ENC1的克隆,原核表达与数种细胞系表达谱分析

以人 3月胎脑总RNA为模板 ,用RT PCR的方法得到了ENC1(ectoderm neuralcortex 1)基因的cDNA ,经测序证实该cDNA的长度为 180 0bp ,包含ENC1的完整编码区 .将之克隆入pGEX 4T 1载体构建重组表达质粒 ,转化大肠杆菌BL2 1表达 ,经Sepharose 4B纯化得到目的蛋白 .通过Northern印迹和RT PCR检验了该基因在数种细胞系中的表达 ,结果表明其在神经胶质母细胞瘤细胞系U2 5 1中有较高的表达 ,而在包括神经母细胞瘤细胞系SH SY5Y的其他数种细胞系中无表达 .与在正常生理状态下神经系统中两种细胞的表达情况相反 .这种分布不同的情况提示了ENC1在这两种不同来源的肿瘤的发生发展中具有不同作用     

人高迁移率族蛋白B1，高迁移率族蛋白B1 A box和B box的原核表达、 纯化及高迁移率族蛋白B1多克隆抗血清的制备

目的:获取重组人高迁移率族蛋白B1(HMGB1),HMGB1Abox和Bbox的纯化蛋白,制备HMGB1的多克隆抗血清。方法:采用PCR方法扩增人HMGB1,HMGB1的Abox和Bbox目的基因片段,构建原核表达载体,进行原核表达与蛋白纯化,然后用HMGB1免疫新西兰大白兔,制备多克隆抗血清。采用ELISA检测抗血清效价,用免疫组化检测HMGB1在小鼠肝损伤组织中的表达。结果:成功构建了人HMGB1,HMGB1的Abox和Bbox原核表达载体pET28-HMGB1、pET28-Abox、pET28-Bbox,在E.coli BL21中表达,镍亲和层析柱提纯,获取纯净目的蛋白。HMGB1免疫新西兰大白兔后,抗血清效价为1:2,000,000,具有高度特异性。免疫组化显示小鼠坏死肝组织HMGB1表达增加。结论:本研究获得了人HMGB1以及HMGB1的Abox和Bbox的纯化蛋白,制备了人HMGB1的多克隆抗血清,为HMGB1的结构、组织表达谱及其功能的研究奠定了基础。     

人高迁移率族蛋白B1，高迁移率族蛋白B1Abox和Bbox的原核表达、纯化及高迁移率族蛋白B1多克隆抗血清的制备

目的：获取重组人高迁移率族蛋白B1（HMGB1）,HMGB1Abox和Bbox的纯化蛋白,制备HMGB1的多克隆抗血清。方法：采用PCR方法扩增人HMGB1,HMGB1的Abox和Bbox目的基因片段,构建原核表达载体,进行原核表达与蛋白纯化,然后用HMGB1免疫新西兰大白兔,制备多克隆抗血清。采用ELISA检测抗血清效价,用免疫组化检测HMGB1在小鼠肝损伤组织中的表达。结果：成功构建了人HMGB1,HMGB1的Abox和Bbox原核表达载体pET28-HMGB1、pET28一Abox、pET28-Bbox,在E．co1iBL21中表达,镍亲和层析柱提纯,获取纯净目的蛋白。HMGB1免疫新西兰大白兔后,抗血清效价为1：2,000,000,具有高度特异性。免疫组化显示小鼠坏死肝组织HMGB1表达增加。结论：本研究获得了人HMGB1以及HMGB1的Abox和Bbox的纯化蛋白,制备了人HMGB1的多克隆抗血清,为HMGB1的结构、组织表达谱及其功能的研究奠定了基础。     

