Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells

SF2, an activity necessary for 5' splice site cleavage and lariat formation during pre-mRNA splicing in vitro, has been purified to near homogeneity from HeLa cells. The purest fraction contains only two related polypeptides of 33 kD. This fraction is sufficient to complement an S100 fraction, which contains the remaining splicing factors, to splice several pre-mRNAs. The optimal amount of SF2 required for efficient splicing depends on the pre-mRNA substrate. SF2 is distinct from the hnRNP A1 and U1 snRNP a polypeptides, which are similar in size. Endogenous hnRNA copurifies with SF2, but this activity does not appear to have an essential RNA component. SF2 appear to be necessary for the assembly or stabilization of the earliest specific prespliceosome complex, although in the absence of other components, it can bind RNA in a nonspecific manner. SF2 copurifies with an activity that promotes the annealing of complementary RNAs. Thus, SF2 may promote specific RNA-RNA interactions between snRNAs and pre-mRNA, between complementary snRNA regions, and/or involving intramolecular pre-mRNA helices. Other purified proteins with RNA annealing activity cannot substitute for SF2 in the splicing reaction.     

Development and application of monoclonal antibodies for detection and analysis of aquareoviruses

Monoclonal antibodies (mAbs) play an important role in detection of aquareoviruses. Three mAbs against grass carp reovirus (GCRV) were prepared. Isotyping revealed that all three mAbs were of subclass IgG2b. Western blot assay showed that all three mAbs reacted with GCRV 69 kDa protein (the putative VP5). In addition to the 69 kDa protein of GCRV, mAb 4B6 also recognize a 54 kDa protein. All three mAbs were used for detecting aquareovirus by Western blot assay and indirect immunofluorescence assay (IFA). All of them reacted with GCRV, and mAb 4A3 could also react with turbot Scophthalmus maximus reovirus (SMReV) and largemouth bass Microptererus salmonides reovirus (MsReV). Viral antigens were only observed in the cytoplasm of infected cells. Finally, syncytia formation was observed with light microscopy and fluorescence microscopy using fluorescein labelled 4A3 mAb at various times post-infection. Syncytia were observed at 36 hr post-infection (hpi) by light microscopy and at 12 hpi by fluorescence microscopy. The immunofluorescence based assay allowed earlier detection of virus than observation of virus-induced cytopathic effect (CPE) assay in inoculated cell cultures. The sensitivity and specificity of these mAbs may be useful for diagnosis and monitoring of aquareoviruses.     

Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A

Spliceostatin A (SSA) is a stabilized derivative of a Pseudomonas bacterial fermentation product that displays potent anti-proliferative and anti-tumor activities in cancer cells and animal models. The drug inhibits pre-mRNA splicing in vitro and in vivo and binds SF3b, a protein subcomplex of U2 small nuclear ribonucleoprotein (snRNP), which is essential for recognition of the pre-mRNA branch point. We report that SSA prevents interaction of an SF3b 155-kDa subunit with the pre-mRNA, concomitant with nonproductive recruitment of U2 snRNP to sequences 5' of the branch point. Differences in base-pairing potential with U2 snRNA in this region lead to different sensitivity of 3' splice sites to SSA, and to SSA-induced changes in alternative splicing. Indeed, rather than general splicing inhibition, splicing-sensitive microarray analyses reveal specific alternative splicing changes induced by the drug that significantly overlap with those induced by knockdown of SF3b 155. These changes lead to down-regulation of genes important for cell division, including cyclin A2 and Aurora A kinase, thus providing an explanation for the anti-proliferative effects of SSA. Our results reveal a mechanism that prevents nonproductive base-pairing interactions in the spliceosome, and highlight the regulatory and cancer therapeutic potential of perturbing the fidelity of splice site recognition.     

Monoclonal antibodies specific for the two types of wall-forming bodies of Eimeria tenella macrogametes (Coccidia, Apicomplexa)

Two monoclonal antibodies (mAbs) raised against the macrogamonts of Eimeria tenella identified antigens located in the wall-forming bodies of type I (WF I) and type II (WF II) by indirect immunofluorescence and by immunoelectron microscopy. With these mAbs, the involvement of both types of wall-forming body at the protein level in the formation of the inner and outer oocyst walls of E. tenella was shown by indirect immunofluorescence assay. On Western blots of pure macrogamont, mAb E1D8 against WF I reacted with a series of bands between 42 kDa and 105 kDa. In pure, unsporulated extract, this mAb recognized a complex of bands between 26 kDa and 153 kDa. mAb E2E5 against WF II, on Western blots of pure extract of macrogamonts, recognized an antigen of 51 kDa. Later in the development, after the formation of the inner oocyst wall, mAb E2E5 reacted with three polypeptide of 23, 25 and 30 kDa. Proteolytic processing may be forwarded as the mechanism regulating the distinct regulation protein involved in the oocyst wall.     

