Anti-neuraminidase monoclonal antibody and application thereof
A technology of antibodies and amino acids, applied in applications, antibodies, anti-enzyme immunoglobulins, etc.
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[0128] Antibody preparation
[0129] The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences for the light and heavy chains can be fused together to form single-chain antibodies.
[0130] Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
[0131] In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
[0132] At present, the DNA sequence encoding the antibody of the ...
Embodiment 1
[0154] Example 1 Monoclonal Antibody Screening
[0155] 4-week-old SPF grade female BALB / c mice were selected, and each mouse was injected with 50ul of NA plasmid containing 50ug, or 50ul of PBS (control group), and then received electric shocks on both sides of the intramuscular injection needle hole. Immunize once every 2 weeks with the same dose and volume of plasmid solution, and immunize 3 times in total. Seven days after the 3 times of immunization, the blood was collected by docking the tail, and the anti-NA antibody titer in the serum was detected. Mice with higher antibody titers were directly immunized with 50ug of NA plasmid in the tail vein, and 3 days later, the spleen cells of the mice were collected for cell fusion. After the fusion of splenocytes and myeloma cells SP2 / 0, aliquot into 96-well cell culture plates, 37°C, 5% CO 2 Cultured in an incubator. Observe the cell state under an inverted microscope, and aspirate the supernatant for antibody detection aft...
Embodiment 2
[0157] Preparation and purification of embodiment 2 monoclonal antibody
[0158] 8-week-old BALB / C female mice, each mouse was inoculated intraperitoneally with PBS or the hybridoma cells prepared in Example 1, and each mouse was injected with 1×10 7 / 0.5ml. After an interval of 5 days, ascitic fluid was collected. Ascites was purified by protein G affinity column, and the collected eluate was put into a dialysis bag and dialyzed against PBS solution.
[0159] The dialyzed eluate was assayed for its concentration, aliquoted, and frozen at -70°C. The purity of the purified antibody was identified by SDS-PAGE gel.
[0160] 12% of SDS-PAGE identification results such as figure 1 As shown, the purity of the antibody can reach more than 95%, and the heavy chain and light chain of the antibody can be seen at about 57kD and 24kD, and its molecular weight is consistent with the theoretical value.
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