Human single-chain antibody 4c-scfv against enzymatic activity of botulinum toxin type b and its application
A 4c-scfv, botulinum toxin technology, applied in the direction of antibodies, applications, anti-toxins, etc., can solve problems such as immune response or infectious diseases
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Embodiment 1
[0044] Example 1: Synthesis of Botulinum Toxin Type B Light Chain Gene
[0045] According to the complete gene sequence of botulinum toxin type B reported by Genbank, the light chain gene was designed and synthesized, and EcoR was inserted at the same time and Nde Restriction site, clone the gene fragment into pMD-19T vector, and transform it into JM109.
Embodiment 2
[0046] Embodiment 2: Botulinum toxin type B light chain gene is connected with PET-28a carrier
[0047]EcoR and Nde Double-digested plasmid pMD-19T-Bont-B, the digested product was analyzed by agarose gel electrophoresis, the results are shown in figure 2 As shown, the target gene BontB with a size of about 1335 can be seen, which is consistent with the expected size;
[0048] EcoR and Nde The large fragment of the double-digested vector PET-28a and the small fragment of the target gene BontB were recovered and ligated with T4 DNA ligase to obtain the recombinant plasmid PET-28a-BontB, which was transformed into Escherichia coli JM109 competent cells and carried out on agarose plates containing kan resistance. initial screening. Pick a single colony and culture it in LB liquid medium; extract the plasmid with a plasmid recovery kit.
[0049] Insertion of EcoR by synthesis and Nde Botulinum toxin type B light chain gene sequence at the restriction site, and prime...
Embodiment 3
[0054] Embodiment 3: SDS-PAGE result analysis of recombinant protein PET-28a-BontB expression product
[0055] The recombinant plasmid PET-28a-BontB was transformed into the expression strain Escherichia coli BL21 (DE3), and the expression was induced by IPTG. The results of SDS-PAGE showed that the recombinant protein PET-28a-BontB had an obvious expression band at about 50KD, and the size was the same as the theoretical value. match; see Figure 5 ;
[0056] SDS-PAGE electrophoresis was performed on the supernatant and the precipitate of the lysate of the recombinant protein expressing bacteria at the same time. The ratio of the supernatant to the precipitate was 1:10. The results showed that most of the target protein was in the supernatant, and there was also a small amount of the target protein in the corresponding position It is believed that through the optimization of induction conditions, it can be expressed in a soluble form under a suitable induction environment, s...
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