Anti-npt II protein monoclonal antibody and its application
A monoclonal antibody and protein technology, applied in the direction of anti-enzyme immunoglobulin, microbial-based methods, instruments, etc., can solve the problems of non-continuous production, batch-to-batch difference, and poor specificity, and achieve extremely strong specificity and sensitivity Effect
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Embodiment 1
[0028] The preparation of embodiment 1 recombinant PMI protein
[0029] 1. Gene cloning and expression purification
[0030] PCR primers were designed according to the sequence of the npt ii gene, and amplification was carried out with Escherichia coli genomic DNA as a template ( figure 1 ), insert the amplified product into the corresponding restriction site of the pET30a(+) vector, and construct the recombinant expression vector pET30a-npt vector, obtain a pET30a(+) linear fragment of about 5.4k and an insertion of about 790bp after double digestion According to the sequencing results, select the plasmid with no base mutation to obtain the prokaryotic expression vector of npt II gene. Transfer the recombinant vector pET30a-npt II into the expression bacteria, pick a single colony and then culture and amplify it, transfer the overnight bacteria cultured by a single colony to 100ml LB medium at a ratio of 1:100, add the final concentration of 50μg / ml Kanamycin, cultured with...
Embodiment 2
[0031] The establishment of embodiment 2 hybridoma cell lines
[0032] 1. Immunity
[0033] Two polypeptides NPT II-pep1 and NPTII-pep2 chemically synthesized according to the amino acid sequences of Seq ID No: 2 and Seq ID No: 3 in the sequence listing were coupled to KLH carrier protein (Thermo Company) respectively. The cross-linked polypeptide was emulsified with complete Freund's adjuvant (Sigma Company), immunized 4-6 week-old female Balb / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and each mouse was injected subcutaneously in the abdomen. Rats at 6 o'clock, the dose was 60 μg / rat. Immunization was boosted every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Company) at a dose of 30 μg per mouse. 7 days after the third booster immunization, indirect ELISA (wavelength 450nm) was used to detect the polyantibody titer of the anti-immunogen in the mouse serum. 50μg / only.
[0034] 2. Cell Fusion...
Embodiment 3
[0040] Example 3 Preparation of Monoclonal Antibody by Ascites Induction Method
[0041] 1. Ascites preparation
[0042] Cells in the logarithmic growth phase were washed with serum-free medium and suspended, counted to 5×10 5 , 1ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The removed ascites was centrifuged at 4000rpm for 10min at 4°C. Carefully suck out the ascites in the middle and collect in a centrifuge tube, and store at 4°C or -20°C. 2. Purification of monoclonal antibodies
[0043] Antibody was purified from ascitic fluid by HiTrap rProtein A FF (GE Company) affinity chromatography according to the instructions. The purity was identified by SDS-PAGE gel, and the concentration was determined by Bradford method. Purified antibodies were stored at -20°C.
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