A bovine modified antibody with insecticidal activity and its preparation method and application
An insecticidal activity, bovine-derived technology, applied in the field of bovine-derived modified antibodies and their preparation, can solve problems such as no public reports, and achieve the effect of less harm to human body
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Embodiment 1
[0062] Example 1 Determination and synthesis of bovine modified antibody sequence
[0063] 1.1 With reference to the method of Almagro et al. (2006) and slightly improved, the VH and VL sequences of the bovine antibody were obtained from GenBank, using the keywords " Bos taurus immunoglobulin heavy chain variable "("bovine antibody heavy chain variable region", VH) and " Bos taurus "immunoglobulin light chain variable" ("bovine antibody light chain variable region" VL) searched for "protein" ("protein"), and as a result, 126 proteins were found for VH, and 75 proteins were found for VL. Amino acid analysis was performed on VH and VL respectively Sequence alignment and analysis, so as to determine the conserved part, and according to the identification method of IMGT (Lefranc et al ., 2003) (Lefranc, M. P.; Pommie, C.; Ruiz, M.; Giudicelli, V.; Foulquier, E.; Truong, L.; Thouvenin-Contet, V.; Lefranc, G. IMGT uniquenumbering for immunoglobulin and T cell receptor variabl...
Embodiment 2
[0076] Example 2 The expression vector of the bovine modified antibody was replaced by pIT2
[0077] The vector for ligation of the nucleic acid fragments synthesized by GenScript is pUC57 ( Figure 4 ), in order to be able to express on pIT2, a universal vector for phage display, it is double-digested with the Nco I and Not I restriction sites, such as Figure 5 As shown, it can be seen that the complete plasmid was cut into 2 parts, and the smaller 714bp target fragment was recovered using the PCR product gel recovery kit and purified with T4 DNA ligase 16 o Ligated overnight under C, the ligated carrier was pIT2 (such as Image 6 shown), and the connection product was obtained the next day;
[0078] Add 5 µl of the ligation product to 100 µl of TG1 chemically competent cells, mix well, and place on ice for 30 min; then transfer the mixture to 42°C for heat shock for 1 min, and quickly ice-bath for 2 min; then add to the mixture 900 µl 2×TY liquid medium, recover for 1 ho...
Embodiment 3
[0087] Example 3 ELISA identification of bovine modified antibodies
[0088] 3.1 Preparation of Plutella xylostella BBMV
[0089] Referring to Wolfersberger’s experimental method (Wolfersberger, 1987), the midgut BBMV of Plutella xylostella was prepared by Mg-EGTA sedimentation method. The specific method was as follows: the midgut was extracted from the 4th instar larvae of Plutella xylostella, washed in pre-cooled 0.15 M NaCl, Add 3 mL of homogenization buffer to every 500 midguts; after 3 times of repeated homogenization in ice bath, take an appropriate amount and add 24 mM MgCl 2 Then vortex and mix, ice bath and centrifuge, then transfer the supernatant to a new high-speed centrifuge tube and then centrifuge; discard the supernatant, invert the centrifuge tube until the liquid is exhausted, resuspend the pellet in HEPES buffer, and separate After packaging, store at -80°C for use; BBMV protein concentration was determined by the Bradford method.
[0090] 3.2 ELISA ident...
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