谷氨酰胺：6-磷酸果糖酰胺基转移酶多克隆抗体的制备及应用

目的：采用原核表达系统表达和纯化重组谷氨酰胺：6-磷酸果糖酰胺基转移酶（GFAT）,制备GFAT多克隆抗体,并用以研究在糖尿病发生发展过程中GFAT的表达情况。方法：通过生物信息学分析其抗原性和属间同源性,选择需要的基因区域;利用RT-PCR扩增小鼠肝脏cDNA中GFAT基因片段,克隆到表达载体pET28b中;在大肠杆菌BL21（DE3）中诱导表达,并用镍离子螯合柱（Ni-NTA）纯化重组GFAT;用纯化的重组GFAT免疫BALB／c小鼠后得到多克隆抗体;用Western印迹检测正常小鼠、高血糖小鼠及胰岛素抵抗小鼠的组织GFAT的表达。结果：Western印迹分析表明,制备的GFAT抗体具有较高的特异性,可特异性识别重组GFAT和正常小鼠肝脏、肾脏、骨骼肌和肺组织的GFAT;与正常小鼠相比,高脂饲料诱导的胰岛素抵抗小鼠肌肉组织的GFAT表达升高约1．8倍,肝脏组织则略有升高;高血糖小鼠肌肉组织和肝脏组织的GFAT表达也略有上升,但无统计学差异。结论：利用原核表达及Ni-NTA纯化系统可制备小鼠GFAT多克隆抗体;正常小鼠骨骼肌GFAT表达较高;肌肉组织的GFAT表达在胰岛素抵抗小鼠显著性升高,而与小鼠血糖水平无明显相关性。     

抗果蝇MRJ蛋白单克隆抗体的制备和鉴定

制备抗果蝇MRJ蛋白单克隆抗体可用于研究果蝇mrj的生物学功能,使用IPTG诱导重组质粒pET28a-mrj在大肠杆菌Rosetta中表达,重组蛋白经过Ni-IDA凝胶柱亲和纯化后免疫BALB/c小鼠。然后取免疫好的小鼠的脾脏细胞与小鼠骨髓瘤细胞SP2/0融合,经克隆和筛选获得了能分泌抗果蝇MRJ蛋白单克隆抗体的杂交瘤细胞株。腹水制备后获得单克隆抗体,通过ELISA和Western Blot对所获得的抗体进行鉴定,结果表明所制备单克隆抗体能够特异性结合于原核及真核细胞表达的MRJ蛋白,可用于研究mrj基因的生物学功能。     

琥珀酰辅酶A转移酶的表达纯化及抗体制备

目的:琥珀酰辅酶A转移酶(SCOT)是酮体代谢过程中的关键限速酶,此酶缺陷多由SCOT基因突变引起,患者多有酮症酸中毒表现。为了进一步研究SCOT的功能,采用原核表达系统表达并纯化重组SCOT,制备SCOT多克隆抗体。方法:选择蛋鸡、肉鸡模式生物为研究对象,通过生物信息学对其抗原性和属间同源性进行分析,通过RT-PCR从鸡的骨骼肌cDNA中扩增了SCOT基因N端半长片段,克隆到表达载体pET28b中,在大肠杆菌BL21(DE3)中诱导表达,并用镍离子螯合柱(Ni-NTA)纯化重组SCOT;用纯化的重组SCOT免疫小鼠后得到多克隆抗体。结果:Western印迹表明,制备的SCOT抗体具有较高的特异性,可特异性识别鸡的SCOT蛋白,同时可特异性识别小鼠和人的相应SCOT蛋白。结论:SCOT多克隆抗体的制备为后续在鸡、鼠和人中研究SCOT基因提供基础。     

重组小鼠树突状细胞因子DCF1的原核表达和纯化

目的:克隆小鼠重组树突状细胞因子DCF1蛋白进行原核表达、纯化与鉴定.方法:采用PCR从小鼠脑cDNA克隆dcf1基因,构建DCF1原核表达重组质粒(pET30a-DCF1)并转化E.coli的BL21(DE3)菌株.IPTG诱导重组蛋白表达,并在变性条件下经Ni sepharose FF6亲和层析柱纯化,再通过SDS-PAGE和Western blotting鉴定.结果:成功克隆到大小为972bp的小鼠源dcf1基因片段并准确插入表达载体pET30a,0.1 mmol/L IPTG诱导转化菌2h可表达大量的DCF1蛋白,并可经Ni Sepharose FF6柱亲和层析得到高度纯化.结论:成功获得纯化的42kDa重组小鼠DCF1蛋白,为后续进行DCF1蛋白功能研究奠定了基础.     