人精子蛋白17单克隆抗体的制备及特性鉴定

目的:制备抗精子蛋白17(Sp17)的单克隆抗体(mAb)并鉴定其特性。方法:克隆人Sp17cDNA,表达带有6His标记的重组Sp17,用纯化的重组Sp17免疫BALB/c小鼠制备mAb。用ELISA筛选抗体阳性的细胞克隆。用免疫组化染色法及阻断试验鉴定mAb的特异性。结果:获得2株杂交瘤细胞系3C12和3D6,其分泌的mAb的Ig亚类(型)分别为IgG1和IgM(κ),杂交瘤细胞培养上清的ELISA效价分别为1∶64和1∶32;腹水mAb的效价分别为1∶1×105和1∶5×104。用人和大鼠睾丸组织以及人精液精子免疫组化染色及阻断试验证明,抗Sp17mAb具有良好的特异性。抗Sp17mAb也可识别卵巢癌组织中异常表达的Sp17。结论:成功地制备特异性的抗Sp17mAb,为研究该蛋白的功能、天然分布及异常表达奠定了基础。     

Different functional domains on the transferrin receptor molecule defined by monoclonal antibodies.

Three monoclonal antibodies (mAb) FG 1/5, FG 1/6 and FG 2/12, specific for the human transferrin receptor molecule (TR), have been used to define epitopes on the TR molecule and to block natural killer lysis. FG 2/12 mAb but not FG 1/5 or FG 1/6 blocked [125I-] transferrin binding to the cellular receptor. Furthermore, FG 1/5 and FG 1/6 mAbs competed out the binding of each other to the cells but not significantly that of FG 2/12. As expected, the binding of F2/12 but not of FG 1/5 or FG 1/6 was inhibited by transferrin. In addition, FG 2/12 inhibited in a dose-dependent manner the NK activity of purified T3- large granular lymphocyte effector cells against HeLa or Molt-4 cells but not against K-562 or U937 cells. FG 1/5 preferentially inhibited NK activity against HeLa cells and FG 1/6 mAb was completely uneffective. These inhibitions were stronger at low effector to target cell (E:T) ratios than at high E:T ratios, suggesting that NK cells and anti-TR mAbs compete for the same site in the target cell. It was shown that FG 1/5 and FG 2/12 mAbs blocked cells' conjugate formation by acting at the target cell level. Our results confirm the role of TR as a one of the target structures in NK lysis and suggest that the epitope recognized by NK cells is close to but different from the transferrin binding site.     

抗艰难梭菌A毒素单克隆抗体的制备及特性分析

目的 :制备抗艰难梭菌A毒素的单克隆抗体 (mAb)并鉴定其特性。方法 :用纯化的艰难梭菌A毒素免疫BALB/c小鼠 ,将免疫小鼠的脾细胞与骨髓瘤细胞Sp2 / 0融合 ,采用间接ELISA筛选杂交瘤细胞。用ELISA检测mAb腹水的效价、相对亲和力和进行表位分析 ;用Westernblot检测mAb的特异性。结果 :得到 6株杂交瘤细胞株 ,5C10株细胞分泌的mAb为IgG2a ,4B5和 8A1株细胞分泌的mAb为IgG1,其他 3株细胞mAb (2H7、3E9和 6G8)均分泌IgM。中和试验表明 ,所有的mAb均无中和活性。腹水mAb的效价均在 10 -4以上 ,其中mAb 2H7、6G8、5C10、4B5和 8A1具有共同的表位 ,而mAb 3E9识别的位点与其他 5株不同。mAb 8A1和 4B5的相对亲和力>10 5,其他 4株mAb的相对亲和力 >10 4。在非变性条件下 ,PAGE后Westernblot的结果显示 ,6株mAb均可与相对分子质量 (Mr)为 5 5× 10 4的A毒素产生反应 ;而在变性条件下 ,还原与非还原SDS PAGE后Westernblot均显示 ,6株mAb均可与Mr 为 5× 10 4～ 2 4× 10 4的A毒素产生反应。结论 :6株杂交瘤细胞株均能分泌抗艰难梭菌A毒素的特异性mAb ,为艰难梭菌A毒素的研究提供了有利的工具     

