Monoclonal antibody to cry1ah1 protein and its application
A monoclonal antibody and protein technology, applied in the direction of immunoglobulin, anti-bacterial immunoglobulin, instruments, etc., can solve the problems of high false positive and false negative rate, low antibody specificity, and low sensitivity, and achieve sensitivity and specificity High performance, improved detection accuracy, and high sensitivity
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Embodiment 1
[0056] Cry1Ah1 protein, Cry1Aa1 protein (Genebank accession number: AAA22353.1), Cry1Ad1 protein (Genebank accession number: AAA22340.1), Cry1Ab1 protein (Genebank accession number: AAA22330.1), Cry1Ae1 protein (Genebank accession number: AAA22410.1 ), Cry1Ai1 protein (Genebank accession number: AY174873.1) and Cry1Ac1 protein (Genebank accession number: AAA22331.1) amino acid sequences were analyzed with MEGA 7.0 software MEGA 7.0 software, the results are shown in figure 1 . It can be seen from the results that the Cry1Ah1 protein and Cry1Ac1 have the highest amino acid similarity and the closest evolutionary relationship.
Embodiment 2
[0058] Antigen preparation
[0059] The gene sequence Cry1Ah1 (SEQ ID No.1) was connected to the Escherichia coli-Bacillus thuringiensis shuttle expression vector pSTK to obtain pSTK-Cry1Ah1. Firstly, pSTK-Cry1Ah1 was transformed into E. coli strain SCS110 to demethylate the pSTK-Cry1Ah1 DNA; then the demethylated pSTK-Cry1Ah1 was introduced into the Bt crystal-free mutant strain HD73 - Among them, the Bt expression strain HD73-Cry1Ah1 was obtained.
[0060] The Bt expression strain HD73-Cry1Ah1 was planted in beef extract peptone medium, cultured at 230 rpm, 30°C for about 30 hours, and the Cry1Ah1 protein was extracted, and then the protein expression was detected by SDS-PAGE. The results showed that HD73-Cry1Ah1 can express a large amount of 135kDa protein protein.
[0061] Among them, the extraction of Cry1Ah1 protein is as follows:
[0062] (1) Pick a single colony of HD73-Cry1Ah1 and inoculate it into a test tube filled with 5 mL of LB liquid medium containing the cor...
Embodiment 3
[0077] Preparation of monoclonal antibodies
[0078] 1) Animal immunity
[0079] The Cry1Ah1 protein purified in Example 2 was used to subcutaneously immunize 4 SPF BALB / c female mice at an amount of 50 μg / mouse, numbered in sequence: 1 to 4. The first subcutaneous booster immunization; the second subcutaneous booster immunization, the immune dose of protein was 40 μg / bird; the third subcutaneous booster immunization, the protein immune dose was 40 μg / bird; the fourth subcutaneous booster immunization, the protein immune dose 30 μg / monkey; blood was taken from the orbit to measure the serum titer.
[0080] 2) Immunological titer detection
[0081] Steps: Coat Cry1Ah1 protein with coating solution, 2 μg / ml, overnight at 4°C; 2% skimmed milk, block at 37°C for 2 hours; serially dilute serum 2 times from 200 times, blank control (blank) is PBS, negative control (negative) is a 200-fold dilution of negative serum. RESULTS: The No. 4 mouse with the highest immune titer was sele...
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