Biomedical subjects

Ying Huang

Publications and source records attributed to Ying Huang.

At least 55 records · Page 3Linked to original sources

PubMed2006

Comparative proteomic analysis of hypoxia-treated and untreated human leukemic U937 cells.

We reported recently that moderate hypoxia and hypoxia-mimetic agents could induce growth arrest and differentiation of leukemic cells via the mediation of hypoxia-inducible factor 1 alpha (HIF-1alpha), but the exact molecular mechanisms remain largely unknown. In this study, human acute promonocytic leukemic U937 cells were incubated under 2% O2 or in 50 microM of the hypoxia mimetic agent cobalt chloride (CoCl2) and normal oxygen for 24 h, and their protein expression profiles were compared by 2-DE coupled with MALDI-TOF/TOF MS/MS. We identified 62 and 16 proteins that were significantly deregulated by hypoxia and CoCl2 treatment, respectively. These proteins were mainly involved in metabolism, gene expression regulation, signal transduction, cell proliferation, differentiation and apoptosis. As an example, N-myc downstream regulated gene 1 (NDRG1), a putative differentiation-related gene, was up-regulated in both 2% O2- and CoCl2-treated U937 cells. Moreover, enforced HIF-1alpha expression also elevated NDRG1 mRNA and protein in U937 cells. These data will provide some clues for understanding mechanisms by which leukemic cells response to hypoxia.

Apoptosis↗

PubMed2006

Effects of dopamine receptor type 1 and Gs protein alpha subunit gene polymorphisms on blood pressure at rest and in response to stress.

BACKGROUND: Exaggerated blood pressure (BP) response to behavioral stress has been recognized as an independent predictor of increased BP levels in human subjects. The coupling of dopamine receptor type 1 (DRD1) with stimulatory G-proteins plays an important role in BP regulation. Therefore, we investigated whether the A-48G polymorphism of the DRD1 gene and the T393C polymorphism of the Gs protein alpha subunit (GNAS) gene influenced BP levels at rest and in response to stress. METHODS: A total of 912 white and African American (44.5%) twin subjects (17.4 +/- 3.4 years) were genotyped. Systolic and diastolic BP were measured at rest and during two 10-min stress tasks (a social competence interview and a virtual reality car driving simulation test). Regular association analyses and sibpair transmission disequilibrium tests were performed. The two polymorphisms, A-48G (DRD1) and T393C (GNAS), were genotyped by polymerase chain reaction with restriction digestion enzymes. RESULTS: A dominant BP-lowering effect of the -48G allele was observed on diastolic BP at rest (P = .032) and in response to the car driving simulation test (P = .046). The 393C allele was associated with higher systolic BP levels during the social competence interview (P = .024) and the car driving simulation test (P = .016). There were no statistical significances between the two loci for systolic and diastolic BP at rest and during stress. CONCLUSIONS: Our findings suggest that DRD1 and GNAS loci contribute to BP regulation at rest, and in particular, in response to behavioral stress. The BP measurement during behavioral stress may have added value for gene association studies.

Adolescent↗

PubMed2006

Ultrasound-guided hydrostatic reduction of intussusceptions by saline enema: a review of 5218 cases in 17 years.

BACKGROUND: The aim of this study was to evaluate the results of ultrasound (US)-guided hydrostatic reduction (UGSED) of intussusceptions in pediatric patients by saline enema. METHODS: Five thousand two hundred eighteen pediatric patients with intussusceptions treated by UGSED from October 1985 to October 2002 were reviewed retrospectively. RESULTS: The success rate of reduction in 5218 patients was 95.5%. Two hundred thirty-seven patients (4.5%) underwent surgery. Colonic perforation occurred in 9 patients (0.17%). Two infants suffered from milk aspiration because of vomiting during the hydrostatic enema reduction. There was no mortality. CONCLUSIONS: UGSED of intussusceptions avoids radiation exposure. It is reliable and safe. It has high success rate and minimal complications. It is a perfect method for the nonoperative treatment of pediatric intussusception and can be widely used as routine therapy.

Catheterization↗

PubMed2006

Crystal structure of the HP1-EMSY complex reveals an unusual mode of HP1 binding.

