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Bovine-derived reshaped antibody with insecticidal activity as well as preparation method and application thereof

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

Active Publication Date: 2015-12-09
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Cry1Ab toxin anti-idiotypic single-chain antibody can mimic Cry toxin and compete with its receptor protein, and replace the CDR region of anti-idiotypic single-chain antibody with better insecticidal effect with the corresponding CDR region on other animal-derived single-chain antibodies, Interspersed between the 8 FRs of antibodies from various sources, that is, on the basis of animal-derived antibodies, the CDR region genes of animal-derived antibodies are replaced by human-derived antibody CDR region genes, which can quickly obtain modified antibodies from various animal sources, and obtain multiple Insecticidal protein resources from animal sources, using these genetic engineering antibody technologies to explore the possibility of developing new and highly safe insecticidal protein resources, also provides new ideas and directions for the development of new insecticidal whites. At present, the above-mentioned anti- There is no public report on the method of molecular modification of insect genes

Method used

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  • Bovine-derived reshaped antibody with insecticidal activity as well as preparation method and application thereof
  • Bovine-derived reshaped antibody with insecticidal activity as well as preparation method and application thereof
  • Bovine-derived reshaped antibody with insecticidal activity as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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 " Bostaurus immunoglobulinheavychainvariable" ("bovine antibody heavy chain variable region", VH) and " Bostaurus "immunoglobulin lightchain variable" ("bovine antibody light chain variable region" VL) searched for "protein" ("protein"), as a result, 126 proteins were found for VH, and 75 proteins were found for VL, and the amino acid sequences of VH and VL were compared And analysis, so as to determine the conserved part, and according to the identification method of IMGT (Lefranc etal ., 2003) (Lefranc, M.P.; Pommie, C.; Ruiz, M.; Giudicelli, V.; Foulquier, E.; Truong, L.; Thouvenin-Contet, V.; Dev. Comp. Immunol. , 2003,27:55-77.) To determine FR1, FR2, FR3 and FR4 of VH and VL, the VH and VL amino acid seq...

Embodiment 2

[0076] Example 2 The expression vector of bovine 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 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] 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 mix...

Embodiment 3

[0087] ELISA identification of embodiment 3 bovine 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 identific...

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Abstract

The invention determines the sequence of a bovine-derived reshaped antibody by predicating 8 FRs in heavy and light chain variable regions of a bovine-derived antibody through information biology and planting 6 CDRs of an anti-idiotypical single-chain antibody A12, and artificial synthesis is carried out to construct a bovine-derived reshaped antibody bovine-A12-PUC57 of the A12; a PIT2 carrier which is easily subjected to phage expression is replaced to obtain a bovine-derived reshaped antibody bovine-A12-PIT2. An ELISA analysis shows that the bovine-derived reshaped antibody bovine-A12-PIT2 has a certain combining capacity with plutella xylostella midgut BBMV; a biological activity test shows that the bovine-derived reshaped antibody has certain insecticidal activity. By utilizing a method of information biology predication and the artificial synthesis, the reshaped antibody (an insecticidal protein source) originated from animals can be rapidly and efficiently obtained. The possibility of utilizing genetically engineered antibody technologies to explore and develop novel and highly safe insecticidal protein resources provides a novel train of thought and direction for developing novel insecticidal proteins.

Description

technical field [0001] The invention belongs to the field of microbial pesticides, in particular to a bovine modified antibody with insecticidal activity and its preparation method and application. Background technique [0002] from Bacillus thuringiensis ( Bacillus thuringiensis , Bt) Bt toxin protein is currently the most effective insect-resistant resource, but the number of new high-efficiency strains reported is less and less (Bravoetal., 2013; Bravoetal., 2011) and the increase in the use of Bt toxin makes it useful in pest resistance There are ecological risks in terms of medicinal properties and the rise of secondary pests. This situation has prompted the active development of Bt toxin insect-resistant resources with high specific activity and new functions (Bravo and Soberon, 2008; Pigott et al., 2008). Therefore, the screening of insecticidal proteins that mimic Bt toxin and the development of other types of insecticidal proteins, especially protein resources with...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13C12N15/10C12N15/70A01N47/44A01P7/04
Inventor 刘贤金仲建锋张霄刘媛何鑫胡晓丹武爱华徐重新谢雅晶梁晓林曼曼
Owner JIANGSU ACAD OF AGRI SCI
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