C反应蛋白真核表达质粒的构建、表达及其对小鼠血管平滑肌细胞TNF-α表达的影响

目的构建小鼠C-反应蛋白基因重组质粒pIRES-CRP,观测其对血管平滑肌细胞TNF-α表达的影响。方法用RT-PCR法,以小鼠肝组织RNA为模板,扩增获得编码C-反应蛋白(C-reactive protein,CRP)的全长基因。将CRP基因克隆入真核表达质粒pIRES中,构建重组真核表达质粒pIRES-CRP。将重组质粒转染HeLa细胞,通过RT-PCR法检测CRPmRNA的表达,Western blot检测CRP蛋白的表达。将阳性质粒转染小鼠血管平滑肌细胞,通过Western blot检测CRP对TNF-α表达的影响。结果经酶切鉴定、PCR和测序分析结果表明,所克隆的CRP基因与报道结果完全一致,重组体的连接方向正确,阅读框与预期完全一致,真核表达质粒pIRES-CRP构建成功。将重组质粒pIRES-CRP瞬时转染Hela细胞,通过RT-PCR和Western blot证实CRP基因得到表达。将阳性质粒转染小鼠血管平滑肌细胞,TNF-α的表达明显高于空白对照组和空载体组(P<0.01)。结论成功构建小鼠CRP基因重组质粒pIRES-CRP,CRP基因能在人子宫颈癌Hela细胞中正常转录和翻译,表达的蛋白能与CRP的特异抗体反应。CRP能明显上调小鼠血管平滑肌细胞TNF-α的表达,这为解释CRP在冠状动脉的形成过程中起着促炎作用提供了新的实验依据。     

糖基因Glt8d2重组蛋白的表达、纯化及多克隆抗体的制备

目的构建新糖基转移酶基因Glt8d2的原核表达载体,诱导融合蛋白的表达,并对其进行纯化;制备兔抗GltSd2蛋白多克隆抗体并进行鉴定。方法应用RT—PCR技术,以HepG2细胞mRNA为模板,扩增Glt8d2目的基因片段,构建原核表达载体pET-32a（＋）-Glt8d2。转化大肠埃希菌B121,异丙基-D-半乳糖苷诱导并通过SDS—PAGE凝胶电泳分析、Western印迹分析、生物质谱分析证实Gh8d2重组蛋白表达正确。大量表达后利用Ni’亲和柱对表达蛋白进行纯化及柱上复性。纯化蛋白免疫新西兰大白兔,获得抗Gh8d2蛋白的多克隆抗体。以纯化的Glt8d2重组蛋白为抗原,分别以免疫前后的新西兰兔血清作为第一抗体,利用Western印迹和酶联免疫吸附法对多克隆抗体进行特异性分析及效价检测。结果扩增获得Glt8d2基因片段,成功表达了Gh8d2重组蛋白,经SDS—PAGE凝胶电泳和Western印迹分析得到证实。成功获得重组蛋白及兔抗Gh8d2多克隆抗体。ELISA检测证实多克隆抗体效价〉1：320000。免疫组织化学分析显示,该基因编码的糖基转移酶在肝实质细胞内呈胞质模式表达分布。结论利用大肠埃希菌B121能够成功表达Gh8d2重组蛋白,获得高特异性、高效价兔抗Glt8d2重组蛋白的多克隆抗体,为今后研究Glt8d2基因的生物学功能研究奠定了基础。     