Heterochromatin protein-1 (HP1) plays an essential role in both the assembly of higher-order chromatin structure and epigenetic inheritance. The C-terminal chromo shadow domain (CSD) of HP1 is responsible for homodimerization and interaction with a number of chromatin-associated nonhistone proteins, including EMSY, which is a BRCA2-interacting protein that has been implicated in the development of breast and ovarian cancer. We have determined the crystal structure of the HP1beta CSD in complex with the N-terminal domain of EMSY at 1.8 A resolution. Surprisingly, the structure reveals that EMSY is bound by two HP1 CSD homodimers, and the binding sequences differ from the consensus HP1 binding motif PXVXL. This structural information expands our understanding of HP1 binding specificity and provides insights into interactions between HP1 homodimers that are likely to be important for heterochromatin formation.

Amino Acid Sequence↗

PubMed2006

Apoptosis and proliferation markers in diffusely infiltrating astrocytomas: profiling of 17 molecules.

Caspases and inhibitor of apoptosis proteins (IAPs) are antagonizing key apoptosis regulators. Limited studies of a few IAPs indicated their roles in astrocytomas. However, the overall expression status and significance of apoptosis regulators in astrocytomas is not clear. We examined the expression profile of the caspases (CASP3, 6, 7, 8, 9, 10, and 14), APAF1, SMAC, BCL2, the IAPs (BIRC5/survivin, CIAP1, CIAP2, XIAP, and LIVIN), and the proliferation markers Ki67 and PHH3 in 78 diffusely infiltrating astrocytomas and 24 normal brain samples by immunohistochemistry. Western blotting for major caspases and IAPs and reverse transcription-polymerase chain reaction analyses for IAPs were performed on a subset of 27 fresh samples. Our data showed BIRC5 nuclear labeling index (BIRC5-N) was the apoptosis marker most significantly different in World Health Organization grade II to IV astrocytomas and most strongly associated with proliferative activity. Expression level of other apoptosis-related proteins was modest or low in astrocytomas and did not correlate significantly with tumor grade or proliferation. Apoptosis regulators and proliferation markers were not detected in astrocytes of normal brain by immunostaining. This expression profile suggested involvement of apoptosis regulators in astrocytoma tumorigenesis, but tumor progression was more closely associated with proliferative advantages of which BIRC5 nuclear expression appeared to be a manifestation.

Adult↗

PubMed2006

Neutrotolerant acidophilic Streptomyces species isolated from acidic soils in China: Streptomyces guanduensis sp. nov., Streptomyces paucisporeus sp. nov., Streptomyces rubidus sp. nov. and Streptomyces yanglinensis sp. nov.

The taxonomic status of six neutrotolerant acidophilic streptomycetes isolated from acidic soils in Yunnan Province, China, was established using a polyphasic approach. The morphological and chemotaxonomic characteristics revealed that the isolates belong to the genus Streptomyces. Almost complete 16S rRNA gene sequences of the isolates were determined and aligned with available corresponding sequences of representatives of the family Streptomycetaceae; phylogenetic trees were inferred using four tree-making algorithms. The isolates formed a distinct, albeit heterogeneous, subclade in the Streptomyces 16S rRNA gene tree together with the type strain of Streptomyces yeochonensis, but were readily distinguishable from the latter using DNA-DNA hybridization and phenotypic data. It was evident from the genotypic and phenotypic data that the isolates belonged to four novel Streptomyces species, for which the following names are proposed: Streptomyces guanduensis sp. nov. (type strain 701T=CGMCC 4.2022T=JCM 13274T), Streptomyces paucisporeus sp. nov. (type strain 1413T=CGMCC 4.2025T=JCM 13276T), Streptomyces rubidus sp. nov. (type strain 13c15T=CGMCC 4.2026T=JCM 13277T) and Streptomyces yanglinensis sp. nov. (type strain 1307T=CGMCC 4.2023T=JCM 13275T); isolates 317 and 913 belong to this latter species.

Bacterial Typing Techniques↗

PubMed2006

Streptacidiphilus oryzae sp. nov., an actinomycete isolated from rice-field soil in Thailand.

The taxonomic position of ten acidophilic actinomycetes isolated from an acidic rice-field soil was established using a polyphasic approach. 16S rRNA gene sequences determined for the isolates were aligned with corresponding sequences of representatives of the genera Kitasatospora, Streptacidiphilus and Streptomyces and phylogenetic trees were inferred using four tree-making algorithms. The isolates had identical sequences and formed a distinct branch at the periphery of the Streptacidiphilus 16S rRNA gene tree. The chemotaxonomic and morphological properties of representative isolates were consistent with their assignment to the genus Streptacidiphilus. The isolates shared nearly identical phenotypic profiles that readily distinguished them from representatives of the established species of Streptacidiphilus. It is evident from the genotypic and phenotypic data that the isolates form a homogeneous group that corresponds to a novel species in the genus Streptacidiphilus. The name proposed for this new taxon is Streptacidiphilus oryzae sp. nov.; the type strain is strain TH49(T) (=CGMCC 4.2012(T) = JCM 13271(T)).

