Cloning and functional verification of PhAEP gene, a key enzyme for biosynthesis of heterophyllin A in Pseudostellaria heterophylla / 中国中药杂志

This paper aimed to study the role of asparagine endopeptidase(AEP) gene in the biosynthesis mechanism of cyclic peptide compounds in Pseudostellaria heterophylla. The transcriptome database of P. heterophylla was systematically mined and screened, and an AEP gene, tentatively named PhAEP, was successfully cloned. The heterologous function verification by Nicotiana benthamiana showed that the expression of the gene played a role in the biosynthesis of heterophyllin A in P. heterophylla. Bioinformatics analysis showed that the cDNA of PhAEP was 1 488 bp in length, encoding 495 amino acids with a molecular weight of 54.72 kDa. The phylogenetic tree showed that the amino acid sequence encoded by PhAEP was highly similar to that of Butelase-1 in Clitoria ternatea, reaching 80%. The sequence homology and cyclase active site analysis revealed that the PhAEP enzyme may specifically hydrolyse the C-terminal Asn/Asp(Asx) site of the core peptide in the HA linear precursor peptide of P. heterophylla, thereby participating in the ring formation of the linear precursor peptide. The results of real-time quantitative polymerase chain reaction(RT-qPCR) showed that the expression level of PhAEP was the highest in fruits, followed by in roots, and the lowest in leaves. The heterophyllin A of P. heterophylla was detected in N. benthamiana that co-expressed PrePhHA and PhAEP genes instantaneously. In this study, the PhAEP gene, a key enzyme in the biosynthesis of heterophyllin A in P. heterophylla, has been successfully cloned, which lays a foundation for further analysis of the molecular mechanism of PhAEP enzyme in the biosynthesis of heterophyllin A in P. heterophylla and has important significance for the study of synthetic biology of cyclic peptide compounds in P. heterophylla.

C/EBPβ/AEP Signaling Drives Alzheimer's Disease Pathogenesis / 神经科学通报·英文版

Alzheimer's disease (AD) is the most common type of dementia. Almost two-thirds of patients with AD are female. The reason for the higher susceptibility to AD onset in women is unclear. However, hormone changes during the menopausal transition are known to be associated with AD. Most recently, we reported that follicle-stimulating hormone (FSH) promotes AD pathology and enhances cognitive dysfunctions via activating the CCAAT-enhancer-binding protein (C/EBPβ)/asparagine endopeptidase (AEP) pathway. This review summarizes our current understanding of the crucial role of the C/EBPβ/AEP pathway in driving AD pathogenesis by cleaving multiple critical AD players, including APP and Tau, explaining the roles and the mechanisms of FSH in increasing the susceptibility to AD in postmenopausal females. The FSH-C/EBPβ/AEP pathway may serve as a novel therapeutic target for the treatment of AD.

Reducing Acrylamide Exposure: A Review of the Application of Sulfur-Containing Compounds - A Caribbean Outlook

Acrylamide, a known neurotoxin, reproductive toxin, genotoxin, probable carcinogen, hepatotoxin, and immunotoxin, has sparked intense curiosity due to its prominent presence in thermally processed, carbohydrate-rich foods. Acrylamide formation occurs via the Maillard reaction at temperatures ≥100ºC. Thorough investigations on acrylamide mitigation through the application of sulfur-containing compounds to raw materials, and during food processing have been conducted. Although prominent results in acrylamide reduction have been observed, limitations are considered. These limitations involve the social and economic challenges of a population, such as the Caribbean. This study seeks to answer just how effective the application of sulfur-containing compounds is in reducing acrylamide exposure, especially when this applies to a developing region.

Development of a UPLC-MS/MS method for the therapeutic monitoring of L-asparaginase

This study aimed to develop a UPLC-MS/MS method for determining plasma levels of L-aspartic acid and L-asparagine and the activity of L-asparaginase. L-aspartic acid, L-asparagine, and L-aspartic acid-2,3,3-d3 were extracted from human plasma by protein precipitation with sulfosalicylic acid (30%, v/v). The plasma samples were analyzed using an Imtakt Intrada amino acid analysis column with 25 mM ammonium formate and 0.5% formic acid in acetonitrile as the mobile phase with step gradient method at a flow rate of 0.5 mL/min. The injection volume was 5 µL, and the total run time was 15 min. Inter- and intra-batch accuracies (%) ranged from 96.62–106.0% for L-aspartic acid and 89.85–104.8%, for L-asparagine, and the coefficient of variation (CV%) did not exceed 7%. The validation results for L-aspartic acid and L-asparagine satisfied the specified criterion, however, the results for L-asparaginase activity assay showed a borderline validity. This study could be a foundation for further development of therapeutic drug monitoring systems using UPLC-MS/MS.