小鼠精胺氧化酶的原核表达与鉴定

目的:原核表达和分离纯化小鼠精胺氧化酶(SMO)。方法:采用RT-PCR法从小鼠胚胎干细胞(ES细胞)RNA中克隆小鼠SMOcDNA,构建SMO原核表达质粒并转染大肠杆菌BL21(DE3)菌株,经IPTG诱导,将表达的小鼠SMO重组蛋白在变性条件下经Ni-NTA树脂亲和层析纯化和透析复性。结果:在大肠杆菌中高表达出小鼠SMO重组蛋白;纯化并透析复性后的重组SMO具备快速氧化特异性底物精胺的酶活性。结论:建立了原核表达和纯化有活性小鼠SMO的实验方法。     

小鼠睾丸特异性基因Vad1.2重组蛋白的构建及多克隆抗体制备

目的：构建小鼠睾丸特异性基因Vad1.2重组蛋白,制备多克隆抗体。方法：将小鼠睾丸组织Vad1.2转录本行RT-PCR扩增,通过DNA重组技术插入克隆载体p ET15b,进行酶切及DNA序列分析。将表达载体转化入大肠杆菌BL21（DE3）RIL感受态细胞,10 mmol/L IPTG诱导表达重组蛋白,通过10%SDS-PAGE、Western blotting及质谱分析进行鉴定。随后对重组Vad1.2蛋白进行纯化和进一步鉴定。最后,制备兔抗小鼠Vad1.2多克隆抗体,并通过Vad1.2-EGFP质粒转染GC-2spd（ts）细胞对其特异性进行验证。结果：大肠杆菌BL21（DE3）RIL细胞中诱导表达并纯化的小鼠Vad1.2重组蛋白构建正确并经Western blotting和质谱分析得到证实。所制备的多克隆抗体可特异性识别GC-2spd（ts）细胞中过表达的Vad1.2-EGFP融合蛋白。结论：成功构建小鼠Vad1.2重组蛋白,并制备多克隆抗体,为Vad1.2基因的进一步研究奠定了实验基础。     

小鼠附睾分泌的防御素Defb48的原核表达及纯化

目的:获取小鼠附睾分泌的防御素Defb48的基因并原核表达、纯化,为研究其功能奠定基础。方法:提取小鼠附睾组织的总RNA,用RT-PCR技术扩增出不含信号肽的Defb48编码序列,将2段Defb48编码序列串联克隆至原核表达载体pET28(a)中,经酶切和测序鉴定正确的重组质粒转化大肠杆菌Rosetta(DE3),IPTG诱导表达重组蛋白;用镍柱进行重组蛋白的纯化,然后进行SDS-PAGE和Western印迹分析鉴定。结果:获得实验所需小鼠附睾分泌的防御素Defb48的基因序列,测序结果与已发表的基因序列一致;在大肠杆菌Rosetta(DE3)中表达了His-Defb48融合蛋白,经Western印迹分析,在相对分子质量18 000处有特异的蛋白条带,镍柱纯化后获得纯度较好的融合蛋白。结论:克隆了小鼠附睾分泌的防御素Defb48基因,并在大肠杆菌Rosetta(DE3)中表达纯化出融合蛋白,为制备其多克隆抗体,进而进一步研究Defb48的功能奠定了基础。     

