Coiled-coil domain containing protein 8 (CCDC8) having inhibition on HIV-1 and application thereof
A coiled-coil structure, HIV-1 technology, applied in the field of biomedicine, can solve the side effects of HIV, can not completely cure AIDS, drug resistance and other problems
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Embodiment 1
[0024] Example 1, HIV-1 resistance experiment of CCDC8
[0025] First, the plasmid pTT5-SH5-CCDC8 expressing CCDC8 was constructed. The vector pTT5-SH5 was constructed by Yves Durocher, Biotechnology Research Institute, Montreal, Canada and the vector sequence was provided. Using the method of reverse transcription polymerase chain reaction (RT-PCR), the primers are TT-CCDC8-BamHI and TT-CCDC8-EcoRI (as shown in Table 1) to amplify CCDC8, and the amplified product is treated with restriction endonuclease After digestion with BamHI and EcoRI, clone into pTT5-SH5 vector. The CCDC8 sequence was determined and confirmed by Invitrogen (SEQIDNo.1), and the protein sequence was translated according to the triple codon rule (SEQIDNo2).
[0026] Then, HEK293T cells (CRL-11268, ATCC) were cultured in DMEM medium supplemented with 10% serum and penicillin in a 37°C incubator containing 5% carbon dioxide. After recovery, the cells are cultured for two weeks and can be used after being ...
Embodiment 2
[0034] Example 2, CCDC8 can cause polyubiquitination and degradation of HIV-1 Gag
[0035] CCDC8 interacts with Gag, OBSL1 and Cul7. Checking the literature shows that CCDC8 is related to 3M syndrome, a genetic disease. 3M syndrome is an autosomal recessive genetic disease, mainly characterized by short stature but normal intelligence. Using exome sequencing, 70% of patients had mutations in the E3 ligase Cul7, 25% had mutations in OBSL1, and 5% had mutations in CCDC8. 3M syndrome and in vitro co-immunoprecipitation experiments linked CCDC8, OBSL1 and Cul7 together, suggesting that they are in a cellular pathway. We also found that OBSL1 or Cul7 alone had no anti-HIV-1 effect ( Image 6 A). Our co-immunoprecipitation experiments also showed that HIV-1Gag interacted with CCDC8 ( Image 6 B), Cul7 also interacts with CCDC8 and OBSL1 ( Image 6 C). The specific method is as follows. After 48 hours of transfection, the cells were lysed with the following buffer solution (20...
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