Biological Activity of Anti-idiotypic Single Chain Fragment Variable Antibody Against Cry1B by Site-directed Mutagenesis

doi:10.13560/j.cnki.biotech.bull.1985.2021-0197

Abstract

Abstract:

The long-term application of Bacillus thuringiensis（Bt）toxins leads to ecological risk in pest resistance and so on,which promots the development of Bt resources with high specific activity and new functional genes. Our previous studies revealed that the preparation technique of anti-idiotypic（anti-Id）single chain fragment variable（scFv）was a new way to develop novel insecticidal protein. However,the binding ability of the obtained Bt anti-Id scFv C7 and brush-border membrane vesicles（BBMV）of Cnaphalocrocis medinalis was not high,and needed further modification and improvement. The molecular mimicking technology including homology model and molecular docking was used for C7 and the receptor aminopeptidase N in the BBMV of C. medinalis,and hot spot residues on the binding region of the C7 and the aminopeptidase N was predicted. The hot spot residues of the C7 were used to construct a saturation mutant antibody library,and solid-phase screening was to produce mutant Y124G. BIAcore analysis indicated that the binding ability of Y124G and BBMV increased. Bioassay indicated that the insecticidal activity to C. medinalis increased. This study lays the foundation for molecular modification of antibody,and provids new idea for the creation of new biological pesticides.

Key words: Cry1B, molecular modification, site-directed mutagenesis, insecticidal activity, surface plasmon resonance

ZHONG Jian-feng, LI Xing-kui, XU Chong-xin, ZHANG Xiao, LIU Xian-jin. Biological Activity of Anti-idiotypic Single Chain Fragment Variable Antibody Against Cry1B by Site-directed Mutagenesis[J]. Biotechnology Bulletin, 2021, 37(10): 186-195.

Figures/Tables 11

Fig. 1 Amino acid sequence alignment of anti-Id scFv C7 and D5 against Cry1B toxin

Table 1 Primer sequences for the construction of site-directed mutagenesis library

引物名称 Primer	序列 Sequence（5'-3'）
LMB3	CAGGAAACAGCTATGAC
pHENseq	CTATGCGGCCCCATTCA
NNK_VH	CCCCAGTAGTCAAANNKNNKACCAGATTTCGC
NNK_VL	CTCCTGATCTATNNKGCATCCNNKTTGCAAAGTGG

Fig. 2 Three-dimensional structure models of scFv-C7 and CmAPN A:Three-dimensional structure model of scFv-C7. B:3D structure model of CmAPN. C:Ramachandran plot for scFv-C7 model D:Ramachandran plot for CmAPN model

Fig. 3 Molecular docking and the interaction hot spot analysis between scFv-C7 and CmAPN A:Molecular docking of scFv-C7 and CmAPN. B:Predicted hot spot residues on binding interface from scFv-C7

Fig. 4 Saturation mutagenesis of hot spot amino acids on docking interface based on scFv-C7 A:Amplification of VH mega-primer. B:Full-length amplification of scFv-C7 with H-CDR3 saturation mutagenesis site. C:Amplification of VL mega-primer. D:Full-length amplification of scFv-C7 with H-CDR3 and L-CDR2 saturation mutagenesis sites. M:DNA marker. 1:VH mega-primer. 2:PCR product with H-CDR3 saturation mutagenesis. 3:VL mega-primer. 4:PCR product with H-CDR3 and L-CDR2 saturation mutagenesis sites

Fig.5 Verification of correction from constructed satura-tion mutant antibody library M:DNA marker. CK:Blank control. 1-10:Ten pieces of randomly picked monoclones

Table 2 Enrichment and screening of scFv-C7 mutant antibody library

富集轮数 Enriching rounds	包被蛋白 Coating antigen protein	包被浓度 Coating antigen concentration /（μg·mL^-1）	投入量 Input	产出量 Output	产出/投入 Output/Input
1	BBMV	100	2.0×10⁶	6.0×10²	3.0×10^-4
2	BBMV	50	1.8×10⁶	3.5×10³	1.9×10^-3
3	BBMV	25	2.1×10⁶	7.5×10³	3.6×10^-3

Fig. 6 ELISA analysis of monoclonal phage for mutant antibodies

Fig.7 Gene cloning（A）and amino acid sequence alignment（B）of mutant scFv antibodies M:2 000 bp DNA marker. CK:Blank control. 1- 4 refers to 3A5,3G2,2F7 and C7 gene,respectively

Fig. 8 SPR analysis of mutant scFv antibodies binding to CmBBMV CK-:Negative control scFv-D5. CK+:Positive control（Cry1B toxin）

Table 3 Bioassay of mutant scFv antibodies against C. medinalis larvae

样品 Samples	校正死亡率Corrected mortality/%
样品 Samples	24 h	48 h	72 h
CK^-	0.00 ±0.00c	0.00 ±0.00d	4.44±1.11d
CK⁺	35.56±2.94a	62.22±1.11a	76.67±5.09a
C7	25.56±2.94b	27.78±1.11c	42.22±2.94c
Y124G	21.11±2.22b	34.44±1.11b	56.67±1.92b

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