Avaliação da resistência de formas mutantes da enzima L-asparaginase a proteases séricas humanas / Evaluation of L-asparaginase mutant forms resistance to human serum proteases

O tratamento para a Leucemia Linfoblástica Aguda LLA utiliza, entre outros fármacos, a enzima L-asparaginase (ASNase) proveniente da bactéria Escherichia coli. Reações imunológicas estão entre os problemas do tratamento com ASNase, e a formação de anticorpos contra essa proteína pode impedir o sucesso no tratamento. Duas cisteíno proteases lisossomais estão relacionadas com a degradação de ASNase nos seres humanos, a Catepsina B (CTSB) e Asparagina Endopeptidase (AEP). Em estudos prévios do nosso grupo obteve-se mutantes de ASNase resistentes a degradação por CTSB e/ou AEP in vitro. Nesse trabalho avaliamos essas mutantes quanto a sua citotoxicidade em linhagens celulares de leucemia e conduzimos estudos in vivo, aplicando as proteoformas de ASNases em camundongos Balb C para avaliar a atividade asparaginase sérica das enzimas ao longo do tempo, bem como obter informações sobre a formação de anticorpos contra essas proteoformas. Nos ensaios de citotoxicidade, duas das proteoformas testadas tiveram efeito citotóxico semelhante a forma selvagem, enquanto uma outra proteoforma tem a citotoxicidade sensivelmente reduzida. Já nos ensaios in vivo, uma proteoforma demonstrou meia vida sérica maior da atividade asparaginásica, e duas proteoformas causaram reduzida formação de anticorpos. Juntos, esses resultados colaboram para a obtenção de uma nova geração de ASNases com melhor biodisponibilidade, e efeitos adversos reduzidos, gerando a possibilidade de menores doses e frequência de aplicações

The Treatment for Acute Lymphoblastic Leukemia (ALL) includes the biopharmaceutical L-asparaginase (ASNase) from Escherichia coli. Immunological reactions are among the problems of treatment using ASNase, and the antibodies formation protein may prevent success in treatment. Lysosomal cysteine proteases are related to ASNase degradation, Cathepsin B (CTSB) and Asparagine Endopeptidase (AEP). In previous studies, ASNase mutants resistant to CTSB and / or AEP degradation in vitro were obtained. In this work, mutants were evaluated in cytotoxicity in ALL cell lines and, in vivo studies, applying doses of the wild and mutant ASNases in Balb C mice to evaluate serum asparaginase activity of the enzymes over time, as well as to obtain information on the formation of antibodies against these proteoforms. Regarding to the cytotoxicity, two proteoforms among the tested had similar cytotoxicity than the wild-type. While another proteoform had the cytotoxicity severely reduced. One proteoform have demonstrated greater serum half-life of asparaginase activity, while two other mutants caused reduced antibody formation. Together, these results collaborate to obtain a new generation of ASNases with increased bioavailability and reduced side effects, generating the possibility of lower doses and frequency of applications

Quantitative comparison of 68Ga-NGR and 18F-FDG uptake in well-differentiated hepatocellular carcinoma bearing mice / 中华核医学与分子影像杂志

