马铃薯蔗糖磷酸合成酶StSPS1的原核表达及抗体制备

doi:10.7668/hbnxb.20194198

摘要/Abstract

摘要：

为了对马铃薯蔗糖磷酸合成酶(SPS)编码蛋白StSPS1进行原核表达及多克隆抗体制备。从四倍体马铃薯品种川芋10号的块茎中克隆出了StSPS1基因,该基因编码区全长为3 165 bp,编码蛋白的长度为1 055 aa。随后基于构建的His标签融合表达载体PET30a-StSPS1,进行了StSPS1蛋白的诱导、变性、纯化、复性及兔免疫试验。结果发现,StSPS1蛋白分子量约为119.62 ku,在可溶性上清中的表达极少,主要在不溶的沉淀中表达,最佳的诱导条件为37 ℃下用0.5或1.0 mmol/L的IPTG诱导4 h。由于StSPS1为包涵体蛋白,故对其进行包涵体变性处理,并利用His标签纯化出了与目的条带大小相符的蛋白,同时通过His抗体进行了蛋白质免疫印迹(WB)试验,发现在119.62 ku处检测到目标条带,说明StSPS1包涵体蛋白纯化成功。最后,通过将透析复性后的StSPS1蛋白注射进兔子皮下组织中,成功免疫出2个StSPS1的抗体,经过WB鉴定发现2个抗体均能在抗原和川芋10号叶片的总蛋白中杂出目标条带。综上,对马铃薯StSPS1蛋白进行了诱导及纯化,并成功制备了StSPS1蛋白的兔多克隆抗体。

关键词: 马铃薯, 蔗糖磷酸合成酶, StSPS1, 原核表达, 多克隆抗体制备

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

蔡诚诚, 李罗品, 温和, 刘石锋, 王强, 李立芹, 王西瑶. 马铃薯蔗糖磷酸合成酶StSPS1的原核表达及抗体制备[J]. 华北农学报, 2023, 38(6): 11-17. doi: 10.7668/hbnxb.20194198.

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.

http://www.hbnxb.net/CN/Y2023/V38/I6/11

图/表 9

表1 本研究中使用的引物序列

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

图1 川芋10号中StSPS1的克隆及pBM23连接菌落PCR鉴定箭头.StSPS1目的条带;M.DNA Marker(图3同);#1、#2、#3.3个重组单菌落。

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.

图2 川芋10号中StSPS1基因及蛋白序列比对分析 A.StSPS1差异基因序列比对;B.StSPS1差异蛋白序列比对。

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.

图3 pET30a-StSPS1重组质粒构建及鉴定 A.pET30a-StSPS1载体简要结构;B.pET30a-StSPS1质粒酶切验证。

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

图4 不同IPTG浓度下的StSPS1蛋白诱导结果 1.诱导前的条带;2—4.1.0,0.5,0.1 mmol/L浓度的IPTG诱导的条带。箭头指向目的条带。M.蛋白 Marker。图5—8同。

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。

图5 不同温度及变性处理下的StSPS1蛋白诱导结果 1—2.16,37 ℃诱导后的可溶性上清;3—4.16,37 ℃下包涵体变性后的上清。

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 ℃.

图6 StSPS1蛋白纯化、复性及鉴定 A.Ni-NTA亲和纯化后的结果:1.破碎后的蛋白,2.流出液,3—4.洗脱后的蛋白;B.StSPS1蛋白透析结果;C.StSPS1蛋白His抗体的WB检测结果。

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.

图7 2个StSPS1抗体在抗原中的WB鉴定

Fig.7 WB identification in antigen with two StSPS1 antibodys

图8 2个StSPS1抗体在川芋10号叶片中的WB鉴定

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

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