Abstract
BackgroundNever 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).ResultsThe 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.ConclusionsAn 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.
Highlights
Never in mitosis gene-A (NIMA)-related expressed kinase 2 (NEK2) is a serine/threonine protein kinase regulated by the cell cycle
The pET30a-NEK2 recombinant plasmid was transformed into E. coli trans5ɑ cells, and PCR confirmed that the positive clones were consistent with the size of the NEK2 gene (Fig. 1a, lane 2)
Expression and identification of the NEK2 fusion protein The recombinant strain was induced by IPTG, and the results showed that the precipitated components of the recombinant strain after IPTG induction exhibited specific clear bands at approximately 58 kDa (Fig. 1c), indicating that the induced recombinant human NEK2 protein mainly existed in the form of inclusion bodies. (See figure on page.) Fig. 1 Cloning and Expression of NEK2 in prokaryotic system. a The construction of the pET30a-NEK2 vector
Summary
Never in mitosis gene-A (NIMA)-related expressed kinase 2 (NEK2) is a serine/threonine protein kinase regulated by the cell cycle. Never in mitosis Gene-A (NIMA)-related expressed kinase 2 (NEK2), a serine/threonine protein kinase regulated by the cell cycle, is localized in centrosomes [1]. NEK2 regulates the cell cycle and mitosis through centrosome separation during mitosis. Abnormal expression of NEK2 can lead to chromosomal division abnormalities, resulting in chromosome instability and the formation of polyploid cells, promoting tumorigenesis [3]. Many studies reported that NEK2 was highly expressed in a variety of malignant tumors, such as liver cancer [5], gastric cancer [6], head and neck squamous cell carcinoma [7], and bladder
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