Actinobacteria↗

PubMed2006

Actinoalloteichus hymeniacidonis sp. nov., an actinomycete isolated from the marine sponge Hymeniacidon perleve.

A polyphasic study was undertaken to establish the taxonomic status of a Gram-positive, aerobic actinomycete, strain HPA177(T), isolated from a marine sponge, Hymeniacidon perleve. The organism formed branching, non-fragmenting vegetative hyphae and produced black pigment. Chemotaxonomic characteristics were consistent with its assignment to the genus Actinoalloteichus. Analysis of the 16S rRNA gene sequence showed that strain HPA177(T) formed a robust clade with type strains of the genus Actinoalloteichus, but was distinct from them. A number of phenotypic characteristics also readily distinguished strain HPA177(T) from species of the genus Actinoalloteichus with validly published names. On the basis of the above data, it is proposed that strain HPA177(T) represents a novel species, Actinoalloteichus hymeniacidonis sp. nov. The type strain of Actinoalloteichus hymeniacidonis is HPA177(T) (=CGMCC 4.2500(T)=JCM 13436(T)).

Actinomycetales↗

PubMed2006

Pseudonocardia oroxyli sp. nov., a novel actinomycete isolated from surface-sterilized Oroxylum indicum root.

A high-G+C-content, Gram-positive bacterium, strain D10(T), was isolated from the root of Oroxylum indicum, a Chinese medicinal plant. Based on 16S rRNA gene sequence analysis, strain D10(T) was a member of the genus Pseudonocardia and was most closely related, albeit loosely, to Pseudonocardia halophobica. Morphological and chemotaxonomic characteristics support the affiliation of strain D10(T) to the genus Pseudonocardia. Results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain D10(T) from related Pseudonocardia species. Strain D10(T) (=CGMCC 4.3143(T)=DSM 44984(T)) therefore represents a novel species, for which the name Pseudonocardia oroxyli sp. nov. is proposed.

Actinomycetales↗

PubMed2006

All-trans retinoic acid inhibited angiotensin II-induced increase in cell growth and collagen secretion of neonatal cardiac fibroblasts.

AIM: To determine whether all-trans retinoic acid (atRA) acts to modulate angiotensin II (Ang II)-induced cardiac fibroblast cell growth and collagen secretion. METHODS: Cultured neonatal rat cardiac fibroblasts (CF) were used in the experiment. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect cell growth of the CF; and immunocytochemistry and Western blotting were used to measure the production and secretion of collagen and the expression of transforming growth factor-beta1 (TGF-beta1) by the CF. RESULTS: atRA (10(-7) to 10(-5) mol/L) inhibited the Ang II-induced increase in cell growth of CF (P<0.05). Ang II stimulated the secretion of collagen types I and III by the CF. This effect was blocked by AT(1) receptor antagonist losartan (10(-6) mol/L), but not by AT2 receptor antagonist PD123319 (up to 10(-6) mol/L). Exposure of CF to atRA (10(-5) mol/L) attenuated the Ang II-induced increase in the secretion of collagen types I and III (P<0.05). atRA (10(-5) mol/L) also blocked the Ang II-induced increase in the expression of TGF-beta1. CONCLUSION: atRA inhibits the Ang II-induced increase in cell growth and collagen secretion of neonatal rat CF. The effect of atRA is possibly mediated by lowering the TGF-beta1 level. These observations support the notion that atRA is a potential candidate for the prevention and therapy of cardiac remodeling.

Angiotensin II↗

PubMed2006

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 use different receptor complexes to enter T cells.

