Swine-derived reshaped antibody with insecticidal activity, and preparation method and application thereof
An insecticidal activity, pig-derived technology, applied in the field of pig-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 porcine modified antibody sequences
[0063] 1.1 Referring to the method of Almagro et al. (2006) and slightly improved, the VH and VL sequences of porcine-derived antibodies were obtained from GenBank, using the keywords " Susscrofa "immunoglobulinheavychainvariable" ("Pig-derived antibody heavy chain variable region", VH) and " Susscrofa "immunoglobulinlightchainvariable" ("pig-derived antibody light chain variable region" VL) searched for "protein" ("protein"), the results were 337 proteins were found in VH, 94 proteins were found in VL, and the amino acid sequences of VH and VL were compared respectively. and analysis to identify conserved parts and identify them according to the IMGT method (Lefranc etal ., 2003) (Lefranc, M.P.; Pommie, C.; Ruiz, M.; Giudicelli, V.; Foulquier, E.; Truong, L.; Thouvenin-Contet, V.; Lefranc, G. IMGTuniquenumberingforimmunoglobulinandTcellreceptorvariabledomainsandIgsuperfamilyV-likedomains...
Embodiment 2
[0076] Example 2 The expression vector of porcine modified antibody is 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 the general vector pIT2 for phage display, use the NcoI and NotI enzyme cutting sites to carry out double digestion, such as Figure 5 As shown, it can be seen that the complete plasmid was cut into 2 parts, and the smaller 735bp target fragment was recovered using a 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] Take 5 μl of the ligation product and add it to 100 μl TG1 chemically competent cells, mix well, and place on ice for 30 minutes; then transfer the mixture to 42°C for heat shock for 1 minute, and quickly ice-bath for 2 minutes; then add 900 μl 2×TY liquid culture to the mixt...
Embodiment 3
[0087] Example 3 ELISA Identification of Porcine Modified Antibody
[0088] 3.1 Preparation of Plutella xylostella BBMV
[0089] Referring to Wolfersberger’s experimental method (Wolfersberger, 1987), the Mg-EGTA sedimentation method was used to prepare Plutella xylostella midgut BBMV. The specific method was: take the 4th instar larvae of Plutella xylostella, extract the midgut, wash it in pre-cooled 0.15M NaCl, and wash it every Add 3mL of homogenization buffer to 500 midguts; after 3 times of repeated homogenization in ice bath, take an appropriate amount and add 24mM 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 identificat...
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