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Expression and Purification of Human Coagulation Factor X in Mammalian CHO-DG44 Cells
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作者 Jinwu CHEN Yi LI +4 位作者 Mei LIU Sainan WANG Zilong XIAO Junjie XIA Lulu QI 《Agricultural Biotechnology》 CAS 2023年第3期50-54,共5页
[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different ... [Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX. 展开更多
关键词 Recombinant human coagulation factor x(rhFx) Eukaryotic expression MTx affinity chromatography
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XAF1 is frequently methylated in human esophageal cancer 被引量:10
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作者 Xiang-Yu Chen Qiao-Yu He Ming-Zhou Guo 《World Journal of Gastroenterology》 SCIE CAS CSCD 2012年第22期2844-2849,共6页
AIM: To explore epigenetic changes in the gene encod- ing X chromosome-linked inhibitor of apoptosis-associ- ated factor 1 (XAF1) during esophageal carcinogenesis. METHODS: Methylation status of XAF1 was detected ... AIM: To explore epigenetic changes in the gene encod- ing X chromosome-linked inhibitor of apoptosis-associ- ated factor 1 (XAF1) during esophageal carcinogenesis. METHODS: Methylation status of XAF1 was detected by methylation-specific polymerase chain reaction (MSP) in four esophageal cancer cell lines (KYSE30, KYSE70, BICl and partially methylated in TE3 cell lines), nine cases of normal mucosa, 72 cases of pri- mary esophageal cancer and matched adjacent tissue. XAF1 expression was examined by semi-quantitative reverse transcriptional polymerase chain reaction and Western blotting before and after treatment with 5-aza- deoxycytidine (5-aza-dc), a demethylating agent. To investigate the correlation of XAF1 expression and methylation status in primary esophageal cancer, immu- nohistochemistry for XAF1 expression was performed in 32 cases of esophageal cancer and matched adjacent tissue. The association of methylation status and clini-copathological data was analyzed by logistic regression. RESULTS: MSP results were as follows: loss of XAF1 expression was found in three of four esophageal cell lines with promoter region hypermethylation (com- pletely methylated in KYSE30, KYSE70 and BIC1 cell lines and partially in TE3 cells); all nine cases of normal esophageal mucosa were unmethylated; and 54/72 (75.00%) samples from patients with esophageal can- cer were methylated, and 25/72 (34.70%) matched adjacent tissues were methylated (75.00% vs 34,70%, z2 = 23.5840, P = 0.000). mRNA level of XAF1 mea- sured with semi-quantitative reverse transcription poly- merase chain reaction was detectable only in TE3 cells, and no expression was detected in KYSE30, KYSE70 or BIC1 cells. Protein expression was not observed in KYSE30 cells by Western blotting before treatment with 5-aza-dc. After treatment, mRNA level of XAF1 was detectable in KYSE30, KYSE70 and BIC1 cells. Protein expression was detected in KYSE30 after treatment with 5-aza-dc. Immunohistochemistry was performed on 32 cases of esophageal cancer and adjacent tissue, and demonstrated XAF1 in the nucleus and cytoplasm. XAF1 staining was found in 20/32 samples of adjacent normal tissue but was present in only 8/32 samples of esophageal cancer tissue (Z2= 9.143, P = 0.002). XAF1 expression was decreased in cancer samples compared with adjacent tissues. In 32 cases of esophageal can- cer, 24/32 samples were methylated, and 8/32 esopha- geal cancer tissues were unmethylated. XAF1 staining was found in 6/8 samples of unmethylated esophageal cancer and 2/24 samples of methylated esophageal cancer tissue. XAF1 staining was inversely correlated with XAF1 promoter region methylation (Fisher's exact test, P = 0.004). Regarding methylation status and clinicopathological data, no significant differences were found in sex, age, tumor size, tumor stage, or metas- tasis with respect to methylation of XAF1 for the 72 tis- sue samples from patients with esophageal cancer. CONCLUSION: XAF1 is frequently methylated in eso- phageal cancer, and XAF1 expression is regulated by promoter region hypermethylation. 展开更多
关键词 x chromosome-linked inhibitor of apoptosis-associated factor 1 Esophageal cancer METHYLaTION Methylation-specific polymerase chain reaction Semi-quantitative reverse transcriptional polymerase chainreaction
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Isolated coagulopathy without classic CRAB symptoms as the initial manifestation of multiple myeloma: A case report 被引量:1
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作者 Ya Zhang Fang Xu +2 位作者 Jing-Jing Wen Lin Shi Qiao-Lin Zhou 《World Journal of Clinical Cases》 SCIE 2022年第12期3822-3827,共6页
BACKGROUND Multiple myeloma patients usually present with CRAB symptoms(hypercalcemia,renal disease,anemia and bone diseases)as initial manifestations.Bleeding symptoms are less common,most of which result from thromb... BACKGROUND Multiple myeloma patients usually present with CRAB symptoms(hypercalcemia,renal disease,anemia and bone diseases)as initial manifestations.Bleeding symptoms are less common,most of which result from thrombocytopenia or infiltration of plasmacytoma.Relatively,coagulopathy is not so common,especially isolated coagulopathy without CRAB manifestations,which is very rare.Herein,we report a 54-year old female who was hospitalized for intermittent and recurrent mild oral mucosal hemorrhage without other bleeding symptoms for almost one month or typical myeloma features.CASE SUMMARY Two months before admission,the patient underwent implantation of a permanent pacemaker due to sick sinus syndrome.Prothrombin time and activated partial thromboplastin time were significantly prolonged.Factor X deficiency was demonstrated to account for the coagulation dysfunction.An M protein peak was shown by serum protein electrophoresis.26.11%of abnormal plasma cells were detected in bone marrow by flow cytometry,expressing CD38,CD138,CD56 and intracellular immunoglobulin Kappa light chain.Bone marrow biopsy also proved the presence of abnormal plasma cells,but Congo red stain was negative.The patient was finally diagnosed with multiple myeloma IgA-κtype.A literature review indicated that factor X deficiency was highly related to amyloidosis.Before bleeding signs,the patient had cardiac arrhythmia,enlargement of the heart,and progressive heart failure;thus,cardiac amyloidosis was suspected.CONCLUSION Bleeding related to coagulation dysfunction is uncommon in multiple myeloma,especially as the initial manifestation.Amyloidosis is a well-recognized cause of isolated acquired factor X deficiency. 展开更多
关键词 Multiple myeloma Coagulation function HEMORRHaGE factor x Deficiency Case report
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Unitary Bases and Noncommutative Wavelets 被引量:4
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作者 WeiGUO 《Acta Mathematica Sinica,English Series》 SCIE CSCD 2003年第3期591-594,共4页
In this paper, we construct new unitary bases in noncommutative wavelets.
关键词 Type x1 factor Trace Von Neumann algebra Irrational rotation algebras
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