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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (1): 259-264.doi: 10.3724/SP.J.1006.2022.03010

• RESEARCH NOTES • Previous Articles    

Preparation and application of polyclonal antibody against SSIIb protein from maize

YU Guo-Wu(), QING Yun, HE Shan, HUANG Yu-Bi*()   

  1. National Demonstration Center for Experimental Crop Science Education, College of Agronomy, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
  • Received:2020-02-11 Accepted:2021-06-08 Online:2022-01-12 Published:2021-06-17
  • Contact: HUANG Yu-Bi E-mail:13862@sicau.edu.cn;yubihuang@sohu.com
  • About author:First author contact:** Contributed equally to this work
  • Supported by:
    National Major Project for Transgenic Organism Breeding(2016ZX08003-001);National Natural Science Foundation of China(31501322);Special Postdotoral Research Project of Sichuan Province(03130104);Scientific Research Foundation for Returned Overseas Chinese Scholars(00124300)

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Abstract:

Antibody is an important reagent for protein function research. It is difficult to carry out protein function research due to the lack of corresponding antibody. Maize starch synthase IIa and starch synthase IIb (SSIIb) belong to starch synthase, which are mainly responsible for the extension of amylopectin. However, the studies of starch synthase IIb were still in lack. In this study, we immunized New Zealand white rabbits with recombinant GST fusion protein (455-704 aa) at the C-terminal of starch synthase IIb as antigen and prepared polyclonal antibody against SSIIb protein. Western blot showed that the polyclonal antibody of SSIIb could recognize not only SSIIb-C (455-704 aa) antigen, but also SSIIb protein in endosperm of maize at different developmental stages. Western blot analysis using the polyclonal antibody revealed that the expression pattern of SSIIb protein in maize endosperm increased first and then decreased from 10 to 30 days after pollination, and the highest expression level was found at 15 days after pollination. Semi-quantitative RT-PCR results indicated that the transcription level of SSIIb was the highest at 20 days after pollination. These results indicated that the specific polyclonal antibody against SSIIb was successfully prepared, and it could specifically recognize the endogenous SSIIb protein of maize by Western blot, which laid a foundation for further study of the function of SSIIb protein.

Key words: SSIIb, polyclonal antibody, prokaryotic expression, Western blot

Fig. 1

RT-PCR amplification of SSIIb-C (455-704 aa) gene fragment and validation of recombinant PGEX-6p-1-SSIIb-C (455-704 aa) vector by restriction enzyme digestion A: domain analysis of SSIIb protein in maize; B: construction of pGEX-6p-1-SSIIb-C (455-704 aa) vector; C: PCR amplification of SSIIb-C (455-704 aa) gene fragment, M: marker DL 2000; 1: water as negative control; 2: PCR amplification of ZmSSIIb-C (455-704 aa) using maize 20 DAP endosperm cDNA as template; D: identification positive recombinant clone by BamH I and EcoR I; M: maker DL 5000; 1: pGEX-6p-1-SSIIb-C (455-704 aa) digested by BamH I and EcoR I; 2: pGEX-6p-1-SSIIb-C (455-704 aa) recombinant plasmid."

Fig. 1

RT-PCR amplification of SSIIb-C (455-704 aa) gene fragment and validation of recombinant PGEX-6p-1-SSIIb-C (455-704 aa) vector by restriction enzyme digestion A: domain analysis of SSIIb protein in maize; B: construction of pGEX-6p-1-SSIIb-C (455-704 aa) vector; C: PCR amplification of SSIIb-C (455-704 aa) gene fragment, M: marker DL 2000; 1: water as negative control; 2: PCR amplification of ZmSSIIb-C (455-704 aa) using maize 20 DAP endosperm cDNA as template; D: identification positive recombinant clone by BamH I and EcoR I; M: maker DL 5000; 1: pGEX-6p-1-SSIIb-C (455-704 aa) digested by BamH I and EcoR I; 2: pGEX-6p-1-SSIIb-C (455-704 aa) recombinant plasmid."

Fig. 2

Induced expression and purification of GST-SSIIb-C (455-704 aa) fusion protein A: the expression of GST-SSIIb-C (455-704 aa) fusion protein was detected by Coomassie brilliant blue staining; B: the purification of GST-SSIIb-C (455-704 aa) fusion protein was detected by Coomassie brilliant blue staining. 1-4: purified GST-SSIIb-C (455-704 aa) fusion protein); BSA was used as loading control."

Fig. 2

Induced expression and purification of GST-SSIIb-C (455-704 aa) fusion protein A: the expression of GST-SSIIb-C (455-704 aa) fusion protein was detected by Coomassie brilliant blue staining; B: the purification of GST-SSIIb-C (455-704 aa) fusion protein was detected by Coomassie brilliant blue staining. 1-4: purified GST-SSIIb-C (455-704 aa) fusion protein); BSA was used as loading control."

Fig. 3

Specificity and sensitivity analyses of polyclonal antibody against recombinant GST-SSIIb-C (455-704 aa) protein Lane 1-6: the purified GST-SSIIb-C (455-704 aa) protein was used as an antigen, and the loading amount for each channel were 400, 200, 100, 50, 25, and 12.5 ng, respectively. The ratio of antibody dilution was 1:2000."

Fig. 3

Specificity and sensitivity analyses of polyclonal antibody against recombinant GST-SSIIb-C (455-704 aa) protein Lane 1-6: the purified GST-SSIIb-C (455-704 aa) protein was used as an antigen, and the loading amount for each channel were 400, 200, 100, 50, 25, and 12.5 ng, respectively. The ratio of antibody dilution was 1:2000."

Fig. 4

Application of ZmSSIIb antibody in protein expression analysis of SSIIB protein at different development stages of endosperm and semi-quantitative RT-PCR analysis of ZmSSIIb gene expression A: the relative expression of ZmSSIIb protein in maize endosperm at different stages after pollination. β-actin was used as loading control; ZmSSIIb antibody dilution ratio was 1:500. β-actin is 1:2000; B: semi-quantitative expression analysis of ZmSSIIb gene in different stages of development of maize endosperm. ZmTXN was used as reference gene. DAP: days after pollination."

Fig. 4

Application of ZmSSIIb antibody in protein expression analysis of SSIIB protein at different development stages of endosperm and semi-quantitative RT-PCR analysis of ZmSSIIb gene expression A: the relative expression of ZmSSIIb protein in maize endosperm at different stages after pollination. β-actin was used as loading control; ZmSSIIb antibody dilution ratio was 1:500. β-actin is 1:2000; B: semi-quantitative expression analysis of ZmSSIIb gene in different stages of development of maize endosperm. ZmTXN was used as reference gene. DAP: days after pollination."

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