Objective To quantitatively compare the diagnostic capability of 68Ga-NGR and 18F-FDG in well-differentiated hepatocellular carcinoma (HCC) bearing mice by microPET/CT imaging.Methods The in vitro cellular uptake,in vivo microPET/CT imaging and biodistribution studies of 68Ga-NGR and 18F-FDG were quantitatively compared in SMMC-7721-based well-differentiated HCC.The human fibrosarcoma (HT-1080) and human colorectal adenocarcinoma (HT-29) cells/xenografts were respectively used as positive and negative reference groups for CD13.The expression of CD13 was qualitatively verified by immunohistostaining.The levels of CD13 and glucose-6-phosphatase (G6Pase) were semi-quantitatively analyzed by Western blot test for all 3 types of tumors.Two-sample t test was used for data analysis.Results The in vitro cellular uptake showed that the 68Ga-NGR uptake in SMMC-7721 and HT-1080 cells was higher than that in HT-29 cells,and the 68Ga-NGR uptake was higher than 18F-FDG uptake in SMMC-7721 cells.The in vivo micro-PET/CT imaging results revealed that the uptake of 68Ga-NGR in SMMC-7721 tumor was (2.17±0.21) ％ID/g,remarkably higher compared to (0.73±0.26) ％ID/g of 18F-FDG uptake (t =8.826,P＜0.01).The tumor/liver ratio of 68Ga-NGR was 2.05±0.16,which was 2.03-fold higher than that of 18F-FDG.In the HT-1080 tumors,the uptakes of 68 Ga-NGR and 18F-FDG were both high,and the values were (2.46±0.23) ％ID/g,(3.47±0.31) ％ID/g.The uptake of 68Ga-NGR was significantly lower than that of 18F-FDG in HT-29 tumors:(0.67±0.20) ％ID/g vs (3.17±0.29) ％ID/g;t=4.221,P＜0.01.Western blot and immunohistostaining results were as follows:HT-1080(CD13+,G6Pase-),SMMC-7721(CD13+,G6Pase+),HT-29 (CD13-,G6Pase-).Conclusions The uptake of 68Ga-NGR is higher than 18F-FDG uptake in SMMC-7721 tumor bearing mice,therefore it is worthwhile to consider the feasibility of clinical translation for PET/CT in diagnosis of HCC.Furthermore,because of the difference in 68Ga-NGR and 18F-FDG avidities in tumors with different molecular phenotypes of CD13 and G6Pase,there is an underlying potential for molecular imaging in the determination of molecular phenotypes.

Quantitative comparison of 68Ga-NGR and 18F-FDG uptake in well-differentiated hepatocellular carcinoma bearing mice / 中华核医学与分子影像杂志

Objective To quantitatively compare the diagnostic capability of 68Ga-NGR and 18F-FDG in well-differentiated hepatocellular carcinoma (HCC) bearing mice by microPET/CT imaging.Methods The in vitro cellular uptake, in vivo microPET/CT imaging and biodistribution studies of 68Ga-NGR and 18F-FDG were quantitatively compared in SMMC-7721-based well-differentiated HCC.The human fibrosarcoma (HT-1080) and human colorectal adenocarcinoma (HT-29) cells/xenografts were respectively used as positive and negative reference groups for CD13.The expression of CD13 was qualitatively verified by immunohistostaining.The levels of CD13 and glucose-6-phosphatase (G6Pase) were semi-quantitatively analyzed by Western blot test for all 3 types of tumors.Two-sample t test was used for data analysis.Results The in vitro cellular uptake showed that the 68Ga-NGR uptake in SMMC-7721 and HT-1080 cells was higher than that in HT-29 cells, and the 68Ga-NGR uptake was higher than 18F-FDG uptake in SMMC-7721 cells.The in vivo microPET/CT imaging results revealed that the uptake of 68Ga-NGR in SMMC-7721 tumor was (2.17±0.21) %ID/g, remarkably higher compared to (0.73±0.26) %ID/g of 18F-FDG uptake (t=8.826, P<0.01).The tumor/liver ratio of 68Ga-NGR was 2.05±0.16, which was 2.03-fold higher than that of 18F-FDG.In the HT-1080 tumors, the uptakes of 68Ga-NGR and 18F-FDG were both high, and the values were (2.46±0.23) %ID/g, (3.47±0.31) %ID/g.The uptake of 68Ga-NGR was significantly lower than that of 18F-FDG in HT-29 tumors: (0.67±0.20) %ID/g vs (3.17±0.29) %ID/g;t=4.221, P<0.01.Western blot and immunohistostaining results were as follows: HT-1080(CD13+, G6Pase-), SMMC-7721(CD13+, G6Pase+), HT-29(CD13-, G6Pase-).Conclusions The uptake of 68Ga-NGR is higher than 18F-FDG uptake in SMMC-7721 tumor bearing mice, therefore it is worthwhile to consider the feasibility of clinical translation for PET/CT in diagnosis of HCC.Furthermore, because of the difference in 68Ga-NGR and 18F-FDG avidities in tumors with different molecular phenotypes of CD13 and G6Pase, there is an underlying potential for molecular imaging in the determination of molecular phenotypes.

