cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen

The Ku antigen is a DNA-associated nuclear protein recognized by sera from patients with autoimmune diseases. It consists of two polypeptides of 86 and 70 kDa. cDNA clones encoding the 86-kDa subunit of the Ku antigen were isolated by probing lambda gt11 recombinant cDNA expression libraries with a monoclonal antibody specific for this protein. The amino acid sequence deduced from the cDNA comprises 732 amino acids and corresponds to a protein with molecular weight of 81.914. Nineteen residues at the NH2 terminus determined by protein sequencing corresponded to the sequence deduced from the cDNA. The predicted amino acid sequence contains a region with repeating leucine residues similar to the "leucine zipper" structure observed in the c-myc, v-myc, and c-fos oncogene products. The largest cDNA hybridized to 2.7- and 3.4-kilobase poly(A)+ mRNAs from HeLa cells. The cDNA clones expressed fusion proteins immunoreactive with the monoclonal antibody and sera from patients with autoimmune diseases.     

Cell-surface streptavidin fusion protein for rapid selection of transfected mammalian cells

We developed a series of eight mammalian cell surface marker fusion genes by using the streptavidin gene from Streptomyces avidinii. These fusion genes are useful and non-growth-toxic selection markers for rapid-harvest transfected mammalian cells. Two streptavidin constructs were used; the longer fragment contains the native bacterial signal sequence, which the shorter fragment lacks. For expression of the streptavidin antigen on the surface of mammalian cells, streptavidin was flanked by a mammalian signal sequence and a transmembrane domain (from mouse H2-K or Kit); some constructs also contained the gene for enhanced green fluorescent protein (EGFP). We transfected a series of plasmids encoding the fusion proteins into HeLa cells and determined that the transfected cells produced the fusion protein on their cell surfaces. To separate transfected cells from nontransfected cells, we incubated cells with a polyclonal antibody against streptavidin, and antibody-bound cells were harvested by the use of paramagnetic beads coupled with the corresponding secondary antibody. We obtained highly pure populations of transfected cells; this result also confirmed the production of the fusion protein on the cell surface. Cell growth assays revealed that none of the transfected fusion genes or their products adversely affected the proliferation of HeLa cells. Our results indicate that the fusion constructs we developed and the immunomagnetic separation protocol we used are valuable tools for various transfection applications. In particular, the constructs containing EGFP are advantageous because transfection efficiency can be assessed without additional treatment of cells.     

Biochemical analysis of a human epithelial surface antigen: differential cell expression and processing

Epithelial surface antigen (ESA) is a glycoprotein with a distribution in vivo that is largely confined to human epithelial cells. Previous studies using a mouse monoclonal antibody (MH99) detecting ESA had shown that the antigen immunoprecipitated from most epithelial cancer cell lines has two chains (38,000 and 32,000 Da) when separated under reducing conditions and only one (38,000 Da) under nonreducing conditions. We now show that the 38-kDa band observed under nonreducing conditions consists of two species, one a 38-kDa single chain protein and the other a disulfide-linked dimer consisting of the 32-kDa chain bonded to a previously unrecognized 6-kDa chain. Pulse-chase studies have shown that ESA is synthesized as a 34-kDa protein which is glycosylated to a 38-kDa glycoprotein containing both high mannose and complex carbohydrate chains. With longer chase periods, a 32-kDa species also appears. Peptide mapping, together with the pulse-chase data, suggests that the 32- and 6-kDa species are formed from the 38-kDa protein, probably by limited proteolysis. Epithelial cell lines differ in their ratios of 38/32-kDa species, some cell lines having only the 38-kDa form. Incubation of radiolabeled extracts of cells having only the 38-kDa protein with unlabeled extracts of the other cell types resulted in progressive conversion of the 38-kDa species to the 32- and 6-kDa forms. Only cell lines expressing both forms of ESA are able to carry out this cleavage of the 38-kDa protein. This is a novel mechanism for generating cell-type related differences in cell surface glycoprotein expression. Finally, sequential immunoprecipitation experiments showed that the antigen detected by Ab MH99 is closely related or identical to that detected by Ab 17-1A, a previously described colon cancer antigen.     

Sperm antigen 6 is the murine homologue of the Chlamydomonas reinhardtii central apparatus protein encoded by the PF16 locus

A cDNA encoding sperm antigen 6 (Spag6), the murine homologue of the Chlamydomonas reinhardtii PF16 protein-a component of the flagella central apparatus-was isolated from a mouse testis cDNA library. The cDNA sequence predicted a 55.3-kDa polypeptide containing 8 contiguous armadillo repeats with 65% amino acid sequence identity and 81% similarity to the Chlamydomonas PF1 protein. An antipeptide antibody generated against a C-terminal sequence recognized a 55-kDa protein in sperm extracts and localized Spag6 to the principal piece of permeabilized mouse sperm tails. When expressed in COS-1 cells, Spag6 colocalized with microtubules. The Spag6 gene was found to be highly expressed in testis and was mapped using the T31 radiation hybrid panel to mouse chromosome 16. Mutations in the Chlamydomonas PF16 gene cause flagellar paralysis. The presence of a highly conserved mammalian PF16 homologue (Spag6) raises the possibility that Spag6 plays an important role in sperm flagellar function.     

Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. Sequence identity with the Fas antigen.

The APO-1 antigen as defined by the mouse monoclonal antibody anti-APO-1 was previously found to be expressed on the cell surface of activated human T and B lymphocytes and a variety of malignant human lymphoid cell lines. Cross-linking of the APO-1 antigen by anti-APO-1 induced programmed cell death, apoptosis, of APO-1 positive cells. To characterize the APO-1 cell surface molecule and to better understand its role in induction of apoptosis, the APO-1 protein was purified to homogeneity from membranes of SKW6.4 B lymphoblastoid cells by solubilization with sodium deoxycholate, affinity chromatography with anti-APO-1 antibody, and reversed phase high performance liquid chromatography. Each purification step was followed by an APO-1-specific solid phase enzyme-linked immunosorbent assay using the monoclonal antibody anti-APO-1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the APO-1 antigen was found to be a membrane glycoprotein of 48-kDa. Endoproteinase-cleaved peptides of the APO-1 protein were subjected to amino acid sequencing, and corresponding oligonucleotides were used to identify a full-length APO-1 cDNA clone from an SKW6.4 cDNA library. The deduced amino acid sequence of APO-1 showed sequence identity with the Fas antigen, a cysteine-rich transmembrane protein of 335 amino acids with significant similarity to the members of the tumor necrosis factor/nerve growth factor receptor superfamily. The APO-1 antigen was expressed upon transfection of APO-1 cDNA into BL60-P7 Burkitt's lymphoma cells and conferred sensitivity towards anti-APO-1-induced apoptosis to the transfectants.     

三重表达肺结核DNA疫苗在细胞水平的检测

目的 检测共表达siRNA、抗原基因与hIL-12的新型肺结核DNA疫苗在人胚肾293细胞中表达。方法 在已构建含有靶向抗调亡基因Mcl-1L的siRNA、结核分枝杆菌抗原基因Ag85B-ESAT6 (PVAE)、hIL-12的新型肺结核DNA疫苗pVAX-siRNA-PVAE-IL-12基础之上,将抗原基因与增强型绿色荧光蛋白（EGFP）基因融合,得到真核表达重组质粒pVAX1-siRNA-PVAE-EGFP-hIL12。并将siRNA单元用靶向EGFP基因的siEGFP代替,得到pVAX1-siEGFP-PVAE-EGFP-hIL12重组表达载体。用重组质粒转染人胚肾细胞293,分别观察融合抗原基因与siEGFP的表达。以ELISA测定培养细胞上清中hIL-12的表达。结果 经酶切鉴定和测序证实肺结核DNA疫苗改造成功。转染细胞中检测到绿色荧光,证实抗原表达。对照组检测到siEGFP的表达。转染48 h后检测出细胞上清中hIL-12的量为1571.63 pg/mL;72 h后检测出细胞上清中hIL-12的量为2392.25pg/mL。结论 已构建的肺结核质粒DNA疫苗能在真核细胞中有效表达siRNA、抗原基因与hIL-12。为进一步研究该DNA疫苗抗肺结核的免疫治疗和基因治疗效果打下基础。     

Production of a species specific monoclonal antibody against a 56-kDa Mycobacterium marinum antigen which is potentially useful in culture identification

A monoclonal antibody (MoAb) designated 1H11 has been produced against a 56-kDa antigen from Mycobacterium marinum. This MoAb has been shown to be species specific by both enzyme-linked immunosorbent assay and Western blot. The relationship of this 56 kDa to other known mycobacterial antigens is currently under investigation. MoAb 1H11 recognised bacilli taken from cultures by both immunofluorescence and immunoperoxidase staining. This MoAb 1H11 may provide a simple method for rapid identification of M. marinum from cultures of clinical isolates.     

