CENP-E combined protein and encoding gene and application
A CENP-E, binding protein technology, used in applications, gene therapy, genetic engineering, etc., can solve problems such as spindle checkpoint inactivation and chromosomal instability
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Embodiment 1
[0042] Embodiment 1, the acquisition of CENP-E binding protein CENP-V gene
[0043] Extract the total RNA of human testis tissue, synthesize its cDNA by reverse transcription and use it as a template, in the primer CENP-VF (upstream primer): 5'-ATGAACCAGCCGTGCAACTCGATGGAG-3' and CENP-VR (downstream primer): 5'-CTAGTCTAGGATGTCGCCACATTCCAG Under the guidance of -3', the wild-type human CENP-E binding protein CENP-V gene was amplified by PCR. After the reaction, the PCR amplification product was ligated into the vector pMD 18-T (TaKaRa Company), and then the ligated product was transformed into Escherichia coli (E.coli) DH5α competent cells, positive clones were screened, plasmids were extracted, and the target fragment was obtained. The recombinant plasmid named pGEM-CENP-V was sequenced, and the sequencing results showed that the wild-type human CENP-V gene had the nucleotide sequence of SEQ ID NO: 2 in the sequence listing, and SEQ ID NO: 2 in the sequence listing It consists...
Embodiment 2
[0044] Embodiment 2, the detection of CENP-V periodic expression in the cell
[0045] 1. Construction of wild-type CENP-V gene eukaryotic expression vector
[0046] The recombinant plasmid pGEM-CENP-V containing the wild-type CENP-V gene was double-digested with restriction endonucleases EcoR I and Xho I, the 1572bp target gene fragment was recovered and purified, and then ligated into the vector pEGF-C1 (Clontech Company), the ligation product was transformed into Escherichia coli DH5α competent cells, positive clones were screened, and plasmids were extracted to obtain the eukaryotic expression vector of the wild-type CENP-V gene, which was named pEGF-CENP-V.
[0047] 2. Construction of cell lines stably expressing CENP-V and its periodic expression detection
[0048] 1. Construction of cell lines stably expressing CENP-V
[0049] The eukaryotic expression plasmid of the wild-type CENP-V gene obtained in step 1 was transfected into Hela cells with Lipofectamine 2000 (Invit...
Embodiment 3
[0052] Example 3, Cell cycle localization of CENP-V and antibody detection of CENP-V
[0053] 1. Preparation of CENP-V Antibody
[0054] 1. Preparation of antigen
[0055] The coding sequence of the full-length amino acid residues of CENP-V was ligated into the HIS expression vector pET28a (Invitrogen Company), the ligation product was transformed into E. Screened on the LB resistance plate of the protein, pick out the single clone grown out, inoculate it into 5mL LB liquid medium, shake the bacteria at 37℃ for 12-18 hours, and then inoculate into 500mL at the ratio of 1:100 In LB liquid medium, shake culture at 37°C and 250rpm. When the OD value at 600nm reaches 0.6, add 0.2mg / LIPTG and induce at 37°C for 3 hours. After the cultivation, the cells were collected by centrifugation, resuspended in PBS, and the cells were lysed using a pressure breaker, and the protease inhibitor PMSF (purchased from Bio Basic Inc.) was added at the same time, and then centrifuged at 12000rpm f...
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