生物技术通报 ›› 2021, Vol. 37 ›› Issue (10): 186-195.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0197
仲建锋1(), 李兴奎2, 徐重新1, 张霄1, 刘贤金1()
收稿日期:
2021-02-20
出版日期:
2021-10-26
发布日期:
2021-11-12
作者简介:
仲建锋,男,博士,副研究员,研究方向:农产品质量安全与生物控制技术;E-mail: 基金资助:
ZHONG Jian-feng1(), LI Xing-kui2, XU Chong-xin1, ZHANG Xiao1, LIU Xian-jin1()
Received:
2021-02-20
Published:
2021-10-26
Online:
2021-11-12
摘要:
Bt毒素的长期应用使其在害虫抗药性等方面存在生态风险,这一现状促使高特异活性和新功能Bt抗虫资源的积极开发。前期研究发现抗独特型单链抗体制备技术是开发新型杀虫蛋白的一个新途径。然而获得的Bt Cry1B抗独特型单链抗体C7与昆虫BBMV结合能力不高,需进一步改造提高。通过分子模拟技术对C7与稻纵卷叶螟BBMV上的受体氨肽酶N进行同源建模、分子对接,并预测C7与氨肽酶N结合区域的热点残基。利用C7的热点残基构建饱和突变抗体库,并采用固相筛选方法,得到突变体Y124G。BIAcore分析表明Y124G与BBMV的结合能力增加,生物测定发现对稻纵卷叶螟的杀虫活性提高。这为抗体分子改造技术的应用奠定了基础,也为新型生物农药创制提供了新的思路。
仲建锋, 李兴奎, 徐重新, 张霄, 刘贤金. Cry1B抗独特型单链抗体的定点突变及生物活性分析[J]. 生物技术通报, 2021, 37(10): 186-195.
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.
引物名称 Primer | 序列 Sequence(5'-3') |
---|---|
LMB3 | CAGGAAACAGCTATGAC |
pHENseq | CTATGCGGCCCCATTCA |
NNK_VH | CCCCAGTAGTCAAANNKNNKACCAGATTTCGC |
NNK_VL | CTCCTGATCTATNNKGCATCCNNKTTGCAAAGTGG |
表1 饱和突变抗体库构建使用的引物序列
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 |
图2 ScFv-C7和CmAPN三维结构的同源建模 A:ScFv-C7的三维结构模拟;B:CmAPN三维结构模拟;C:ScFv-C7的Ramachandran图;D:CmAPN的Ramachandran图
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
图3 scFv-C7和CmAPN的分子对接及互作热点分析 A:ScFv-C7和CmAPN的分子对接;B:ScFv-C7在接触界面上的热点残基
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
图4 基于scFv-C7对接界面热点氨基酸的饱和突变 A:VH“大引物”的扩增;B:带有H-CDR3饱和突变位点的scFv-C7全长扩增;C:VL“大引物”的扩增;D:带有H-CDR3和L-CDR2饱和突变的scFv-C7全长扩增;M:DNA分子量;1:VH“大引物”;2:带有H-CDR3饱和突变的PCR产物;3:VL“大引物”;4:带有H-CDR3和L-CDR2饱和突变位点的PCR产物;
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
图5 构建的饱和突变抗体库正确性验证 M:DNA分子量;CK:空白对照;1-10:10个随机挑取的单克隆
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
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 |
表2 scFv-C7突变抗体库的富集和筛选
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 |
图7 突变单链抗体的基因克隆(A)与氨基酸序列比对(B) M:2 000 bp DNA maker;CK:空白对照;1-4分别表示3A5、3G2、2F7、C7基因
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
图8 SPR分析突变单链抗体与CmBBMV的结合 CK-:阴性对照scFv-D5;CK+:阳性对照Cry1B毒素
Fig. 8 SPR analysis of mutant scFv antibodies binding to CmBBMV CK-:Negative control scFv-D5. CK+:Positive control(Cry1B toxin)
样品 Samples | 校正死亡率Corrected mortality/% | ||
---|---|---|---|
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 |
表3 突变单链抗体对稻纵卷叶螟的生物测定
Table 3 Bioassay of mutant scFv antibodies against C. medinalis larvae
样品 Samples | 校正死亡率Corrected mortality/% | ||
---|---|---|---|
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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