Optimization of asparaginase production from Zymomonas mobilis by continuous fermentation / Otimização da produção de asparaginase de Zymomonas mobilis por fermentação contínua

A asparaginase é uma enzima usada em tratamento clínico como agente quimioterapêutico e em tecnologia de alimentos na prevenção de formação de acrilamida em alimentos fritos e assados. Asparaginase é industrialmente produzida por micro-organismos, principalmente bactérias gram negativas. Zymomonas mobilis é uma bactéria gram negativa que utiliza glicose, frutose e sacarose como fonte de carbono e é conhecida por sua eficiência para produzir etanol, sorbitol, levana, ácido glicônico, e mais recentemente, tem despertado interesse no uso desse micro-organismo na produção de asparaginase. Este trabalho teve como objetivo otimizar a produção de asparaginase de Z. mobilis por fermentação contínua, pelo uso do delineamento experimental e da metodologia da superfície de resposta, testando as variáveis: sacarose, extrato de levedura e asparagina. A condição ótima alcançada, com produção de 117,45 UI L-1 foi na taxa de diluição 0,20 h-1, utilizando 0,5 g L-1 de extrato de levedura, 20 g L-1 de sacarose e 1,3 g L-1 de asparagina. Observou-se que a relação carbono:nitrogênio (1:0,025) exerceu forte influência na resposta da atividade de asparaginase. A utilização de Z. mobilis por fermentação contínua demonstrou ser uma alternativa promissora na produção biotecnológica da asparaginase.

Asparaginase is an enzyme used in clinical treatments as a chemotherapeutic agent and in food technology to prevent acrylamide formation in fried and baked foods. Asparaginase is industrially produced by microorganisms, mainly gram-negative bacteria. Zymomonas mobilis is a Gram-negative bacterium that utilizes glucose, fructose and sucrose as carbon source and has been known for its efficiency in producing ethanol, sorbitol, levan, gluconic acid and has recently aroused interest for asparaginase production. Current assay optimizes the production of Z. mobilis asparaginase by continuous fermentation using response surface experimental design and methodology. The studied variables comprised sucrose, yeast extract and asparagine. Optimized condition obtained 117.45 IU L-1 with dilution rate 0.20 h-1, yeast extract 0.5 g L-1, sucrose 20 g L-1 and asparagine 1.3 g L-1. Moreover, carbon:nitrogen ratio (1:0.025) strongly affected the response of asparaginase activity. The use of Z. mobilis by continuous fermentation has proved to be a promising alternative for the biotechnological production of asparaginase.

Optimal labeling conditions of 68Ga-DOTA-iNGR and its biodistribution in mice / 中华核医学与分子影像杂志

Objective To explore the optimal conditions of preparing 68Ga-DOTA-iNGR (NGR peptide containing CendR motif),to evaluate its biodistribution in normal mice and to perform microPET imaging in tumor-bearing nude mice.Methods 68Ga fresh eluent (200 μl,92.5-129.5 MBq) obtaining with 68Ge-68Ga radionuclide generator was used to label DOTA-iNGR.The optimal conditions of labeling including pH,temperature,reacting time and concentration of DOTA-iNGR were determined.Then,the in vitro and in vivo stability and octanol/water partition coefficient of 68Ga-DOTA-iNGR were further analyzed.The biodistribution in normal Kunming mice was examined at 10,20,40,60 and 120 min after injection of 68Ga-DOTA-iNGR.Nude mice bearing HT-1080 (CDl3-positive) and HT-29 (CDl3-negative) tumors were established and underwent microPET imaging at 1 h after the intravenous injection of 68Ga-DOTA-iNGR.Data were analyzed using independent-sample t test.Results The optimal conditions of labeling was mixing 2 μg DOTA-iNGR peptide with 200 μl 68Ga (92.5-129.5 MBq) at pH 4.0,temperature 90-100 ℃ for 5-10 min.Under this condition,labeling rate reached (97.5± 1.3)％.The radiochemical purity of 68Ga-DOTA-iNGR in both saline (room temperature) and mouse serum (37 C) were both above 95％ after 4 h incubation,and the radiochemical purity in urine was greater than 85％ after 1 h metabolism in vivo.The partition coefficient was-2.71±0.18.In normal mice,majority of 68Ga-DOTA-iNGR was excreted from kidneys with a rapid clearance from blood.The in vivo microPET imaging showed that 68Ga-DOTA-iNGR was remarkably accumulated in the CD13-positive HT-1080 tumor.Conclusions Labeling DOTA-iNGR with 68Ga under our condition is a simple and efficient procedure with high labeling rate and high specificity.The product 68Ga-DOTA-iNGR has high stability,ideal biodistribution,and specific binding to CD13-positive tumor,which means that it's a very promising molecular probe for noninvasively detecting CD13-positive tumor.