Characterization of a nuclear 20S complex containing the survival of motor neurons (SMN) protein and a specific subset of spliceosomal Sm proteins

Spinal muscular atrophy (SMA) is a neurodegenerative disease of motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. Cytoplasmic SMN directly interacts with spliceosomal Sm proteins and facilitates their assembly onto U snRNAs. Nuclear SMN, in contrast, mediates recycling of pre-mRNA splicing factors. In this study, we have addressed the function of SMN in the nucleus. We show that a monoclonal antibody directed against SMN inhibits pre-mRNA splicing. Interestingly, the mode of inhibition suggests a novel role for SMN in splicing that occurs prior to, or in addition to, its role in recycling. Using biochemical fractionation and anti-SMN immunoaffinity chromatography, we identified two distinct nuclear SMN complexes termed NSC1 and NSC2. The biochemical properties and protein composition of NSC1 were determined in detail. NSC1 migrates in sucrose gradients as a U snRNA-free 20S complex containing at least 10 proteins. In addition to SMN, these include the SMN-interacting protein 1 (SIP-1), the putative helicase dp103/Gemin3, the novel dp103/Gemin3-interacting protein GIP1/Gemin4 and three additional proteins with apparent masses of 43, 33 and 18 kDa, respectively. Most surprisingly, NSC1 also contains a specific subset of spliceosomal Sm proteins. This shows that the SMN-Sm protein interaction is not restricted to the cytoplasm. Our data imply that nuclear SMN affects splicing by modulating the Sm protein composition of U snRNPs.     

Characterization of two monoclonal antibodies to Epstein-Barr virus diffuse early antigen which react to two different epitopes and have different biological function.

Five monoclonal antibodies (mAbs) were identified using immunofluorescence that were specific for the Epstein-Barr virus (EBV) encoded 52/50 kDa early antigen (EA-D) protein complex. Evidence to suggest that these mAbs react with the same 52/50 kDa EA-D protein was obtained by Western blotting, immunoprecipitation and ELISA. Two of the mAbs, 90E2 and 214A9, neutralized EBV DNA polymerase activity. The 214A9 mAb also inhibited the activity of bacteriophage T4 DNA polymerase while the 90E2 mAb did not. These data suggest that the 90E2 and 214A9 mAbs recognize two different epitopes on the 52/50 kDa EA-D protein. The high frequency of recovery of hybridomas producing anti 52/50 kDa EA-D mAbs suggest that this protein may have an important role in EBV pathogenesis/replication.     

Combination of Clk family kinase and SRp75 modulates alternative splicing of Adenovirus E1A

SR proteins are non-snRNP splicing factors harbouring a domain rich in Arg-Ser repeats, which are extensively phosphorylated by several kinases. We performed a comparative study of different SR kinases, including SRPK, Clk, PRP4 and DYRK, and found that only Clks efficiently altered 5' splice site selection of Adenovirus E1A. The phosphorylation state of SR proteins was examined using a phospho-SR specific antibody mAb1H4 and a 75 kDa protein was most evidently hyperphosphorylated by Clks. Administration of TG003, a specific inhibitor for the Clk family members, specifically and rapidly induced dephosphorylation of 75 kDa SR protein. Imaging with mRFP-SRp75 in living cells revealed that its nuclear distribution was rapidly altered upon inhibition of the Clk activity by TG003. Co-transfection experiments demonstrated that HA-tagged SRp75 was hyperphosphorylated by Clk family members, but not by other SR kinases. These results indicate that Clks specifically hyperphosphorylate SRp75. Furthermore, SRp75 over-expression promoted the selection of 12S 5' splice site in E1A pre-mRNA, which is stimulated by co-expression of Clks. These results suggest that the specific combination of SR protein and SR kinase plays a distinct role in alternative splicing through dynamic balance of phosphorylation.     

