Preparation of an anti-NEK2 monoclonal antibody and its application in liver cancer
Abstract Background Never in mitosis gene-A (NIMA)-related expressed kinase 2 (NEK2) is a serine/threonine protein kinase regulated by the cell cycle. The purpose of this study was to obtain NEK2 protein to prepare an anti-NEK2 monoclonal antibody (mAb) and explore the application of the anti-NEK2 m...
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| Format: | Article |
| Language: | English |
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BMC
2021-10-01
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| Series: | BMC Biotechnology |
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| Online Access: | https://doi.org/10.1186/s12896-021-00717-3 |
| _version_ | 1818897020177350656 |
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| author | Qiuli Chen Hui Li Lichao Yang Sha Wen Xuejing Huang Jiajuan Liu Xiaoping Guo Bing Hu Gang Li Min He |
| author_facet | Qiuli Chen Hui Li Lichao Yang Sha Wen Xuejing Huang Jiajuan Liu Xiaoping Guo Bing Hu Gang Li Min He |
| author_sort | Qiuli Chen |
| collection | DOAJ |
| description | Abstract Background Never in mitosis gene-A (NIMA)-related expressed kinase 2 (NEK2) is a serine/threonine protein kinase regulated by the cell cycle. The purpose of this study was to obtain NEK2 protein to prepare an anti-NEK2 monoclonal antibody (mAb) and explore the application of the anti-NEK2 mAb of therapeutic and diagnostic in hepatocellular carcinoma (HCC). Results The NEK2 gene sequence was cloned from the normal liver cell line HL7702, and the full-length NEK2 gene sequence was cloned into the prokaryotic expression vector pET30a and transformed into Escherichia coli BL21 (DE3) cells. The recombinant fusion protein was obtained under optimized conditions and injected in BALB/c mice to prepare an anti-NEK2 mAb. By screening, we obtained a stable hybridoma cell line named 3A3 that could stably secrete anti-NEK2 mAb. Anti-NEK2 3A3 mAb was purified from ascites fluid. The isotype was IgG1, and the affinity constant (Kaff) was 6.0 × 108 L/mol. Western blot, indirect enzyme-linked immunosorbent assay (iELISA), immunofluorescence and immunocytochemical analyses showed that the mAb could specifically recognize the NEK2 protein. MTT assays showed that the mAb 3A3 could inhibit the proliferation of HCC cells. KEGG pathway analysis showed that NEK2 might affected pathways of the cell cycle. Moreover, NEK2-related genes were mainly enriched in the S and G2 phases and might act as tumor-promoting genes by regulating the S/G2 phase transition of HCC cells. Conclusions An anti-NEK2 mAb with high potency, high affinity and high specificity was prepared by prokaryotic expression system in this study and may be used in the establishment of ELISA detection kits and targeted treatment of liver cancer. |
| first_indexed | 2024-12-19T19:09:32Z |
| format | Article |
| id | doaj.art-7f49bb4c670b4901a5f1bbf2c6c163b3 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-12-19T19:09:32Z |
| publishDate | 2021-10-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-7f49bb4c670b4901a5f1bbf2c6c163b32022-12-21T20:09:20ZengBMCBMC Biotechnology1472-67502021-10-0121111210.1186/s12896-021-00717-3Preparation of an anti-NEK2 monoclonal antibody and its application in liver cancerQiuli Chen0Hui Li1Lichao Yang2Sha Wen3Xuejing Huang4Jiajuan Liu5Xiaoping Guo6Bing Hu7Gang Li8Min He9School of Public Health, Guangxi Medical UniversitySchool of Public Health, Guangxi Medical UniversitySchool of Public Health, Guangxi Medical UniversityLaboratory Animal Center of Guangxi Medical UniversityLaboratory Animal Center of Guangxi Medical UniversityLaboratory Animal Center of Guangxi Medical UniversityLaboratory Animal Center of Guangxi Medical UniversityLaboratory Animal Center of Guangxi Medical UniversitySchool of Public Health, Guangxi Medical UniversitySchool of Public Health, Guangxi Medical UniversityAbstract Background Never in mitosis gene-A (NIMA)-related expressed kinase 2 (NEK2) is a serine/threonine protein kinase regulated by the cell cycle. The purpose of this study was to obtain NEK2 protein to prepare an anti-NEK2 monoclonal antibody (mAb) and explore the application of the anti-NEK2 mAb of therapeutic and diagnostic in hepatocellular carcinoma (HCC). Results The NEK2 gene sequence was cloned from the normal liver cell line HL7702, and the full-length NEK2 gene sequence was cloned into the prokaryotic expression vector pET30a and transformed into Escherichia coli BL21 (DE3) cells. The recombinant fusion protein was obtained under optimized conditions and injected in BALB/c mice to prepare an anti-NEK2 mAb. By screening, we obtained a stable hybridoma cell line named 3A3 that could stably secrete anti-NEK2 mAb. Anti-NEK2 3A3 mAb was purified from ascites fluid. The isotype was IgG1, and the affinity constant (Kaff) was 6.0 × 108 L/mol. Western blot, indirect enzyme-linked immunosorbent assay (iELISA), immunofluorescence and immunocytochemical analyses showed that the mAb could specifically recognize the NEK2 protein. MTT assays showed that the mAb 3A3 could inhibit the proliferation of HCC cells. KEGG pathway analysis showed that NEK2 might affected pathways of the cell cycle. Moreover, NEK2-related genes were mainly enriched in the S and G2 phases and might act as tumor-promoting genes by regulating the S/G2 phase transition of HCC cells. Conclusions An anti-NEK2 mAb with high potency, high affinity and high specificity was prepared by prokaryotic expression system in this study and may be used in the establishment of ELISA detection kits and targeted treatment of liver cancer.https://doi.org/10.1186/s12896-021-00717-3NEK2Prokaryotic expressionMonoclonal antibodyHepatocellular carcinomaCell cycle |
| spellingShingle | Qiuli Chen Hui Li Lichao Yang Sha Wen Xuejing Huang Jiajuan Liu Xiaoping Guo Bing Hu Gang Li Min He Preparation of an anti-NEK2 monoclonal antibody and its application in liver cancer BMC Biotechnology NEK2 Prokaryotic expression Monoclonal antibody Hepatocellular carcinoma Cell cycle |
| title | Preparation of an anti-NEK2 monoclonal antibody and its application in liver cancer |
| title_full | Preparation of an anti-NEK2 monoclonal antibody and its application in liver cancer |
| title_fullStr | Preparation of an anti-NEK2 monoclonal antibody and its application in liver cancer |
| title_full_unstemmed | Preparation of an anti-NEK2 monoclonal antibody and its application in liver cancer |
| title_short | Preparation of an anti-NEK2 monoclonal antibody and its application in liver cancer |
| title_sort | preparation of an anti nek2 monoclonal antibody and its application in liver cancer |
| topic | NEK2 Prokaryotic expression Monoclonal antibody Hepatocellular carcinoma Cell cycle |
| url | https://doi.org/10.1186/s12896-021-00717-3 |
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