人SUMO-3基因原核表达及多克隆抗体制备

构建人SUMO-3基因的原核表达载体pET41a(+)-SUMO-3,表达重组GST-SUMO-3融合蛋白,制备人SUMO-3多克隆抗体。试验结果显示,通过PCR方法从重组质粒pEYFP-SUMO-3中克隆到的SUMO-3 N端93个氨基酸的基因序列与NCBI上提供的序列一致,重组质粒pET41a(+)-SUMO-3构建成功;重组pET41a(+)-SUMO-3在E.coli.BL21 (DE3) pLysS中表达GST-SUMO-3融合蛋白,分子量为44.0 kDa,与预期分子量一致;采用亲和层析纯化融合蛋白GST-SUMO-3并免疫家兔,获得人SUMO-3抗体;Western blot 检测显示该抗体可以特异性识别SUMO-3,ELISA检测结果成阳性,抗体效价约为1: 20000。实验结果为进一步研究人SUMO-3及SUMO第二类家族的功能提供了有用工具。     

Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor alpha

A gene encoding human tumour necrosis factor alpha (TNF-alpha) has been chemically synthesized, cloned and expressed to yield a biologically active protein in Escherichia coli. The 480-bp gene was assembled by enzymic ligation of 32 oligonucleotides, cloned directly into M13mp18 for sequence verification and expressed in the broad host range high-level expression vector pMMB66EHST. Expressed recombinant TNF-alpha was shown to have the correct molecular weight, processed N-terminal sequence, antibody cross-reactivity and tumour cell killing activity. The expression product of the synthetic gene has been purified to homogeneity by a two-step ion-exchange procedure and the purified material shown to be active.     

非天然氨基酸突变的小鼠RANKL蛋白原核表达及抗血清制备

目的:研究非天然氨基酸突变的小鼠RANKL(m RANKL)蛋白的原核表达,并以表达的蛋白制备抗m RANKL蛋白的抗血清。方法:从小鼠的骨髓中提取总RNA,经PCR扩增m RANKL的胞外段,逆转录合成目的 DNA片段。将目的基因中编码第234位酪氨酸的密码子(TAT)突变成可编码非天然氨基酸p-硝基苯丙氨酸(p NO2Phe)的琥珀密码子(TAG),构建p ET28a-p NO2Phe234m RANKL重组表达载体,与p EVOL质粒共转化至表达菌E.coli BL-21(DE3),诱导表达并纯化。以纯化的蛋白免疫小鼠,制备小鼠抗m RANKL抗血清。采用ELISA测定抗血清效价,用Western Blot测定其特异性。结果:RT-PCR扩增出750bp的RANKL基因,并成功构建了p ET28a-p NO2Phe234m RANKL重组表达载体;p-硝基苯丙氨酸突变的m RANKL蛋白获得成功表达和纯化;以纯化的蛋白免疫小鼠,获得抗m RANKL抗血清,ELISA检测效价为1:6400,Western Blot结果显示该抗血清既可与p-硝基苯丙氨酸突变的m RANKL结合,也可与野生型m RANKL结合。结论:原核表达并纯化了p-硝基苯丙氨酸突变的m RANKL,制备了小鼠抗m RANKL的抗血清,为进一步研究阻断RANKL-RANK通路的新方法奠定了基础。     

A potent mitogenic lectin from the mycelia of a phytopathogenic fungus, <Emphasis Type="Italic">Rhizoctonia bataticola</Emphasis>, with complex sugar specificity and cytotoxic effect on human ovarian cancer cells