Thermodynamic study of asparagine and glycyl-asparagine using computational methods

This work aimed to develop an ab initio procedure for accurately calculating pKa values and applied it to study the acidity of asparagine and glycyl-asparagine. DFT methods with B3LYP composed by 6-31+G(d) basis set were applied for calculating the acidic dissociation constant of asparagine and glycyl-asparagine. The formation of intermolecular hydrogen bonds between the available species and water was analyzed using Tomasi,s method. Results showed that in alkaline solutions, the cation, anion and neutral species of asparagine and glycyl-asparagine were solvated with one, two, three and four molecules of water, respectively. There was an excellent similarity between the experimentally attained pKa values and the theoretically ones in this work.

Synthesis of poly asparagine derivatives and its cytotoxicity study / 天津医药

Objective To synthesize poly asparagine derivatives and to evaluate its safety at the cellular level, which provide research platform for its potential application as drug carrier. Methods Polysuccinimide was synthesized by ther?mal polymerization of L-polyaspartic acid, and the target product of PSI-Phe-EA was obtained by the ring-opening reaction of polysuccinimide using L-phenylalanine methyl ester hydrochloride and ethanol amine. The structure of PSI-Phe-EA were characterized by 1H NMR. The rate of ring-opening of PSI was calculated by internal standard method of 1H NMR. The change of hydrophilicity was studied by the comparison of solubility. The cytotoxicity and morphology modification by PSI-Phe-EA at designate concentrations was investigated by MTT method and inverted microscopy respectively. The effects on cell cycles were analyzed by flow cytometry after propidium iodide (PI) staining. Results 1H NMR results confirmed the structure of PSI-Phe-EA and the ring-openning rate of PSI was 40%. The hydrophilicity of PSI-Phe-EA was greatly in?creased upon ring opening using ethanol amine. MTT test showed that the cell survival rates of NIH 3T3 and HepG2 cells were higher than 80%under the examined concentration (<100 mg/L). Inverted microscopy showed that 50 mg/L of PSI-Phe-EA treatment had no adverse effects on cell morphology. Cell cycle analysis indicated that PSI-Phe-EA treatment had no in?fluence on cell cycles of NIH 3T3 and HepG2 cell lines. Conclusion PSI-Phe-EA showed high hydrophilicity without sig?nificant effects on the cells survival, cells morphology and cell cycles. It is a kind of safe polymer material.

Autistic children exhibit distinct plasma amino acid profile.

In order to ascertain whether autistic children display characteristic metabolic signatures that are of diagnostic value, plasma amino acid analyses were carried out on a cohort of 138 autistic children and 138 normal controls using reverse-phase HPLC. Pre-column derivatization of amino acids with phenyl isothiocyanate forms phenyl thio-carbamate derivates that have a λmax of 254 nm, enabling their detection using photodiode array. Autistic children showed elevated levels of glutamic acid (120 ± 89 vs. 83 ± 35 mmol/L) and asparagine (85 ± 37 vs. 47 ± 19 mmol/L); lower levels of phenylalanine (45 ± 20 vs. 59 ± 18 mmol/L), tryptophan (24 ± 11 vs. 41 ± 16 mmol/L), methionine (22 ± 9 vs. 28 ± 9 mmol/L) and histidine (45 ± 21 vs. 58 ± 15 mmol/L). A low molar ratio of (tryptophan/large neutral amino acids) × 100 was observed in autism (5.4 vs 9.2), indicating lesser availability of tryptophan for neurotransmitter serotonin synthesis. To conclude, elevated levels of excitatory amino acids (glutamate and asparagine), decreased essential amino acids (phenylalanine, tryptophan and methionine) and decreased precursors of neurotransmitters (tyrosine and tryptophan) are the distinct characteristics of plasma amino acid profile of autistic children. Thus, such metabolic signatures might be useful tools for early diagnosis of autism.

