Prokaryotic Expression and Antibody Preparation of Potato Sucrose Phosphate Synthase StSPS1

doi:10.7668/hbnxb.20194198

Abstract

Abstract:

To express the protein StSPS1 encoding potato sucrose phosphate synthase(SPS)in E.coli and prepare polyclonal antibodies.The StSPS1 gene was cloned from the tubers of tetraploid potato variety Chuanyu 10,with a total coding region of 3 165 bp and a protein length of 1 055 aa.Subsequently,based on the constructed His tag fusion expression vector PET30a-StSPS1,the protein induction,denaturation,purification,renaturation,and rabbit immune tests of StSPS1 protein were carried out.The results showed that the molecular weight of StSPS1 protein was approximately 119.62 ku,and its expression was minimal in soluble supernatant,mainly in insoluble precipitates.The optimal induction conditions were induced with 0.5 or 1.0 mmol/L IPTG at 37 ℃ for 4 h.Due to StSPS1 being an inclusion body protein,it was subjected to inclusion body denaturation and purified using His tags to match the size of the target band.At the same time,a protein immunoblotting(WB)test was performed using His antibodies,and the target band was detected at 119.62 ku,indicating the successful purification of StSPS1 inclusion body protein.Finally,by injecting the dialyzed and refolded StSPS1 protein into the subcutaneous tissue of rabbits,two antibodies against StSPS1 were successfully immunized.After WB identification,it was found that both antibodies could hybridize target bands in the antigen and the total protein of leaves in Chuanyu 10.In summary,potato StSPS1 protein was induced and purified,and rabbit polyclonal antibodies against StSPS1 protein were successfully prepared.

Key words: Potato, Sucrose phosphate synthase, StSPS1, Prokaryotic expression, Polyclonal antibody preparation

CAI Chengcheng, LI Luopin, WEN He, LIU Shifeng, WANG Qiang, LI Liqin, WANG Xiyao. Prokaryotic Expression and Antibody Preparation of Potato Sucrose Phosphate Synthase StSPS1[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(6): 11-17. doi: 10.7668/hbnxb.20194198.

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Figures/Tables 9

Tab.1 Primers used in this study

名称 Name	引物(5'—3') Primer(5'—3')
StSPS1	StSPS1-F:ATGGCGGGAAACGATTGGAT
	StSPS1-R:GGGTATTATCCTTTGAGTACCG
M13	M13-F:TGTAAAACGACGGCCAGC
	M13-R:AGGAAACAGCTATGACC
T7	T7-F:TAATACGACTCACTATAGGG
	T7-R:CCAAGGGGTTATGCTAG
StSPS1-30a	StSPS1-30a-F:CGCCATATGATGGCGGGAAACGATTG
	StSPS1-30a-R:CCGCTCGAGTCCTTTGAGTACCGCTAG

Fig.1 Cloning of StSPS1 in Chuanyu 10 and the colony PCR identification of pBM23 construction The arrow.The target band of StSPS1;M.DNA Marker (The same as Fig.3);#1,#2 and #3.3 recombinant single colony.

Fig.2 Sequence alignment analysis of StSPS1 gene and protein in Chuanyu 10 A.Differential gene sequence alignment of StSPS1;B.Differential protein sequence alignment of StSPS1.

Fig.3 Construction and identification of pET30a-StSPS1 recombinant plasmid A.Brief structure of pET30a-StSPS1 vector;B.Enzyme digestion of pET30a-StSPS1 plasmid.

Fig.4 StSPS1 protein induction results at different IPTG concentrations 1.The band before induction;2—4.The sediment band induced by 1.0,0.5,and 0.1 mmol/L concentrations of IPTG,respectively.The arrow points to the objective band.M.Protein Marker.The same as Fig.5—8。

Fig.5 StSPS1 protein induction under different temperatures and denaturation treatments 1—2.The soluble supernatant was induced at 16,37 ℃;3—4.The denatured supernatant of the inclusion body at 16,37 ℃.

Fig.6 Purification,renaturation,and identification of StSPS1 protein A.Results of Ni-NTA affinity purification:1.The protein after ultrasonication,2.The effluent,3—4.The eluted protein;B.Dialysis result of StSPS1 protein;C.WB result to StSPS1 protein by using His antibody.

Fig.7 WB identification in antigen with two StSPS1 antibodys

Fig.8 WB in leaves of Chuanyu 10 with two StSPS1 antibodys

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