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A technology of ZZ2004 and hybridoma cell lines, applied in the field of bioengineering, can solve the problems of lack of detection methods and achieve the effect of high antibody content, short production cycle and strong specificity
Inactive Publication Date: 2012-09-19
HENAN INST OF SCI & TECH
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Problems solved by technology
At present, there is still a lack of effective detection methods for this virus, and in the establishment of detection methods, it is particularly critical to obtain effective antibodies
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Embodiment 1
[0024] Embodiment 1 hybridoma cell line preparation
[0025] (1) Immunization of mice
[0026] The duck-derived coronavirus ZZ2004 was inoculated in the allantoic cavity of 10-day-old SPF chicken embryos for reproduction. After 72 hours, the chicken embryo virus allantoic fluid was collected and gradient centrifuged, respectively: 4000rpm, 30 minutes, supernatant; 8000rpm , 30 minutes, take the supernatant; 200000g, 4 hours, dilute the precipitate with normal saline, measure the viral protein content with a spectrophotometer to be 3.5 mg / ml, then according to the virus amount of 50 micrograms of viral protein / only and 200 microliters / only The amount of immune antigen calculated is 14 microliters, diluted with normal saline to 100 microliters, and then fully emulsified with 100 microliters of adjuvant, and the emulsified immune antigen is used to subcutaneously multi-point on the back of the mouse. Immunization, immunization in 4 times.
[0027] (2) cell fusion
[0028] Perf...
Embodiment 2
[0034] The preparation of embodiment 2 ascites monoclonal antibody
[0035]①The expanded hybridoma cells CH / HN / 2004 were injected into the peritoneal cavity of Balb / c mice, and each mouse was injected with 1×10 6 cells;
[0036] ② After 20 days, ascitic fluid was collected, and the antibody titer was measured to be 1:8.2×10 6 .
Embodiment 3
[0037] Example 3 Neutralizing effect of monoclonal antibody on virus
[0038] The monoclonal antibody obtained in Example 2 was subjected to a chicken embryo neutralization test, and the EID of the virus was first measured 50 =10 -4.88 power, equal to 2.1×10 after conversion -6 power, dilute the virus to 200 EID 50 At the same time, the ascites antibody was diluted to a ratio of 0.1 ml each of the diluted antibody and the diluted virus, and mixed so that the diluted antibody dilutions after mixing were: 1:5, 1:10, 1:20, 1 : 40, 1: 80 and 1: 160, and then the mixed solutions of each gradient were reacted in a 37°C incubator for 1 hour, and the mixed solutions of each antibody dilution were inoculated into 6 chicken embryos, and a virus control (0.1 ml of virus night, 100ELD 50 + 0.1 ml of normal saline) and antibody control (1:5 diluted antibody dilution + 0.1 ml of normal saline), the eggs were illuminated once 24 hours after inoculation, and the dead chicken embryos were ...
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Abstract
The invention relates to a hybridoma cell strain, a duck-origin coronavirus ZZ2004-resisting monoclonal antibody and application of the hybridoma cell strain. The cells are fused, screened and cloned to obtain the hybridoma cell strain of which the preservation number is CGMCC NO.4171; and the screened hybridoma cells are processed by amplification culture, and injected into the abdominal cavity of a multiparous female mouse to obtain a high-titer ascites antibody which can neutralize the duck-origin coronavirus. The monoclonal antibody prepared from hybridoma cell strains has the advantages of low cost, short production cycle, the operation is simple, the content of the produced antibody is high and the specificity of the produced antibody is strong. The invention lays a sound foundation for the establishment of methods for diagnosing duck-origin coronavirus infected poultry, and has important economic value and social meaning.
Description
technical field [0001] The invention relates to the field of bioengineering, in particular to a hybridoma cell line, an anti-duck-derived coronavirus ZZ2004 monoclonal antibody and applications thereof. Background technique [0002] In 1937, coronaviruses (Coronaviruses) were first isolated from chickens. In 1965, the first human coronavirus was isolated. It is named "coronavirus" because it can be observed under an electron microscope that there are obvious rod-shaped particle protrusions on its outer membrane, making it look like the crown of a medieval European emperor. According to the serological characteristics of the virus and the differences in nucleotide sequences, the Coronaviridae family is currently divided into two genera, coronavirus and torovirus. The representative strain of Coronaviridae is Avian infectious bronchitis virus (IBV). The virus is an enveloped, moderately pleomorphic spherical or oval virion with rod-shaped protrusions on the surface. [000...
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