Studies using adherent cell lines have shown that glucose transporter-1 (GLUT-1) can function as a receptor for human T-cell leukemia virus type 1 (HTLV). In primary CD4(+) T cells, heparan sulfate proteoglycans (HSPGs) are required for efficient entry of HTLV-1. Here, the roles of HSPGs and GLUT-1 in HTLV-1 and HTLV-2 Env-mediated binding and entry into primary T cells were studied. Examination of the cell surface of activated primary T cells revealed that CD4(+) T cells, the primary target of HTLV-1, expressed significantly higher levels of HSPGs than CD8(+) T cells. Conversely, CD8(+) T cells, the primary target of HTLV-2, expressed GLUT-1 at dramatically higher levels than CD4(+) T cells. Under these conditions, the HTLV-2 surface glycoprotein (SU) binding and viral entry were markedly higher on CD8(+) T cells while HTLV-1 SU binding and viral entry were higher on CD4(+) T cells. Binding studies with HTLV-1/HTLV-2 SU recombinants showed that preferential binding to CD4(+) T cells expressing high levels of HSPGs mapped to the C-terminal portion of SU. Transfection studies revealed that overexpression of GLUT-1 in CD4(+) T cells increased HTLV-2 entry, while expression of HSPGs on CD8(+) T cells increased entry of HTLV-1. These studies demonstrate that HTLV-1 and HTLV-2 differ in their T-cell entry requirements and suggest that the differences in the in vitro cellular tropism for transformation and in vivo pathobiology of these viruses reflect different interactions between their Env proteins and molecules on CD4(+) and CD8(+) T cells involved in entry.

Adult↗

PubMed2006

Altered proteolysis and global gene expression in hepatitis B virus X transgenic mouse liver.

Hepatitis B virus X (HBX) is essential for the productive infection of hepatitis B virus (HBV) in vivo and has a pleiotropic effect on host cells. We have previously demonstrated that the proteasome complex is a cellular target of HBX, that HBX alters the proteolytic activity of proteasome in vitro, and that inhibition of proteasome leads to enhanced viral replication, suggesting that HBX and proteasome interaction plays a crucial role in the life cycle and pathogenesis of HBV. In the present study, we tested the effect of HBX on the proteasome activities in vivo in a transgenic mouse model in which HBX expression is developmentally regulated by the mouse major urinary promoter in the liver. In addition, microarray analysis was performed to examine the effect of HBX expression on the global gene expression profile of the liver. The results showed that the peptidase activities of the proteasome were reduced in the HBX transgenic mouse liver, whereas the activity of another cellular protease was elevated, suggesting a compensatory mechanism in protein degradation. In the microarray analysis, diverse genes were altered in the HBX mouse livers and the number of genes with significant changes increased progressively with age. Functional clustering showed that a number of genes involved in transcription and cell growth were significantly affected in the HBX mice, possibly accounting for the observed pleiotropic effect of HBX. In particular, insulin-like growth factor-binding protein 1 was down-regulated in the HBX mouse liver. The down-regulation was similarly observed during acute woodchuck hepatitis virus infection. Other changes including up-regulation of proteolysis-related genes may also contribute to the profound alterations of liver functions in HBV infection.

Animals↗

PubMed2006

Improved efficiency of bovine cloning by autologous somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) has been used for the cloning of various mammals. However, the rates of successful, healthy birth are generally poor. To improve cloning efficiency, we report the utilization of an 'autologous SCNT' cloning technique in which the somatic nucleus of a female bovine donor is transferred to its own enucleated oocyte recovered by ovum pick up, in contrast to the routine 'allogeneic SCNT' procedure using oocytes from unrelated females. Our results showed that embryos derived from autologous SCNThave significantly higher developmental competence than those derived from allogeneic SCNT, especiallyat the eight-cell (60 vs 44%), morula (45 vs 36%), and blastocyst (38 vs 23%) stages. The pregnancy and birth rates were also higher for the autologous (39 and 23%), compared to the allogeneic (22 and 6%) SCNT groups. Genome-wide histone3-lysine9 methylation profiles reveal that autologous SCNTembryos have less epigenetic defects than the allogeneic SCNTembryos. This study indicates that autologous SCNT can improve the efficiency of bovine cloning with less reprogramming deficiency.

Animals↗

PubMed2006

[Time-resolved fluoroimmunoassay of carcino-embryonic antigen and preparation of its diagnostic reagent].

AIM: To establish a two-site time-resolved fluoroimmunoassay (TRFIA) for detection of carcino-embryonic antigen(CEA) based on the direct sandwich technique(One monoclonal antibody(mAb) is immobilized on the surface of microtiter plate strip wells, another mAb is labelled with Eu(3+)) and prepare its diagnostic reagent. METHODS: The sandwich TRFIA for detection of serum CEA level using anti-CEA mAbs was used. RESULTS: The measure range of CEA-TRFIA was (1-560) microg/L. The analytical sensitivity was 0.28 mug/L. The intra- and inter-assay coefficients of variation (CV) were 7.2%-8.6% and 8.9%-13.2%, respectively. There was no cross-reaction to AFP, CA12-5, CA19-9 and albumin. Cross reactivity to CA15-3 was 1.62 microg/L. The correlation coefficient of blood samples detected by CEA-TRFIA and commercially available CEA CLIA (chemiluminescent immunoassay) kit (Roche) was 0.946. CONCLUSION: CEA-TRFIA is a new immunoassay which has several advantages including high sensitivity, specificity and a wide working range. It is suitable for routine clinical use.