EpCAM蛋白的表达、纯化及其单克隆抗体的制备

目的:原核表达EpCAM蛋白并制备抗EpCAM特异性单克隆抗体,初步鉴定相应单克隆抗体的特性。方法:PCR扩增EpCAM基因胞外区,将目的基因亚克隆至载体pET-28a(+),转化至大肠埃希菌株BL21,IPTG诱导表达,组氨酸亲和层析法纯化表达产物。纯化蛋白免疫BALB/c小鼠,将成功免疫的小鼠脾细胞与骨髓瘤SP2/0细胞融合,经ELISA筛选得到分泌特异性抗EpCAM的单克隆抗体的细胞株,免疫BALB/c小鼠进一步制备相应的单克隆抗体,并通过Western blot(蛋白质印记)和FACS(流式细胞分析)鉴定单抗的特异性及生物学活性。结果:成功构建重组表达载体pET28a-EpCAM并在大肠杆菌中获得表达,经His-tag亲和层析法获得纯化的EpCAM重组蛋白。EpCAM重组蛋白免疫的BALB/c小鼠的脾细胞与SP2/0细胞融合、筛选,获得两株稳定分泌EpCAM抗体的杂交瘤细胞株,分别命名为4B2、2F2并免疫BALB/c小鼠获得相应的单克隆抗体。Western blot结果显示4B2腹水纯化所得单抗能够识别FaDu细胞系(人咽鳞癌细胞)中的EpCAM蛋白,但2F2未能识别FaDu细胞中的变性的EpCAM蛋白。FACS结果显示两者均能和FaDu细胞中天然的EpCAM蛋白结合。讨论:成功制备了抗EpCAM的单克隆抗体,并能够识别人咽鳞癌细胞系FaDu中表达的EpCAM,为进一步研究EpCAM抗体在肿瘤治疗中的作用提供基础。     

Isoforms of alanine aminotransferases in human tissues and serum--differential tissue expression using novel antibodies

Serum alanine aminotransferase (ALT) is used as a clinical marker of hepatotoxicity. Two forms of ALT have been identified, ALT1 and ALT2, encoded by separate genes. The cellular and tissue distribution of the different ALT proteins has not been characterized in humans, and their relative contribution to serum is unknown. Here, we describe the development of novel isoenzyme specific ALT1 and ALT2 antibodies and the expression of the enzymes in human cells and organs. In normal human tissue, high expression of ALT1 was found in liver, skeletal muscle and kidney and low levels in heart muscle and not detectable in pancreas. High ALT2 reactivity was detected in heart and skeletal muscle, while no ALT2 expression was found in liver or kidney. Using immunohistochemistry, strong ALT1 reactivity was found in hepatocytes, renaltubular epithelial cells and in salivary gland epithelial cells, while ALT2 was expressed in adrenal gland cortex, neuronal cell bodies, cardiac myocytes, skeletal muscle fibers and endocrine pancreas. Immunoprecipitation using ALT antibodies on normal human serums showed ALT1 to be mainly responsible for basal ALT activity. Together, the results points to a differential expression of ALT1 and ALT2 in human organs and substantiate a need for investigations regarding the possible impacts on ALT measurements.     

Differential expression of HSPA1 and HSPA2 proteins in human tissues; tissue microarray-based immunohistochemical study

In the present study we determined the expression pattern of HSPA1 and HSPA2 proteins in various normal human tissues by tissue-microarray based immunohistochemical analysis. Both proteins belong to the HSPA (HSP70) family of heat shock proteins. The HSPA2 is encoded by the gene originally defined as testis-specific, while HSPA1 is encoded by the stress-inducible genes (HSPA1A and HSPA1B). Our study revealed that both proteins are expressed only in some tissues from the 24 ones examined. HSPA2 was detected in adrenal gland, bronchus, cerebellum, cerebrum, colon, esophagus, kidney, skin, small intestine, stomach and testis, but not in adipose tissue, bladder, breast, cardiac muscle, diaphragm, liver, lung, lymph node, pancreas, prostate, skeletal muscle, spleen, thyroid. Expression of HSPA1 was detected in adrenal gland, bladder, breast, bronchus, cardiac muscle, esophagus, kidney, prostate, skin, but not in other tissues examined. Moreover, HSPA2 and HSPA1 proteins were found to be expressed in a cell-type-specific manner. The most pronounced cell-type expression pattern was found for HSPA2 protein. In the case of stratified squamous epithelia of the skin and esophagus, as well as in ciliated pseudostratified columnar epithelium lining respiratory tract, the HSPA2 positive cells were located in the basal layer. In the colon, small intestine and bronchus epithelia HSPA2 was detected in goblet cells. In adrenal gland cortex HSPA2 expression was limited to cells of zona reticularis. The presented results clearly show that certain human tissues constitutively express varying levels of HSPA1 and HSPA2 proteins in a highly differentiated way. Thus, our study can help designing experimental models suitable for cell- and tissue-type-specific functional differences between HSPA2 and HSPA1 proteins in human tissues.     