人P2X7真核表达载体的构建及稳定转染细胞株的建立

目的：构建人P2X7基因的真核表达载体,并通过转染获得稳定表达P2X7分子的HEK293细胞株。方法：以人脑组织P2X7cDNA为模板扩增出P2X7基因,插入到真核表达载体pEGFP-N1中,构建重组质粒pEGFP-N1/P2X7。用X-fect试剂盒将重组质粒转染HEK293细胞,通过G418辅助荧光筛选建立稳定表达P2X7-EGFP细胞株。经流式细胞仪、Western blot和激光共聚焦显微镜检测,了解人P2X7在HEK293细胞中的表达水平及细胞内定位。结果：重组质粒pEGFP-N1/P2X7构建正确,建立了稳定表达人P2X7的HEK293细胞系。Western blot和流式细胞仪检测证实,P2X7在HEK293细胞系中成功表达,激光共聚焦显微镜检测显示P2X7-EGFP定位在细胞膜上。结论：重组载体pEGFP-N1/P2X7构建成功并建立了稳定表达人P2X7的HEK293细胞系,为进一步研究P2X7离子通道结构和功能奠定基础。     

Functional EGFP-dystrophin fusion proteins for gene therapy vector development

Transfection and transduction studies involving the use of the full-length dystrophin (11 kb) or the truncated mini-gene (6 kb) cDNAs are hampered by the large size of the resulting viral or non-viral expression vectors. This usually results in very low yields of transgene-expressing cells. Moreover, the detection of the few transgene-expressing cells is often tedious and costly. For these reasons, expression vectors containing the enhanced green fluorescent protein (EGFP) fused with the N-termini of mini- and full-length human dystrophin were constructed. These constructs were tested by transfection of Phoenix cells with Effectene, resulting after 48 h in a green fluorescent signal in 20% of cells. Analysis of the cell extracts by immunoblotting with the use of a monoclonal antibody specific to the dystrophin C-terminus confirmed the expression of EGFP-mini- (240 kDa) and EGFP-full-length human dystrophin (450 kDa) fusion proteins. Moreover, following the in vivo electroporation of the plasmids containing the EGFP-mini- and full-length dystrophin in mouse muscles, both fluorescent proteins were observed in cryostat sections in their normal location under the plasma membrane. This indicates that the fusion of EGFP to dystrophin or mini-dystrophin did not interfere with the normal localization of the protein. In conclusion, the fusion of EGFP provides a good tool for the search of the best methods to introduce mini- or full-length dystrophin cDNA in the cells (in vitro) or muscle fibers (in vivo) for the establishment of a treatment by gene therapy of Duchenne muscular dystrophy patients.     

Green fluorescent antibodies: novel in vitro tools

We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.     

Pemphigus vulgaris antibody identifies pemphaxin. A novel keratinocyte annexin-like molecule binding acetylcholine

Because pemphigus vulgaris (PV) IgGs adsorbed on the rDsg3-Ig-His baculoprotein induced blisters in neonatal mice, it was proposed that anti-desmoglein 3 (Dsg 3) autoantibody causes PV. However, we found that rDsg3-Ig-His absorbs autoantibodies to different antigens, including a non-Dsg 3 keratinocyte protein of 130 kDa. This prompted our search for novel targets of PV autoimmunity. The PV IgG eluted from a 75-kDa keratinocyte protein band both stained epidermis in a pemphigus-like pattern and induced acantholysis in keratinocyte monolayers. Screening of a keratinocyte lambdagt11 cDNA library with this antibody identified clones carrying cDNA inserts encoding a novel molecule exhibiting approximately 40% similarity with annexin-2, named pemphaxin (PX). Recombinant PX (rPX-His) was produced in Escherichia coli M15 cells, and, because annexins can act as cholinergic receptors, its conformation was tested in a cholinergic radioligand binding assay. rPX-His specifically bound [(3)H]acetylcholine, suggesting that PX is one of the keratinocyte cholinergic receptors known to be targeted by disease-causing PV antibodies. Preabsorption of PV sera with rPX-His eliminated acantholytic activity, and eluted antibody immunoprecipitated native PX. This antibody alone did not cause skin blisters in vivo, but its addition to the preabsorbed PV IgG fraction restored acantholytic activity, indicating that acantholysis in PV results from synergistic action of antibodies to different keratinocyte self-antigens, including both acetylcholine receptors and desmosomal cadherins.     

A poliovirus minireplicon containing an inactive 2A proteinase is expressed in vaccinia virus-infected cells.