Stem–loop 4 of U1 snRNA is essential for splicing and interacts with the U2 snRNP-specific SF3A1 protein during spliceosome assembly

The pairing of 5′ and 3′ splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. Here, we report a new interaction between stem–loop 4 (SL4) of the U1 snRNA, which recognizes the 5′ splice site, and a component of the U2 small nuclear ribonucleoprotein particle (snRNP) complex, which assembles across the intron at the 3′ splice site. Using a U1 snRNP complementation assay, we found that SL4 is essential for splicing in vivo. The addition of free U1-SL4 to a splicing reaction in vitro inhibits splicing and blocks complex assembly prior to formation of the prespliceosomal A complex, indicating a requirement for a SL4 contact in spliceosome assembly. To characterize the interactions of this RNA structure, we used a combination of stable isotope labeling by amino acids in cell culture (SILAC), biotin/Neutravidin affinity pull-down, and mass spectrometry. We show that U1-SL4 interacts with the SF3A1 protein of the U2 snRNP. We found that this interaction between the U1 snRNA and SF3A1 occurs within prespliceosomal complexes assembled on the pre-mRNA. Thus, SL4 of the U1 snRNA is important for splicing, and its interaction with SF3A1 mediates contact between the 5′ and 3′ splice site complexes within the assembling spliceosome.     

抗人层黏连蛋白单克隆抗体的制备及特性鉴定

目的:制备抗人层黏连蛋白(laminin,LN)单克隆抗体(mAb)并鉴定其特性。方法:以人LN免疫BALB/c小鼠,采用杂交瘤技术制备抗人的mAb;同时采用间接ELISA法柃测mAb的腹水效价及mAb的相对亲和力;采用ELISA法鉴定mAb的Ig亚类、进行表位分析及特异性鉴定。结果:获得4株可分泌特异性mAb的杂交瘤细胞2A3、2C6、3G7和4H2,其腹水mAb 的效价为3.6 × 104～2.1×106;4株mAb的Ig亚类为IgG1,轻链均属κ型;相对亲和力2C6在1012以上,2A3、3G7和4H2在106以上;其中2株与1个表位结合,另2株与另外的1个表位结合。结论:成功地制备出抗人LN的mAb,为进一步研究LN在一些疾病中的作用提供了工具。     

人HMGB1 B box蛋白的表达、鉴定及其单克隆抗体的制备

目的:表达人HMGB1 B box蛋白,制备其单克隆抗体(mAb),为进一步研究HMGB1 B box在免疫调节和抗感染免疫中的作用奠定基础。方法:将pET28-HMGB1 B box转化DH5α摇菌表达,使用His标记的蛋白纯化柱纯化、鉴定后免疫BALB/c小鼠,取免疫小鼠的脾细胞与Sp2/0细胞进行常规融合,经间接ELISA筛选阳性克隆,获得分泌人HMGB1 B box蛋白mAb的杂交瘤细胞株,通过ELISA、Western blot等方法鉴定其特性(mAb的效价、Ig类别及特异性)。结果:成功地建立了2株稳定分泌抗人HMGB1 B box的mAb细胞株,分别命名为1D2F4E3和2D4E3A2。2株mAb的免疫球蛋白类型均为IgG,Western blot显示,2株mAb均能与HMGB1 B box发生特异性反应,其滴度为1×106,mAb1 D2F4E3和2D4E3A2的A450值分别为0.324±0.093和0.296±0.085。结论:获得了2株分泌HMGB1 B box mAb的细胞株,为HMGB1 B box蛋白的生物学功能研究奠定了基础。     

Variable region sequences of autoantibodies from mice with experimental systemic lupus erythematosus

We have sequenced nine monoclonal antibodies (mAb) derived from C3H.SW mice in which experimental systemic lupus erythematosus (SLE) was induced. The hybridomas were selected for binding to DNA or to HeLa nuclear extract (NE). Three mAb were found to bind DNA, and are shown to exhibit sequence characteristics of pathogenic anti-DNA antibodies. One, mAb 2C4C2, is shown to use a heavy chain V region gene (V_H) identical to the V_H of anti-DNA mAb isolated from other lupus-prone mice, namely (NZB × NZW)F₁. The light chain V region gene (V_L) of mAb 2C4C2 is 98 % homologous to the V_L of another anti-DNA mAb, also isolated from (NZB × NZW)F₁ mice. The other two anti-DNA mAb, 5G12-4 and 5G12-6, share 93 % of their V_H sequences with that of mAb 2C4C2. Six mAb bound proteins of HeLa NE. Four of these six antibodies were found to use the VH124 V_H and V-L7 V_L. The nine mAb use a total of five V_H and four V_L germ-line genes, demonstrating that the autoantibodies induced in mice with experimental SLE do not originate from one B cell clone. Three of these nine V_H and V_L were identical in sequence to germ-line genes, while at least three others had somatic mutations. The latter suggests that the above autoantibodies arise in mice by both usage of existing (pre-immune) B cells, and through an antigen-driven process. Furthermore, it appears that autoantibodies found in mice with experimental SLE use genetic elements similar to those used by mAb that were isolated from mouse strains which develop lupus spontaneously.     

