对银鲫HIRA蛋白的组织特异性表达分析发现,该蛋白仅在成熟卵巢组织中特异表达,故表明HIRA可能对鱼类卵子发生和/或早期胚胎发育具有重要作用.     

GST-Ccd1融合蛋白的表达、纯化及多克隆抗体制备

目的：利用大肠杆菌DH5α表达GST—Ccd1融合蛋白,并用亲和层析分离纯化,进行动物免疫制备多克隆抗体。方法：利用本室构建好的pGEX-5X-1-Ccd1-N原核表达重组质粒,转化大肠杆菌DH5α,经IPTG诱导表达,在大肠杆菌表达系统中获得可溶性表达。经谷胱甘肽Sepharose 4B介质填充的层析柱分离纯化蛋白,制备抗原免疫动物,得到Ccd1的兔源多克隆抗体。结果：ELISA结果显示血清抗体效价可以达到1∶40 000。免疫组化分析表明自制的抗体能特异性与Ccd1蛋白相互作用,可以用于实验分析。结论：制备了效价高特异性良好的抗Ccd1多克隆抗体,经实验验证获得的抗体能够满足针对Ccd1的免疫印迹和免疫组化检测的实验要求,为今后深入研究Ccd1表达的组织分布、细胞内定位及其生物学功能提供了有用的实验工具。     

Immunoneutralization of endometrial monoclonal nonspecific suppressor factor beta (MNSFbeta) inhibits mouse embryo implantation in vivo

Successful embryo implantation and pregnancy in mammals depends on the establishment of immune tolerance between the maternal immune system and fetal cells. Monoclonal nonspecific suppressor factor beta (MNSFbeta), a cytokine produced by suppressor T cells in various tissues, possesses an antigen-nonspecific immune-suppressive function, and may be involved in the regulation of the uterine immune response during embryo implantation. In this study, anti-MNSFbeta IgG administered directly into the uterine lumen, significantly inhibited mouse embryo implantation in a dose-dependent manner in vivo, and this effect was reversed by co-administration of recombinant MNSFbeta. The effects of anti-MNSFbeta IgG on the gene pattern profiles in mouse uterine tissues were examined by cDNA microarray and several changes were confirmed by real-time PCR. Anti-MNSFbeta IgG caused up-regulation (> or = 2-fold) of 71 known genes and 17 unknown genes, and decreased expression (> or = 2-fold) of 74 known genes and 43 unknown genes, including several genes previously associated with embryo implantation or fetal development. Most of the known genes are involved in immune regulation, cell cycle/proliferation, cell differentiation/apoptosis, and lipid/glucose metabolism. These results demonstrate that MNSFbeta plays critical roles during the early pregnancy via multiple pathways.     

Overproduction, preparation of monoclonal antibodies and purification of E. coli asparagine synthetase A.

In order to explore the structure--function relationship of the Escherichia coli asparagine synthetase A it was necessary to devise a system for overexpression of the gene and purification of the gene product. The E. coli asparagine synthetase A structural gene was fused to the 3' end of the human carbonic anhydrase II structural gene and overexpressed in E. coli. The gene product, a 66 kDa fusion protein, which exhibited asparagine synthetase activity, was purified in a single step by affinity chromatography and used as the antigen for the production of monoclonal antibodies. The monoclonal antibodies were screened by ELISA. Colonies were chosen which were positive for purified fusion protein and negative for purified human carbonic anhydrase II. The E. coli asparagine synthetase A gene was then overexpressed and the gene product was used without purification for the final screen. The antibodies selected were used for immunoaffinity chromatography to purify the recombinant overexpressed E. coli asparagine synthetase A. Thus, a procedure is now available so that asparagine synthetase A can be purified to homogeneity in a single step.     

Characterization of N-terminal amino acid sequence of monoclonal nonspecific suppressor factor.