Effect of salt on the growth and metabolism of Glycine max

Soybean plants cultivated with 50, 100 and 200 mM of NaCl, revealed that root growth was less affected by salinity than shoots. Salinity led to a reduction in leaf area and an increase in water content of the roots. These factors could contribute to the adaptation of the plant, improving its hydration. Although nitrate and free amino acid levels were reduced by salt treatment in roots, protein content of leaves was not altered. Salinity led to alterations in xylem amino acid composition, with increases in Ser, Ala, Gaba and Pro and a decrease in Asn. Similar changes were seen for Asn and Ser in roots together with a much stronger increase in Gaba. It is suggested that the decline in Asn reflects its conversion to Ala and Gaba (via Glu) in the roots while the increase in Pro and Gaba could be related to the adaptation of the plant to salinity.

L-asparaginase: A promising enzyme for treatment of acute lymphoblastic leukiemia.

Asparaginases are known to be the cornerstone for treatment of acute lymphoblastic leukemia (ALL) and are used for treatment in all pediatric regimens as well as in the majority of adult treatment protocols. Clinical hypersensitivity reactions against commercially available asparaginase have resulted in failure of asparaginase in treatment of ALL in more than 60% of cases. Thus, it is required to search for serologically different asparaginase from new organisms for the patients exhibiting sensitivity to one formulation of asparaginase, so that they can be switched to another to ensure that they receive the most efficacious treatment regimen possible. The present study report E. coli VRY-15, E. coli VRY-8 and E. coli VRY-14 as potent producer of L-asparaginase. The L-asparaginase obtained from E. coli VRY-15 showed highest specific activity i.e., 19.56 µmol/mg. Attempt was made to purify the enzyme. Molecular weight of purified L-asparaginase obtained from E. coli VRY-15 was found to be 56 K Da as determined using Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE).

Asparagine Synthetase Is Partially Localized to the Plasma Membrane and Upregulated by L-asparaginase in U937 Cells / 华中科技大学学报（医学）（英德文版）

This study investigated the intracellular localization of asparagine synthetase (ASNS) in the relation with chemoresistance in leukemia.pIRES-GFP-ASNS-Flag/Neo expression vector was transiently tansfected into SK-N-MC cells and 297T cells respectively.Immunofluorescence and Western blot analysis were performed for cellular localization of ASNS respectively.U937 cells were treated with L-asparaginase for 48 h and examined for endogenous ASNS expression on plasma membrane by immunofluorescence staining.Immunofluorescence staining showed that the transiently expressed ASNS was partly localized on transfected-SK-N-MC cell surface.Moreover,Western blotting exhibited that ASNS expressed both in cytosol and on plasma membrane of transfected-293T cells.Immunofluo-rescence staining with anti-ASNS-specific monoclonal antibody revealed that endogenous ASNS was localized on the plasma membrane of U937 cells,except for its distribution in the cytosol.In addition,ASNS exhibited a higher expression on plasma membrane after treatment with L-asparaginase as compared with the untreated cells.It was concluded that the subcellular translocation of ASNS may play an important role in L-asparaginase resistance in leukemia cells.

Cloning of asparagine synthetase gene and preparation,identification of the monoclonal antibody against asparagine synthetase / 中华检验医学杂志

Objective To clone human asparagine synthetase(ASNS)gene,express the MS2- ASNS fusion protein through gene engineering and use the purified target protein to immune BALB/C mice, prepare and identify the monoclonal antibody(McAb),which forms the base for studying mechanism of L- asparaginase used as salvage regimen in midline NK/T cell lymphoma nasal type.Methods ASNS gene fragment was amplified by RT-PCR from HepG2 cell line and constructed into prokaryocytic expression vector.Fusion-protein of MS2-ASNS was expressed and used for immunizing BALB/C mice to prepare McAbs against ASNS.Identified the McAb and detected the expression of ASNS in tumor.Results Part of the ASNS reading frame(NCBI,M27396:179-1 420 bp)was cloned and product length of RT-PCR was 1 263 bp.Molecular weight of MS2-ASNS was about 54 700 Da.Two strains of hybridoma secreting ASNS McAbs were obtained.The subtype of the ASNS McAb was IgG2a.Western-blot showed that the McAbs could specifically react with MS2-ASNS fusion protein and ASNS protein in tumor cell lines.ASNS expression was detected by immunocytochemistry and Immunohistochemisrry.Conclusion We have cloned human ASNS gene,obtained the anti-ASNS McAb and examined the expression of ASNS in tumor.