Presplicing complex formation requires two proteins and U2 snRNP

Six fractions derived from a HeLa cell nuclear extract are necessary for the generation of spliced mRNA in vitro. To establish a function for the protein factors present in these fractions, their role in the formation of splicing complexes was analyzed by electrophoresis in native polyacrylamide gels. Two of the fractions are sufficient to assemble the adenovirus major late mRNA precursor into a presplicing complex with characteristics similar to the presplicing complex assembled in nuclear extract. One fraction supplies splicing factor (SF) 1 and at least one small nuclear ribonucleoprotein particle, U2 snRNP. The other fraction contains SF3. Extensive fractionation of this protein has revealed that it is essential for presplicing complex assembly and the splicing reaction.     

抗CP4-EPSPS单克隆抗体的制备和生物学特性的鉴定

目的制备可用于胶体金快速检测试条的抗CP4-EPSPS(5-enolpyruvlshimimate-3-phosphate synthase)单克隆抗体(mAb),并鉴定其特性。方法以重组蛋白CP4-EPSPS免疫BALB/c小鼠,采用杂交瘤技术制备抗CP4-EPSPS的mAb,以间接ELISA法和Western blot进行mAb特异性鉴定;同时采用间接ELISA法鉴定mAb的Ig亚类,检测mAb的效价及相对亲和力,并进行mAb结合表位分析。结果获得2株可分泌特异性mAb的杂交瘤细胞(Ⅲ5A3,Ⅲ13A2)。其抗体亚类均为IgG1;腹水效价分别为1∶106和1∶108;相对亲和力Ⅲ5A3在105以上,Ⅲ13A2在106以上。ELISA相加实验结果显示2株mAb识别相同或相近的抗原表位。结论成功地制备出抗CP4-EPSPS的2株mAb,为建立快速特异检测转基因植物(GMO)的实验方法提供了有力的工具。     

抗禽流感病毒M2蛋白单克隆抗体的制备及鉴定

目的:制备抗禽流感病毒M2蛋白的单克隆抗体(mAb)并进行特性鉴定.方法:利用纯化的融合蛋白GST-M2免疫BALB/c小鼠,然后以GST-M2 和 GST分别作为ELISA抗原进行筛选,选择GST-M2抗原检测强阳性、GST抗原检测阴性的杂交瘤细胞进行克隆,建立能稳定分泌抗AIV M2 mAb的杂交瘤细胞株.mAb的效价采用间接ELISA和琼脂扩散试验(AGP)测定,用夹心ELISA测定Ig亚类,Western blot、抗原捕获ELISA、间接免疫荧光及免疫组化染色法检测 mAb 的特性.结果:得到4株分泌抗禽流感病毒M2蛋白的mAb的杂交瘤细胞株1E1、2F8、4E3 和 5D6,小鼠腹水中抗体的ELISA效价大于210×100、AGP效价大于1:4.抗体亚类鉴定1E1和4E3为IgG2a,2178和5G6为IgG2b.抗原捕获ELISA表明,2F8 wAb能与H5、H9亚型AIV发生特异性反应,而不能与鸡新城疫病毒(NDV)和鸡传染性法氏囊病毒(IBDV)反应.IFA和免疫组化试验表明,2F8 mAb能够与感染了禽流感病毒的MDCK细胞以及鸡体组织细胞发生特异性结合.结论:本研究获得4株抗禽流感病毒M2蛋白的mAb,其中2F8mAb的效价高、特异性强,可作为检测AIV方法的核心试剂.     

Establishment of hybrid cell lines producing monoclonal antibodies to a synthetic peptide from the E1 region of the hepatitis C virus

We aimed at establishing hybridoma cells secreting monoclonal antibodies (mAbs) against E1 synthetic peptide of HCV. BALB/c mice were immunized with HCV E1-synthetic peptide (GHRMAWDMM) and its spleenocytes were fused with the P3NS1 myeloma cell line. Two highly reactive and specific mAbs (10C7 IgG2b mAb, and 10B2 IgG1 mAb) were generated. The target HCV E1 antigen was identified at approximately 38 kDa in serum of infected individuals. A newly developed ELISA detected the target antigen in 90% of sera from HCV RNA infected individuals with a specificity of 84%. So, the generated mAbs may provide promising probes for serodiagnosis of HCV infection.