Hybridoma cell strain and application thereof
A hybridoma cell line and microbial strain technology, which is applied in the field of biomedicine, can solve the problem that the production of canine CR1-L monoclonal antibody has not been reported, etc.
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Embodiment 1
[0024] Using the blastn online program provided by NCBI (National center for biotechnology information), the complement receptor mRNA sequences of humans, chimpanzees, baboons, rabbits and other animals were compared for homology, and the relatively conserved sequences were determined. The subsequent steps are as follows:
[0025] (1) Extracting total RNA from blood cells of healthy beagle dogs;
[0026] (2) RT-PCR amplification of part of canine CR1-L gene cDNA fragment;
[0027] (3) After the reaction is completed, extract the reaction solution and carry out 1% agarose gel electrophoresis. The results are as follows figure 1 As shown, the bands of about 1000 bp, 900 bp and 600 bp were obtained, which were named as A, B, and C, respectively. The PCR products A-C of the CR1-like gene fragment were sequenced, and the sequencing results were compared in the GenBank database for homology comparison. Yes, the results show that both bands A and B are non-specifically amplified, an...
Embodiment 2
[0046] Three 6-week-old Balb / c mice were selected and injected subcutaneously at multiple points on the back. Freund's complete adjuvant was used for the first immunization, and Freund's adjuvant and purified canine Frd-GST-CR1L protein were mixed and emulsified in a volume ratio of 1:1, and 100 ug was injected into each mouse. In the third week and the fifth week, the incomplete Freund's adjuvant and the purified canine Frd-GST-CR1L protein were mixed and emulsified according to the volume ratio of 1:1. . One week after the third immunization, 30 μL of blood was collected from each mouse, and serum titers were detected by ELISA.
[0047] ELISA detection steps are as follows:
[0048] (1) Coating: The corresponding antigen was diluted with the coating buffer CBS at a concentration of 1ug / mL, 100uL / well was added to the ELISA plate, covered with a membrane and sealed, and coated overnight at 4°C.
[0049] (2) Blocking: Discard the solution the next day, add 150 uL / well of bl...
Embodiment 3
[0058] The preparation and purification of canine CR1-L monoclonal antibody, the steps are as follows:
[0059] (1) Preparation of hybridoma cells
[0060] Using CR1-L as the candidate antigen, the mice were re-immunized according to the operation steps of "Animal Immunization" in Example 2. On the 7th day after the third immunization, the spleens of the immunized mice were aseptically separated, and the spleen cell suspension was prepared according to the routine laboratory method, and the concentration of spleen cells was adjusted to 7×10. 8 / mL, for use. The cell samples were sent to Fuyinde Technology Co., Ltd. to prepare and screen hybridoma cells.
[0061] (2) Production and purification of canine CR1-L monoclonal antibody
[0062] ①Tumor cell recovery
[0063] a. Take out the hybridoma cells from liquid nitrogen, thaw them quickly, and transfer them to an ultra-clean bench for use after routine disinfection. The thawed tumor cell suspension was sucked into a 1.5 mL...
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