Monoclonal nonspecific suppressor factor (MNSF), a product of a murine T cell hybridoma, suppresses the antibody response to lipopolysaccharide. In an attempt to clarify the N-terminal sequence, MNSF was prepared and purified by affinity chromatography with the use of an anti-MNSF monoclonal antibody (MO6), and reverse-phase high-pressure liquid chromatography. On the SDS-PAGE, the purified MNSF showed a single band with a molecular weight of 12,000. The N-terminal amino acid sequence of the protein was determined and showed no strong homology to any of the sequences of known biologically active proteins. However, the sequence revealed significant (60%) amino acid identity to transforming growth factor beta 2 (TGF beta 2).     

IFN-gamma enhances the expression of cell surface receptors for monoclonal nonspecific suppressor factor.

Monoclonal nonspecific suppressor factor (MNSF) is a lymphokine derived from a murine T cell hybridoma. The action of MNSF is mediated by specific cell-surface receptors. Since IFN-gamma alters the cellular response to MNSF (M. Nakamura, H. Ogawa, and T. Tsunematsu, J. Immunol. 138, 1799, 1987), we investigated whether IFN-gamma has an effect on the expression of MNSF receptor on target cells. IFN-gamma enhanced the expression of MNSF receptor on both MOPC-31C cells (a murine plasmacytoma line) and EL4 (a murine T lymphoma line). Incubation with IFN-gamma increased the number of specific MNSF-binding sites by about 50 to 90%, with no significant change in binding affinity. IFN-alpha and IFN-beta also increased MNSF binding, although the effect of the saturating amounts was lower than that seen with IFN-gamma. Maximal enhancement of receptor expression was observed after about 15 hr of incubation with IFN-gamma. No demonstrable change occurred in the kinetics of internalization of 125I-MNSF bound to MOPC-31C cells preincubated without or with IFN-gamma.     

重组截短型人源结缔组织生长因子在大肠杆菌中的表达

用RT－PCR法从人胎盘组织中克隆出人源结缔组织生长因子（h-CTGF)cDNA序列867bp,将此cDNA亚克隆至表达载体pET-9a,重组质粒转化BL21（DE3）pLysS,诱导出N端缺失61个氨基酸残基的截短型rh－CTGF,表达量占总菌体蛋白的7％,主要以不溶性包涵体形式存在,采用离心、洗涤和凝胶过滤分离纯化后,行活性检测表明截短型rt-CTGF无刺激增殖活性,并对用原核表达rt-CTGF作了讨论,为制备抗体、CTGF表达调控和功能研究打下了基础。     

Characterization of monoclonal nonspecific suppressor factor (MNSF) with the use of a monoclonal antibody

The secretion of immunoglobulin (Ig) from cultured mononuclear cells by lipopolysaccharide (LPS) stimulation is inhibited by monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by murine T cell hybridoma. In an attempt to develop a murine monoclonal antibody (MAb) with specific reactivity against MNSF, a cell fusion technique that incorporated immune murine splenocytes and HAT-sensitive murine myeloma cells was used. Cross-reactivity experiments confirmed that the MAb (MO6) does not bind to unrelated proteins such as bovine serum albumin, mouse IgG, and murine interferon-gamma (IFN-gamma). There are no effects when anti-IFN-gamma antibodies are used with MNSF. As far as biological activity is concerned, MO6 inhibits in vitro the activity of MNSF in terms of the Ig secretion from cultured lymphocytes. By using MO6, affinity chromatography and immunoblotting were performed. The MNSF on the SDS-PAGE showed a band with m.w. of approximately 70,000, indicating the formation of an aggregate in saline; but after treatment with 0.4 M pyridine-acetic acid buffer, separate bands of 24,000 and 16,000 daltons were evident. Therefore MO6 recognizes 70,000 and both 24,000 and 16,000 daltons. Thus we confirmed by using this MAb and affinity chromatography, the existence of human counterpart, human nonspecific suppressor factor (hNSF), in supernatant from concanavalin A-stimulated T cells. When hNSF was fractionated by high pressure liquid chromatography (HPLC), the activity was found in a region corresponding to 70,000 daltons. However, when fractionated in pyridine-acetic acid buffer, hNSF activity was distributed in a slightly wider range of 15,000 to 30,000 daltons. Physicochemical analysis showed that the purified hNSF is resistant to either heating at 56 degrees C or to 2-mercaptoethanol treatment; however, it is labile to acidification at pH 2.0 and is also sensitive to protease treatment, the characteristics of which were similar to those of murine MNSF. Thus MO6 was confirmed to be a pertinent tool for isolation of hNSF, as well as for murine MNSF.     

