Construction, expression andin vitro biological behaviors of Ig scFv fragment in patients with chronic B cell leukemia
| العنوان: | Construction, expression andin vitro biological behaviors of Ig scFv fragment in patients with chronic B cell leukemia |
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| المؤلفون: | Zhu Lijuan, Liao Wenjun, Lei Ping, Shao Jingfang, Shen Guanxin, Zhang Yue, Wang Zhi-hua, Zhu Huifen |
| المصدر: | Journal of Huazhong University of Science and Technology [Medical Sciences]. 26:157-160 |
| بيانات النشر: | Springer Science and Business Media LLC, 2006. |
| سنة النشر: | 2006 |
| مصطلحات موضوعية: | Genes, Immunoglobulin Heavy Chain, Recombinant Fusion Proteins, Genetic Vectors, Immunoglobulin Variable Region, Biomedical Engineering, lac operon, chemical and pharmacologic phenomena, Biology, Immunoglobulin light chain, Biochemistry, Biomaterials, Plasmid, Escherichia coli, Genetics, medicine, Humans, Cloning, Molecular, Cells, Cultured, B cell, Cell Proliferation, Earth-Surface Processes, Expression vector, respiratory system, medicine.disease, Leukemia, Lymphocytic, Chronic, B-Cell, Molecular biology, Fusion protein, In vitro, medicine.anatomical_structure, B-cell leukemia, Leukocytes, Mononuclear, Electrophoresis, Polyacrylamide Gel, Genes, Immunoglobulin Light Chain, Single-Chain Antibodies |
| الوصف: | The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDS-PAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD= 0.9921 ku), which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of Id-HSP complex vaccine for B-CLL. |
| تدمد: | 1993-1352 1672-0733 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::44daf07902322db0ba3c3bfb864d314dTest https://doi.org/10.1007/bf02895803Test |
| حقوق: | CLOSED |
| رقم الانضمام: | edsair.doi.dedup.....44daf07902322db0ba3c3bfb864d314d |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 19931352 16720733 |
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