Skeletal muscle LIM protein 1 (SLIM1/FHL1) induces alpha 5 beta 1-integrin-dependent myocyte elongation

Skeletal muscle LIM protein 1 (SLIM1/FHL1) contains four and a half LIM domains and is highly expressed in skeletal and cardiac muscle. Elevated SLIM1 mRNA expression has been associated with postnatal skeletal muscle growth and stretch-induced muscle hypertrophy in mice. Conversely, SLIM1 mRNA levels decrease during muscle atrophy. Together, these observations suggest a link between skeletal muscle growth and increased SLIM1 expression. However, the precise function of SLIM1 in skeletal muscle, specifically the role of SLIM1 during skeletal muscle differentiation, is not known. This study investigated the effect of increased SLIM1 expression during skeletal muscle differentiation. Western blot analysis showed an initial decrease followed by an increase in SLIM1 expression during differentiation. Overexpression of SLIM1 in Sol8 or C₂C₁₂ skeletal muscle cell lines, at levels observed during hypertrophy, induced distinct effects in differentiating myocytes and undifferentiated reserve cells, which were distinguished by differential staining for two markers of differentiation, MyoD and myogenin. In differentiating skeletal myocytes, SLIM1 overexpression induced hyperelongation, which, by either plating cells on poly-L-lysine or using a series of peptide blockade experiments, was shown to be specifically dependent on ligand binding to the ₅₁-integrin, whereas in reserve cells, SLIM1 overexpression induced the formation of multiple cytoplasmic protrusions (branching), which was also integrin mediated. These results suggest that SLIM1 may play an important role during the early stages of skeletal muscle differentiation, specifically in ₅₁-integrin-mediated signaling pathways. myoblast; proteins and differentiation     

Differential and special properties of the major human UGT1-encoded gastrointestinal UDP-glucuronosyltransferases enhance potential to control chemical uptake

UDP-glucuronosyltransferase (UGT) isozymes detoxify metabolites, drugs, toxins, and environmental chemicals via conjugation to glucuronic acid. Based on the extended UGT1 locus combined with Northern blot analysis and in situ hybridization, we determined the distribution of UGT1A1 and UGT1A7 through UGT1A10 mRNAs and found them for the first time segmentally distributed in the mucosal epithelia layer of the gastrointestinal tract. Biochemically, recombinant isozymes exhibited pH optima of 5.5, 6.4, 7.6, 8.5, and/or a broad pH range, and activities were found to be unaffected or progressively inhibited by increasing substrate concentrations after attaining Vmax for certain chemicals. Under different optimal conditions, all exhibited wide substrate selections for dietary and environmentally associated chemicals. Evidence also suggests tandem effects of isozymes in the time for completion of reactions when comparing short- and long-term incubations. Moreover, treatment of colon cells with certain diet-associated constituents, curcumin and nordihydroguaiaretic acid, reversibly targets UGTs causing inhibition without affecting protein levels; there is no direct inhibition of control UGT using curcumin as substrate in the in vitro assay. In summary, we demonstrate that UGTs are located in gastrointestinal mucosa, have vast overlapping activities under differential optimal conditions, and exhibit marked sensitivity to certain dietary substrates/constituents, representing a first comprehensive study of critical properties concerning glucuronidating isozymes in alimentary tissues. Additionally, the highly dynamic, complex, and variable properties necessarily impact absorption of ingested chemicals and therapeutic drugs.     

The intracellular distribution and activities of phosphoenolpyruvate carboxykinase isozymes in various tissues of several mammals and birds.