产单核细胞李斯特菌actA基因在大肠杆菌中的表达及其单克隆抗体的研制

利用PCR技术从血清型1/2a的产单核细胞李斯特菌Lm-4株中扩增出actA基因,经克隆筛选和测序鉴定后,构建成该基因的原核表达载体pGEX-6P-1-actA及pET-actA,转入E·coli后,IPTG诱导目的蛋白的表达。SDS-PAGE结果表明,actA基因在两种载体中均获得表达,融合蛋白的大小分别约为120kDa和97kDa。以纯化蛋白为材料进行了ActA单抗的研制,获得4株抗ActA的单克隆抗体杂交瘤细胞株,腹水单抗ELISA效价为1∶5×104~1∶1×105。选取单抗1A5进行Westernblot分析,结果表明单抗1A5能和表达产物进行特异性反应,且与Lm-4多抗血清的Westernblot结果一致。actA基因的原核表达及单抗的研制为研究ActA蛋白的生物学活性及其致病作用奠定了基础。     

Identification of monoclonal nonspecific suppressor factor beta (mNSFbeta) as one of the genes differentially expressed at implantation sites compared to interimplantation sites in the mouse uterus

Successful implantation requires synchronous development of and active dialogue between the maternal endometrium and the implanting blastocyst. While it is well established that appropriate maternal steroid hormones are essential for endometrial preparation for implantation, the molecular events at the actual site of implantation are still little understood. The aims of our studies were to identify genes explicitly expressed or repressed at the sites of implantation by utilising RNA differential display (DDPCR), and to establish the roles of these genes in the implantation process in a mouse model. Ten bands unique in implantation sites compared to interimplantation sites were identified by DDPCR and subsequently confirmed by Northern blotting. One of these bands contained a cDNA fragment that was highly homologous to mouse monoclonal nonspecific suppressor factor beta (MNSFbeta) or Fau. The full cDNA sequence of this gene, obtained by screening a lambdagt11 cDNA library, was essentially the same as MNSFbeta, except that it had much longer 5' untranslated region. Interestingly, both Northern and immunohistochemical analysis showed that the expression of this gene was much lower in implantation sites compared to interimplantation sites on day 4.5 of pregnancy, when embryos first attach to the uterus and initiate implantation, and on day 5.5, when implantation has advanced. These results suggest a role for MNSF during implantation and early pregnancy, possibly through regulating the proliferation and/or differentiation of uterine stromal cells. It may also be involved in the selective production of TH2-type cytokines in implantation sites to regulate the immune system at the maternal-fetal interface.     

小鼠杂交瘤单克隆抗体快速制备技术研究进展

小鼠杂交瘤单克隆抗体来源稳定、后期易制备、产量高,是免疫学中使用最为普遍的抗体.传统的耗时费力的杂交瘤制备技术无法满足日益增长的市场需求.文中从抗原设计筛选、B细胞富集与筛选、骨髓瘤细胞的改造、融合技术的改进、阳性杂交瘤细胞筛选及单克隆抗体性能快速测定中所涉及的快速制备技术方面进行阐述,以期为系统化的小鼠杂交瘤单克隆抗...     

中性白细胞抑制因子在大肠杆菌中的表达与纯化

利用PCR技术扩增编码钩虫中性白细胞抑制因子(NIF)成熟肽的cDNA,克隆于表达载体pET-21a( )。序列分析表明与献报道一致。经IPTG诱导,在大肠杆菌BL21(DE3)plys中实现高效可溶性表达。SDS—PAGE分析结果表明,外源蛋白(相对分子质量28900)约占全菌蛋白的20％。菌体用溶菌酶处理。上清经Q—Sepharose FF阴离子交换、羟基磷灰石层析、Sephacryl S-100凝胶过滤,得到纯度约95％的重组NIF。活性测定结果表明,大肠杆菌表达的重组NIF能有效地抑制中性白细胞粘附。这些结果为利用大肠杆菌制备重组NIF奠定了基础。