Anti-hpv16e7 protein monoclonal antibody 79a11, hybridoma cell line and its preparation method and application
A HPV16E7, hybridoma cell technology, applied in antiviral immunoglobulins, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as trauma, increased psychological and economic burden on patients, and lack of treatment plans , to achieve the effect of good specificity and high sensitivity
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Embodiment 1
[0035] Embodiment 1, preparation HPV16E7 protein
[0036] Design specific primers with 6 histidine tags according to the full-length gene of HPV16E7, the sequences of primers F and R (the underline is the endonuclease) are as follows:
[0037] F: 5'-gc ccatgg accatcaccatcaccatatgcatggagatacacctac-3' (SEQ ID NO. 1);
[0038] R: 5'-gc aagctt ttatggtttctgygaacagatggggcacac-3' (SEQ ID NO. 2).
[0039] Using the total genomic DNA of CaSki cells as a template, the full-length HPV16E7 gene was amplified by PCR. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 minutes, 30 cycles at 94°C for 30s, 35 cycles at 55°C to 70°C for 30s and 72°C for 60s, and extension at 72°C for 7 minutes . NcoⅠ and HindⅢ restriction enzymes cut the PCR product HV16E7 gene fragment and plasmid pET-28a(+), and the correct recombinant plasmid pET-28a(+)-HPV16E7 will be identified to transform the competent cell Rosetta(DE3)pLysS, and the engineering bacteria pET -28a(+)-HPV16E...
Embodiment 2
[0045] Embodiment 2, preparation of anti-HPV16E7 protein monoclonal antibody
[0046] Using hybridoma technology to immunize BALB / c mice with HPV16E7 protein as an antigen, the inguinal lymph node B lymphocytes and spleen cells of the immunized mice were fused with myeloma cells SP2 / 0-Ag14, cultured, screened, and cloned multiple times The hybridoma cell lines of the anti-HPV16E7 protein monoclonal antibody were obtained, and the results are shown in Table 1 and Table 2.
[0047] Table 1 shows the positive polyclonal hybridoma cell line with high titer derived from the spleen, the name of the hybridoma cell line of the monoclonal antibody screened after multiple cloning, and the OD of the antibody in the cell supernatant 450nm Value and antibody subtype, among which the monoclonal antibodies with high titer and very clear subtype include 79A11 (IgM), 38E11 (IgM), 74F3 (subtype is not clear, but mainly IgG2a) and 72E6 (subtype is not clear) . It can be seen that the subtype o...
Embodiment 3
[0070] Embodiment 3, identification of monoclonal antibody
[0071] The monoclonal antibody 79A11 ascites was precipitated with 50% and 33% ammonium sulfate, and then IgM and IgG were separated by molecular sieves, and then the first peak sample of the molecular sieve was purified by IgM column affinity chromatography, and then desalted by molecular sieves. In the 10% SDS-PAGE electrophoresis, lanes 1 and 2 are the samples purified by the comprehensive method of monoclonal antibody 79A11. In lanes 1 and 2, two bands at the molecular weight of 68kDa and 25kDa can be found, which coincide with the heavy chain and light chain of the IgM antibody. The molecular weight of the chains was consistent, and no other impurity bands were found (see figure 2 ). It shows that the purity of the comprehensively purified monoclonal antibody 79A11 is high, and it can be used for the identification and detection of the monoclonal antibody 79A11.
[0072] Nanjing KingScript Biotechnology Co., ...
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