A hybridoma cell line secreting monoclonal antibody against Rift Valley fever virus nss protein and its application
A technology of hybridoma cell lines and monoclonal antibodies, which is applied in antiviral immunoglobulin, analytical materials, biological material analysis, etc., can solve the problem of inability to distinguish infection antibodies from immune antibodies, and achieve good specificity and repeatability Effect
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Embodiment 1
[0021] Example 1 Preparation of recombinant protein
[0022] 1. Construction of recombinant plasmids and protein expression
[0023] According to the Rift Valley fever virus NSs gene sequence (HE687307) published by Genbank, the sequence shown in SEQ ID NO: 1 was obtained. The sequence shown in SEQ ID NO: 1 was synthesized and cloned into pET-28a(+) to obtain the recombinant plasmid pET-28a-NSs. The recombinant plasmid pET-28a-NSs was transformed into E. coli competent cells, and the positive colonies were picked and named pET-28a-NSs(BL21) after correct PCR identification. pET-28a-NSs(BL21) was inoculated into LB liquid medium containing kanamycin and cultured at OD. 600 When reaching 0.6-0.8, 0.5mmol / L IPTG was added, and the expression was induced at 37°C for 5h. The cells were collected and lysed by ultrasonic waves. The supernatant and precipitate of the lysate were collected respectively, and the expression of the recombinant protein was identified by SDS-PAGE gel elec...
Embodiment 2
[0028] Example 2 Establishment of monoclonal antibody hybridoma cell line
[0029] 1. Immunization of BALB / c Mice
[0030] 100 μg of recombinant NSs protein and Freund's adjuvant were mixed and emulsified at a volume ratio of 1:1, and then subcutaneously injected into BALB / c mice at multiple points (100 μg / mice). Afterwards, two booster immunizations were carried out with two weeks between each time and the previous immunization. Each booster immunization was emulsified with a 1:1 mixture of 100 μg recombinant NSs protein and incomplete Freund's adjuvant. A total of 3 immunizations. Two weeks after the third immunization, blood was collected, and the serum titer of the immunized mice was detected by indirect ELISA. The indirect ELISA method was as follows: the recombinant NSs protein was coated on a 96-well microtiter plate at a concentration of 2 μg / mL, 100 μL / well, overnight at 4°C. Wash 3 times with PBST and pat dry; add PBST containing 0.5% BSA (PBS containing 0.5% Twee...
Embodiment 3
[0040] Example 3 Establishment of RVFV NSs blocking ELISA antibody detection method
[0041] 1. Determination of the coating concentration of the antigen and the dilution of the enzyme-labeled monoclonal antibody
[0042] Using square array titration, the recombinant NSs protein was diluted with antigen coating solution (0.05mol / L, pH9.6 carbonate buffer) to 0.5μg / mL, 1μg / mL, 2μg / mL, 4μg / mL concentration, Add 100 μL to each well, incubate overnight at 4°C; wash 3 times with PBST (PBS containing 0.5% Tween-20); add 300 μL PBST containing 0.5% BSA to each well, block at 37°C for 2 h; wash 3 times with PBST; each well Antigen concentration was added with 1:10 diluted RVFV negative and positive serum, incubated at 37°C for 1 h; washed 3 times with PBST; added 100 μL of PBST respectively according to the dilutions of 1:500, 1:1000, 1:1500, 1:2000, 1 :2500, 1:3000 diluted HRP-2D6D10 (the concentration of HRP-2D6D10 before dilution was 1 mg / mL), incubated at 37°C for 1 h; washed 3 t...
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