A lectin with strong mitogenic activity towards human peripheral blood mononuclear cells (PBMCs) and cytotoxic effect on human ovarian cancer cells has been purified from the mycelium of a phytopathogenic fungus, Rhizoctonia bataticola, using ion exchange chromatography and affinity chromatography on asialofetuin-Sepharose. The lectin, termed RBL, is a tetramer of 11-kDa subunits and has unique amino acid sequence at its blocked N-terminus. The purified RBL was blood group nonspecific and its hemagglutination activity was inhibited by mucin (porcine stomach), fetuin (fetal calf serum) and asialofetuin. Glycan array analysis revealed high affinity binding of RBL towards N-glycans and also the glycoproteins containing complex N-glycan chains. Interestingly, the lectin showed high affinity for glycans which are part of ovarian cancer marker CA125, a high molecular weight mucin containing high mannose and complex bisecting type N-linked glycans as well core 1 and 2 type O-glycans. RBL bound to human PBMCs eliciting strong mitogenic response, which could be blocked by mucin, fetuin and asialofetuin demonstrating the carbohydrate-mediated interaction with the cells. Analysis of the kinetics of binding of RBL to PBMCs revealed a delayed mitogenic response indicating a different signaling pathway compared to phytohemagglutinin-L. RBL had a significant cytotoxic effect on human ovarian cancer cell line, PA-1.     

昆虫神经毒素LqhIT2的表达、抗血清制备及活性分析

根据毕赤酵母密码子偏爱性,不改变毒素蛋白质一级结构,设计合成了昆虫神经毒素LqhIT2基因,并分别克隆至大肠杆菌融合表达载体pPET30-a( )和毕赤酵母分泌表达载体pPIC9K.在IPTG的诱导下,神经毒素在大肠杆菌中融合表达,表达产物经镍亲和层析纯化后,用于免疫BALB/c小鼠,制备了特异性较高的抗血清,抗体滴度超过1:128 000.利用制备的抗血清,采用斑点杂交,筛选得到了较高水平分泌表达重组LqhIT2的酵母转化子,摇瓶条件下,毒素表达量约9 mg/L.大肠杆菌表达产物没有生物活性,酵母表达产物经注射蝗虫表现出杀虫活性.     

Alternative splicing of delta-like 1 homolog (DLK1) in the pig and human

Delta-like homolog 1 (DLK1), a paternally imprinted gene with several alternative splicing isoforms, is an important regulator of fetal and postnatal development. We report the sequence of porcine DLK1 (pDLK1) and examine the expression and alternative splicing isoforms in the pig (Sus scrofa) and human. DLK1-A was the sole isoform identified in human tissues and has been shown to be present in mouse and cattle. Surprisingly, DLK1-A was undetected in various tissues from fetal and postnatal pigs. Instead, DLK1-C2 was the most abundant isoform while DLK1-B was expressed to a lesser extent. In fractionated adipose tissue, pDLK1 was most highly expressed in the stromal-vascular cell fraction. In addition, total pDLK1 was highly expressed in fetal adipose tissue but dramatically decreased postnatally. Our data suggests that expression of DLK1-B and -C2 isoforms is sufficient for normal pig development. Furthermore, human and pig samples showed no alterations in species-specific splicing, but expression levels decreased with age, suggesting that regulation of expression, not splicing, is the most likely mechanism controlling the biological function of DLK1.     

Immune response gene 1 deficiency aggravates high fat diet-induced nonalcoholic fatty liver disease via promotion of redox-sensitive AKT suppression

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder worldwide. Immune response gene 1 (IRG1) catalyzes the production of bio-active itaconate, which is actively involved in the regulation of signal transduction. A recent study has found that the expression of IRG1 was significantly down-regulated in obesity-associated fatty liver, but the potential roles of IRG1 in the development NAFLD remain unclear. The present study found that genetic deletion of IRG1 aggravated high fat diet (HFD)-induced metabolic disturbance, including obesity, dyslipidemia and insulin resistance. In addition, HFD induced more severe liver steatosis and higher serum ALT and AST level in IRG1 KO mice, which were accompanied with altered expression of genes involved in lipid uptake, synthesis and catabolism. RNA-seq and immunoblot analysis indicated that deficiency of IRG1 is associated with suppressed activation of AKT, a master metabolic regulator. Mechanistically, IRG1/itaconate enhanced the antioxidative NRF2 pathway and prevented redox-sensitive suppression of AKT. Interestingly, supplementation with 4-octyl itaconate (4-OI), a cell-permeable derivate of itaconate, alleviated HFD-induced oxidative stress, AKT suppression and liver steatosis. Therefore, IRG1 probably functions as a protective regulator in the development of NAFLD and the cell-permeable 4-OI might have potential value for the pharmacological intervention of NAFLD.     