Carcinoembryonic Antigen↗

PubMed2006

[Studies of the possible mechanisms for protective effects of recombinant human growth hormone on intestinal mucosa barrier in rat sepsis].

OBJECTIVE: To investigate the protective effects of recombinant human growth hormone (rhGH) on intestinal mucosal barrier in rat sepsis and explore its possible mechanisms. METHODS: E. coli was injected intraperitoneally to produce rats sepsis models. Forty-two female SD rats were randomly divided into the control group (group C), sepsis group (group S) and treatment group (group T). Group S and group T were further divided into 1 d and 3 d subgroups (T1d,T3d, Sld, S3d), respectively. The expression of IGF-1 mRNA in liver, expression of Bcl-2 protein in intestine, bacteria translocation, the levels of growth hormone(GH) and insulin-like growth factor-1 (IGF-1) in plasma, and the histological appearance of intestine were determined dynamically by means of RT-PCR, radioimmunoassay, immunohistochemical staining and other corresponding methods, respectively. RESULTS: (1) rhGH could significantly attenuate intestinal mucosal injuries and ameliorate bacteria translocation on sepsis rats. (2) The levels of Bcl-2 protein expression in intestine in group T (T1d:2441 +/- 117; T3d: 3628 +/- 235) were obviously higher than those of group S (S1d: 321 +/- 36; S3d: 1873 +/- 57) (P < 0.01). (3) The plasma levels of GH in group T (T1d: 1.28 +/- 0.24 microg/L; T3d: 2.14 +/- 0.48 microg/L) increased markedly than those of group S (S1d: 0.74 -/+ 0.12 microg/L; S3d: 0.60 +/- 0.18 microg/L) (P < 0.01). (4) The plasma levels of IGF-1 in group T (Tld: 168.94 +/- 65.67 microg/L; T3d: 201.56 +/- 64.98 microg/L) elevated significantly than those of group S (Sld: 116.72 +/- 13.96 microg/L; S3d:107.50 +/- 23.53 microg/L) (P < 0.05). (5) The levels of liver IGF-1 mRNA in group T (Tld: 0.98 +/- 0.20; T3d: 1.76 +/- 0.17) were significantly higher than those in group S (S1d: 0.38 +/- 0.09; S3d: 0.46 +/- 0.10) (P < 0.01). CONCLUSION: rhGH conferred protective efficacy in maintaining the integrity of intestinal mucosal barrier against sepsis in rat. The possible mechanisms might involve the rhGH-diminished apoptosis of intestinal mucosa cells and the rhGH-maintained intestinal mucosa barrier via the roles of GH and IGF-1.

Animals↗

PubMed2006

[The role of retropharyngeal lymph node metastasis in staging of nasopharyngeal carcinoma].

OBJECTIVE: The prevalence of retropharyngeal lymph node (RLN) metastasis in nasopharyngeal carcinoma (NPC) is high, but its role in '92 Fuzhou staging system for NPC is uncertain. This study was to identify the prognostic value of RLN metastasis in NPC, and to evaluate its role in the staging system. METHODS: Clinical data of 749 untreated patients with histologically diagnosed NPC, without metastasis, presented between Jan. 1999 and Dec. 1999 at the Department of Radiation Oncology of Cancer Center of Sun Yat-sen University, were reviewed. All patients received enhanced spiral CT scan on the nasopharynx and whole neck before treatment. Cox regression model was used to investigate the prognostic value of RLN metastasis. According to the principle of the staging system, the indices of hazard consistency, hazard discrimination, and distribution were evaluated to identify the role of RLN metastasis in the staging system for NPC. RESULTS: The occurrence rate of RLN metastasis was 51.5%; the occurrence rates were significantly higher in the patients in advanced T stage, N stage, and clinical stage than in the patients in early stages (57.8% vs. 45.2%, P=0.001; 60.3% vs. 47.6%, P=0.001; 57.9% vs. 38.9%, P<0.001). The 5-year overall survival rate and 5-year distant metastasis-freely survival rate were significantly lower in the patients with RLN metastasis than in the patients without RLN metastasis (58.7% vs. 72.2%, P<0.001; 74.5% vs. 84.9%, P<0.001). In multivariate analysis, RLN metastasis was not a prognostic factor for overall survival, but borderline significant difference was observed for distant metastasis-freely survival (P=0.053). The hazard ratios of death and distant metastasis for N0 stage with RLN metastasis were 0.540 and 0.411, respectively, which was similar to those for N1 stage (0.601 and 0.555, respectively). Classifying RLN metastasis to N1 stage improved the hazard consistency in the N classification, but the distribution was unsatisfactory, and the proportion of N1 stage patients was 50.2%. Classifying RLN metastasis to T2 stage improved the hazard discrimination, with good balances of distribution found in T classification, N classification, and clinical staging. CONCLUSIONS: Based on the enhanced CT image, RLN metastasis tends to affect distant metastasis-freely survival of NPC patients. According to the principle of '92 Fuzhou staging system and present patterns of radiotherapy for the primary tumor, classifying RLN metastasis to T2 stage is more reasonable.