It has been difficult to evaluate the role of individual viral proteins in poliovirus replication because a suitable complementation system has not yet been developed. To approach this problem, we constructed a chimeric human immunodeficiency virus type 2 (HIV-2)-gag-poliovirus minireplicon in which regions of the gag gene of HIV-2 were inserted in the poliovirus genome between nucleotides 1174 and 2470. Transfection of this chimeric RNA into HeLa cells results in the replication of the minireplicon and expression of an HIV-2-gag-P1 fusion protein which can be immunoprecipitated with antibodies to HIV-2-gag. Expression of the HIV-2-gag-P1 fusion protein was dependent on replication of the chimeric RNA genome. Although the chimeric HIV-2-gag-poliovirus RNA genome replicated in poliovirus-infected cells, transfection of the chimeric HIV-2-gag-poliovirus genome into vaccinia virus-infected cells resulted in increased replication as measured by analysis of chimeric RNA. The increase in replication correlated with an increase in the expression of the HIV-2-gag-P1 fusion protein in vaccinia virus-infected cells. To characterize this system, we constructed a mutation in the 2A gene to change a cysteine at amino acid 109 to a serine. Expression of the HIV-2-gag-P1 fusion protein was not detected when the HIV-2-gag-poliovirus genome containing the 2A mutation was transfected into HeLa cells, demonstrating the mutation was lethal for replication. When the chimeric genome was transfected into poliovirus-infected cells, no RNA replication or expression of the HIV-2-gag-P1 fusion protein was observed. In contrast, transfection of this genome into vaccinia virus-infected cells resulted in replication of the chimeric RNA and expression of two proteins with larger molecular masses than the HIV-2-gag-P1 proteins, possibly representing HIV-2-gag-P1-2A and HIV-2-gag-P1-2ABC fusion proteins. The transfection of the chimeric HIV-2-gag-poliovirus genome containing the 2A mutation into poliovirus-vaccinia virus coinfected cells resulted in the expression and partial processing of the two larger HIV-2-gag-P1 fusion proteins to give the correct molecular mass for the HIV-2-gag-P1 fusion protein. The 2A mutation was reconstructed back into the full-length infectious cDNA of poliovirus. Transfection of this cDNA into vaccinia virus-infected cells followed by immunoprecipitation with anticapsid antibodies demonstrated the presence of two proteins with molecular masses larger than P1, possibly P1-2A and P1-2ABC fusion proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

两株绿脓杆菌对石油污染土壤的修复作用

本文旨在研究环境条件下微生物对石油污染土壤的修复情况。从矿井周边土样定向筛选出两株绿脓杆菌,摇瓶降解实验发现,两菌混合培养10 d原油降解率达到95.67%,比单菌培养提高至少32%,即两菌对原油降解具有协同作用。根据降解实验结果制备了混合修复菌剂,并且人工构建石油污染场地,展开中试场地修复试验,模拟不同的操作条件下土壤中原油的降解情况。经60 d修复发现,添加了菌剂的场地,石油烃含量下降趋势明显,每克土壤中石油烃含量从初始的0.8%降至0.1%–0.3%,其中额外添加有机肥作为补充碳氮源的场地,总石油烃降解率最高,达到85.28%。而未添加菌剂的对照组石油烃含量仅减少25.85%。     

玉米大斑病菌STK1与EGFP融合基因载体的构建及其在毕赤酵母中的表达

STK1基因是玉米大斑病菌调控分生孢子发育、渗透胁迫调节和致病性的重要MAPK基因。本文首先构建了含有增强型绿色荧光蛋白基因(EGFP)的毕赤酵母GSS115(Pichia pastoris GS115)表达载体p PIC3.5K-EGFP,再以玉米大班病菌模式菌株01-23的菌丝c DNA为模板,PCR扩增STK1基因,克隆到p PIC3.5K-EGFP,构建了STK1-EGFP融合基因的GS115表达载体p PIC3.5K-STK1-EGFP。利用电击转化法将该融合基因表达载体转化到GS115感受态细胞内,利用MD培养基筛选、PCR鉴定,获得了STK1-EGFP融合基因的毕赤酵母转化子。通过RT-PCR和荧光观察,发现STK1基因和EGFP基因均可以高效稳定地表达。另外,在试验中我们还发现,在STK1基因起始密码子前加入Kozak序列可以使STK1-EGFP融合基因的表达强度增强4.8倍。以上研究结果为STK1基因表达蛋白的亚细胞功能定位和抗体制备奠定了基础。     

Molecular cloning and characterization of human kinectin.

We have identified a human cDNA that is homologous to the chicken kinectin, a putative receptor for the organelle motor kinesin. The human cDNA clone hybridized to a single 4.6-kb mRNA species that codes for a protein of 156 kDa molecular mass. The predicted primary translation product contains an N-terminal transmembrane helix followed by a bipartite nuclear localization sequence and two further C-terminal leucine zipper motifs. In addition, the aminoacid sequence revealed a large region (327-1362) of predicted alpha-helical coiled coils. A monoclonal antibody CT-1 raised against a GST-kinectin fusion protein produced a perinuclear, endoplasmic reticulum-like staining pattern in diverse cell types from different species, indicating evolutionary conservation. Monoclonal antibody CT-1 and anti-chicken kinectin antibodies cross-reacted both in Western blotting and immunoprecipitation with a 160-kDa protein, confirming the antigenic identity of this 160-kDa protein with chicken kinectin. Epitope tagging studies revealed that the nuclear localization sequence motif of kinectin is not functional. Furthermore, a truncated kinesin cDNA lacking the N-terminal hydrophobic domain revealed a nonspecific cytoplasmic staining pattern. Together the data suggest that kinectin is an integral membrane protein anchored in the endoplasmic reticulum via a transmembrane domain.