Identification of the human papillomavirus type 6b L1 open reading frame protein in condylomas and corresponding antibodies in human sera.

Genital warts (condylomata acuminata) are among the most frequent sexually transmitted infections. Human papillomavirus type 6 (HPV-6), which is etiologically related to a majority of these lesions, has not been propagated in tissue culture. We generated two forms of HPV-6 viral antigens: a chemically synthesized oligopeptide (referred to as the C-terminal synthetic peptide) corresponding to residues 482 to 495 of the 500-amino-acid-long L1 open reading frame (ORF), and a bacterially expressed 54-kilodalton (kDa) fusion protein containing the N-terminal 13 amino acids encoded by the lambda bacteriophage cII gene followed by one vector-insert junctional residue and 462 amino acids of the L1 ORF sequence (residues 39 to 500). The cII-L1 fusion protein was specifically recognized by an antipeptide serum directed against the N-terminal 13 amino acids derived from the cII gene, an antiserum raised against the C-terminal synthetic peptide, and a genus-specific serum prepared by immunization with disrupted viral capsids. The 54-kDa fusion protein was purified, and the sequence of its first 36 amino acids was determined and found to be as predicted by the DNA sequence. Both the genus-specific anticapsid serum and the antiserum raised against the fusion protein identified authentic L1 ORF proteins in HPV-1-induced (58 kDa) and HPV-6/11-induced (56 kDa) papillomas. The synthetic peptide antiserum recognized the 56- to 58-kDa protein in HPV-6-induced warts, but not in HPV-1- or HPV-11-infected specimens. Using the fusion protein as antigen in immunoassays, we were able to detect the corresponding antibodies in human sera.     

Studies on chemical synthesis of human cystatin A gene and its expression in Escherichia coli

A synthetic gene containing the coding sequence for the human cysteine proteinase inhibitor, cystatin A, was obtained by enzymatic assembly of 20 oligodeoxyribonucleotides which had been chemically synthesized by the solid phase phosphoramidite method. It was cloned into an Escherichia coli plasmid. The expression plasmid for cystatin A was constructed by introducing the synthetic gene downstream of the tac promoter of an E. coli plasmid which is a derivative of pKK223-3 with high copy number. The gene was expressed in E. coli JM109 without IPTG-induction. The expression of cystatin A was detected by SDS-polyacrylamide gel electrophoresis of the E. coli JM109 lysate, followed by immunoblotting using rabbit antiserum raised with human epidermal cystatin A and alkaline phosphatase-conjugated goat anti-rabbit IgG. The result showed that the molecular weight of the expression product is identical with that of the authentic protein and the antigenic properties are also the same. Furthermore, the expression product purified with a CM-papain Sepharose affinity column and FPLC system with a Mono-Q column showed the same inhibitory activity for various cysteine proteinases. Also, purified recombinant cystatin A was found to have identical amino acid composition, NH2-terminal amino acid sequence, and peptide-map on reverse phase HPLC with those of the authentic inhibitor.     

Cloning, characterization and genomic organization of LCC-1 (scya16), a novel human CC chemokine expressed in liver

By homology search of expressed sequence tags (EST) in GenBank a novel member of the CC chemokine family was identified. The full-length sequence of this liver-specific CC chemokine (LCC-1) predicted a mature protein of 97 amino acids with 31-48% identity to other CC chemokines. There was a characteristic amino acid C-term extension when aligned with other chemokines. Northern blot analysis from a panel of human tissues revealed that LCC-1 mRNA expression is restricted to adult and fetal liver. Different polyadenylation results in two mRNA species of 1.5 kb and 0.5 kb in size. LCC-1 is constitutively expressed in human HepG2 hepatoma cells and is induced by hypoxic exposure. The promoter region of the LCC-1 gene contains potential HIF-1 binding sites. The EST for LCC-1 has been previously mapped to the CC chemokine cluster on human chromosome 17q11.2. The organization of the LCC-1 gene (scya16) into three exons interrupted by two introns is identical to that found for other members of the CC chemokine family.     