1. The intracellular distribution and/or activities of phosphoenolpyruvate carboxykinase isozymes were determined in liver, kidney, gastrointestinal mucosa, adipose, skeletal muscle, brain, spleen, lung and heart of fed and fasted rabbits, guinea pigs, rats, chickens and pigeons. 2. Liver and kidney of all species contained the highest enzyme activity/g. 3. Carboxykinase activity/g gastrointestinal mucosa of rabbits was quite high compared to the low activity in guinea pig and rat mucosa and essentially undetectable activity in chicken and pigeon mucosa. 4. Activity/g was high in rat brown adipose. 5. Low carboxykinase activity/g was found in skeletal muscle of all species and in white adipose of guinea pig, rabbit and rat although activity was undetectable in white adipose of chicken and pigeon. 6. Carboxykinase activity was essentially undetectable in brain, spleen, lung and heart of all species.     

Nephroblastoma overexpressed gene (NOV) codes for a growth factor that induces protein tyrosine phosphorylation

NOV (nephroblastoma overexpressed gene) is a member of the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, NOV) family of proteins. These proteins are cysteine-rich and are noted for having growth-regulatory functions. We have isolated the rat NOV gene, and the DNA sequence shares 90% identity with the mouse and 80% identity with the human sequences. The rat NOV gene was expressed in all rat tissues examined, including brain, lung, heart, kidney, liver, spleen, thymus and skeletal muscle. Higher levels of rat NOV mRNA were seen in the brain, lung and skeletal muscle compared to the other tissues. Examination of NOV expression in various human cell lines revealed that NOV was expressed in U87, 293, T98G, SK-N-MC and Hs683 but not in HepG2, HL60, THP1 and Jurkat. The human NOV gene was transfected into 293 cells and the expressed protein purified. When 3T3 fibroblasts were treated with this recombinant NOV protein, a dose-dependent increase in proliferation was observed. Analysis of tyrosine-phosphorylated proteins revealed that when 3T3 cells were treated with NOV, a 221 kDa protein was phosphorylated. These data suggest that NOV can act as a growth factor for some cells and binds to a specific receptor that leads to the phosphorylation of a 221 kDa protein.     

Cloning, expression and characterization of a novel human Ras-related protein that is regulated by glucocorticoid hormone

Ras proteins are a family of guanine nucleotide (GDP and GTP)-binding proteins that play central roles in essential signal transduction pathways. We have isolated in a yeast two-hybrid screen a human cDNA encoding a new protein that is highly homologous (98% identical at the protein level) to mouse DexRas1, a member of the Ras superfamily. The human DexRas1 is expressed in a variety of tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas, with the strongest expression in the heart. Using human fibrosarcoma HT-1080 cells as a model system, we show that the expression of human DexRas1 is stimulated by dexamethasone, suggesting a role of human DexRas1 in dexamethasone-induced alterations in cell morphology, growth and cell-extracellular matrix interactions.     

Expression, purification, and initial characterization of human alanine aminotransferase (ALT) isoenzyme 1 and 2 in High-five insect cells

Alanine aminotransferase (ALT) is a key enzyme for gluconeogenesis as well as a widely used serum marker for liver injury. We have identified two ALT isoenzymes, ALT1 and ALT2, which are encoded by separate genes. In this study, we described the expression, purification and initial characterization of human ALT1 and ALT2 proteins in High-five insect cells. Human ALT1 and ALT2 were expressed as His-tagged fusion proteins by recombinant baculovirus in insect cells and purified into homogeneity in one step by using immobilized Ni²⁺-affinity chromatography. Tag-free ALT1 and ALT2 were obtained by cleavage of enterokinase digestion and used for initial characterization of the enzymes. The specific ALT activity of purified fusion or His-tag-removed ALT1 was about 15-fold higher than that of ALT2 and their enzymatic activities decreased quickly at 37 °C and −20 °C, but were well preserved at −80 °C. Nevertheless, the ALT1 and ALT2 activities remained stable in a buffer containing 25% glycerol. The pH profile was similar between hALT1 and hALT2 in that both enzymes remained fully active between pH 6.5 and 8.0. The purified ALT recombinant proteins can not only be used as a reference protein standard for the ALT assay in clinical chemistry, but also will be useful for understanding the biochemical and biological significance of the isoenzymes and for developing ALT isoform-specific assays for clinical or preclinical diagnostic use.