Adolescent↗

PubMed2006

[Irradiation field design according to locoregional relapse sites after radiotherapy of nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: The lateral faciocervical field, lateral facial field, and cerrobent blocks were employed conventionally in radiotherapy of nasopharyngeal carcinoma (NPC) in Cancer Center of Sun Yat-sen University since 1990s. This study was to explore rationality of irradiated area of lateral faciocervical field and lateral facial field through analyzing the data of some patients presenting locoregional recurrence after conventional radiotherapy. METHODS: Clinical data of the patients, treated in 1999 and presented locoregional failure and had reports of CT examinations before and after radiotherapy and after recurrence, were reviewed. The regions of recurrent lesions were analyzed. Dosimetric analysis of recurrent regions was performed for the patients with available data of irradiating fields. If no less than 95% of the volume of local recurrent lesion was within 95% isodose curve, it was defined as in-field failure; 20% to less than 95% of the volume was within 95% isodose curve as marginal failure; less than 20% of the volume was within 95% isodose curve as out-field failure. RESULTS: Clinical data of 40 patients could be analyzed, and dosimetric analysis of recurrent regions was performed for 15 patients. Local recurrences mainly occurred in the nasopharyngeal cavity and the skull base bone. Dosimetric analysis showed 9 cases of in-field failure, 4 cases of marginal failure, and 2 cases of out-field failure. For the 6 cases of marginal and out-field failure, the main reasons of recurrence were low dose area in target volume due to not strictly following the guidelines of defining field limit, or inappropriately estimating the involved regions of lesions. CONCLUSION: Irradiated area of lateral faciocervical field and lateral facial field employed conventionally in radiotherapy of NPC is rational.

Adult↗

PubMed2006

[Construction and expression of chimeric anti-human CD20 monoclonal antibody].

AIM: To construct the eukaryotic expression vector of chimeric anti-human CD20 monoclonal antibody (mAb) and realize its expression. METHODS: The light- and heavy-chain genes were amplified from hybridoma cell line 1-28 secreting anti-human CD20 mAb by RT-PCR and were cloned to T vector and sequenced. Proteins of mAb 1-28 were separated by reducing SDS-PAGE. Light- and heavy-chain bands were excised from preparative gel, digested by trypsin, and subjected to peptide mass fingerprinting. Software Biolynx and pepeseq were used to evaluate the score of probability. Correctness of the light- and heavy-chain DNA sequences was verified by their protein sequences. Genes of V(H) and V(L) were amplified from T vector and cloned into chimeric antibody expression vector (pCMV-V(H) and pCMV-V(L)), generating the expression vectors of chimeric anti-human CD20 mAb (C1-28) including light chain expression vector C1-28L and heavy chain expression vector C1-28H. The two plasmids were co-transfected into 293T cells with Lipofectamine 2000. RT-PCR was used to detect the transcription at mRNA level. C1-28 expression was detected by Sandwich ELISA and Western blot methods. RESULTS: mAb 1-28's genes were successfully cloned and verified by peptide mass fingerprinting. Eukaryotic expression vectors of anti-human CD20 mAb were constructed and expressed in 293T cells with the expression amount reaching 257 mg/L and the molecular weight consistent with that of human IgG. CONCLUSION: These experiments lay solid foundation for further study on the role of chimeric CD20 antibody in the treatment of non-Hodgkin's lymphoma.

Antibodies, Monoclonal↗