Purification of novel UBAP1 protein and its decreased expression on nasopharyngeal carcinoma tissue microarray

Recently, the UBAPl gene, a putative nasopharyngeal carcinoma (NPC) related gene, which is located at human chromosome 9p21-22 where loss of heterozygosity frequently occurs in NPC, was cloned. The present study aimed to explore the purification approach to UBAP1 protein and its expression pattern in NPC with tissue microarray. The full length coding sequence of UBAP1 was subcloned into a prokaryotic expression vector, pGEX-4T-2, and expressed in Escherichia coli as a GST-fusion protein. With modification of the purification method, GST-UBAP1 fusion protein achieved a high level of purity. The New Zealand rabbit was immunized with the purified fusion protein to prepare polyclonal antiserum following standard protocols. With this antiserum, high-throughput analysis of UBAP1 protein expression using immunohistochemistry was performed on self-made tissue microarrays consisting of 316 nasopharyngeal specimens from 148 NPC and 168 non-cancerous nasopharyngeal epithelia with different morphological features. Consequently, we found that there was a significant decrease in the percentage of positive expression in all NPC on protein levels for UBAP1 (23%), compared to that of the non-NPC (89%) (P<0.01). The result suggests that UBAP1 might be a potential effective diagnosis candidate for NPC and decreased expression of UBAP1 protein is a possible point of dysfunction along the pathogenesis pathway for NPC that may contribute to malignant transformation.     

A new type of flagellin gene in Pseudomonas putida

Previously established PCR amplification and Southern hybridization procedures were developed for the isolation of the 0.8-kb flagellin gene in Pseudomonas putida. The deduced protein sequence has significant homology to the N- and C-terminal sequences of other bacterial flagellins. We propose that P. putida flagellin genes can be divided at least into three size groups: type I (2.0 kb), type II (1.4 kb), and type III (0.8 kb). Type I and type II flagellin genes have been reported. The new 0.8-kb type III gene was expressed in E. coli, and the resulting protein was purified and used to raise polyclonal antibody to study whether this small gene encodes flagellin. The antiserum reacted with purified flagellin monomers from representatives of each flagellin type, as well as proteins of the same sizes in lysates of these organisms, on Western immunoblots. This antiserum was determined to be functional in a motility inhibition assay. Similar results were obtained from antiserum directed against purified type III flagellin, indicating that a new type of flagellin gene in P. putida has been found. Preliminary electron microscopic study revealed that P. putida isolate with the smaller flagellin gene type appeared to have a thinner flagellar filament.     

cDNA cloning and characterization of Drb1, a new member of RRM-type neural RNA-binding protein

Neural RNA recognition motif (RRM)-type RNA-binding proteins play essential roles in neural development. To search for a new member of neural RRM-type RNA-binding protein, we screened rat cerebral expression library with polyclonal antibody against consensus RRM sequences. We have cloned and characterized a rat cDNA that belongs to RRM-type RNA-binding protein family, which we designate as drb1. Orthologs of drb1 exist in human and mouse. The predicted amino acid sequence reveals an open reading frame of 476 residues with a corresponding molecular mass of 53kDa and consists of four RNA-binding domains. drb1 gene is specifically expressed in fetal (E12, E16) rat brain and gradually reduced during development. In situ hybridization demonstrated neuron-specific signals in fetal rat brain. RNA-binding assay indicated that human Drb1 protein possesses binding preference on poly(C)RNA. These results indicate that Drb1 is a new member of neural RNA-binding proteins, which expresses under spatiotemporal control.