生物技术通报 ›› 2022, Vol. 38 ›› Issue (8): 142-149.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1404
收稿日期:
2021-11-10
出版日期:
2022-08-26
发布日期:
2022-09-14
作者简介:
覃雪晶,女,硕士研究生,研究方向:经济林栽培理论与利用;E-mail: 基金资助:
QIN Xue-jing(), WANG Yu-han, CAO Yi-bo, ZHANG Ling-yun()
Received:
2021-11-10
Published:
2022-08-26
Online:
2022-09-14
摘要:
制备青杄(Picea wilsonii)PwHAP5基因的特异性多克隆抗体,为研究PwHAP5基因功能、分子机制奠定基础。经PCR扩增获得青杄花粉PwHAP5基因片段与载体pET-48b相连构建原核表达载体pET-48b-PwHAP5。经诱导产生重组蛋白PwHAP5,通过His-NTA镍柱纯化蛋白产物。蛋白经SDS-PAGE检测后再免疫兔子制备兔抗血清。血清经ProteinA纯化后,通过SDS-PAGE和Western blot检测抗体纯度和特异性。结果表明,成功构建了原核表达载体pET-48b-PwHAP5,制备了分子质量约为45 kD的重组蛋白PwHAP5。获得的青杄PwHAP5兔抗血清经间接ELISA法检测效价达1∶729 000以上;制备的PwHAP5蛋白兔多克隆抗体能特异性识别抗原,效价高,满足后续研究PwHAP5蛋白在青杄生长发育中的功能等需求。
覃雪晶, 王雨涵, 曹一博, 张凌云. 青杄PwHAP5基因原核表达及多克隆抗体制备[J]. 生物技术通报, 2022, 38(8): 142-149.
QIN Xue-jing, WANG Yu-han, CAO Yi-bo, ZHANG Ling-yun. Prokaryotic Expression and Preparation of Polyclonal Antibody of PwHAP5 Gene in Picea wilsonii[J]. Biotechnology Bulletin, 2022, 38(8): 142-149.
引物名称 Primer name | 序列 Sequence(5'-3') | 酶切位点 Enzyme |
---|---|---|
上游引物P1 | CGCGGATCCTATGGATCAGCAGCAGC-CCACAAT | BamH I |
下游引物P2 | CCCAAGCTTACTGCTGCCATGAGGA- GGTG | Hind III |
表1 引物信息
Table 1 Quote sequence information
引物名称 Primer name | 序列 Sequence(5'-3') | 酶切位点 Enzyme |
---|---|---|
上游引物P1 | CGCGGATCCTATGGATCAGCAGCAGC-CCACAAT | BamH I |
下游引物P2 | CCCAAGCTTACTGCTGCCATGAGGA- GGTG | Hind III |
图1 PwHAP5基因片段的PCR扩增及重组质粒鉴定 A:PwHAP5基因的RT-PCR(M:DL2000;1:RT-PCR);B:pET-48b-Pw-HAP5载体的双酶切鉴定(M:DL2000;1:pET-48b-PwHAP5载体BamH I和Hind Ⅲ双酶切产物)
Fig. 1 PCR products of PwHAP5 and identification of the recombinant plasmid A:PCR products of PwHAP5(M:DL2000; 1:RT-PCR). B:Identification of the plasmid pET-48b-PwHAP5(M:DL2000; 1:the plasmid pET-48b-PwHAP5 digested with BamH I and Hind Ⅲ)
图2 目的蛋白PwHAP5表达形式和表达量鉴定 A:目的蛋白表达形式鉴定(1:未经IPTG诱导的重组大肠杆菌pET-48b-PwHAP5/BL21;2:重组大肠杆菌pET-48b-PwHAP5/BL21裂解后的上清液;3:重组大肠杆菌pET-48b-PwHAP5/BL21裂解后的沉淀物);B:目的蛋白SDS-PAGE(1:未经IPTG诱导前的蛋白;2-4:经IPTG诱导4、6、8 h后的蛋白产物)
Fig.2 Identification of expression form and expression amount of target protein PwHAP5 A:Identification on the expression form of target protein(1:Recombinant E. coli pET-48b-PwHAP5/BL2;2:the lysate supernatant of E. coli pET-48b-PwHAP5/BL2;3:the lysate precipitant of E. coli pET-48b-PwHAP5/BL2);B:SDS-PAGE of the target protein(1:the target protein without IPTG induction;2-4:the target protein induced for 4,6 and 8 h after IPTG,respectively)
图3 纯化蛋白SDS-PAGE分析及未免疫兔血清的本底反应 A:Ni-NTA纯化重组蛋白(1:蛋白分子质量标准,2:总蛋白,3-5:分别是第1、2、3次洗脱液中的杂蛋白,6-8:分别是第1、2、3次洗脱液中的重组蛋白);B:本底反应(a,b:两只兔子血清的Western blot检测,1:蛋白分子质量标准,2:兔血清免疫印记检测条带)
Fig.3 SDS-PAGE analysis of purified recombinant protein and Western blotting assay A:Purified recombinant protein via Ni-NTA(1:protein marker;2:the total protein;3-5:the unnecessary proteins in the 1st,2nd and 3rd eluents,respectively;6-8:the necessary proteins in the 1st,2nd and 3rd eluents,respectively). B:Western blotting assay(a and b:serum from 2 different rabbits;1:protein marker;2:the rabbit antiserum)
图6 抗体特异性检测结果 1:PwHAP5蛋白;2:未经诱导的PwHAP5蛋白;3:对照组
Fig. 6 Specificity of rabbit polyclonal antibody of PwHAP5 1:Protein of PwHAP5;2:uninduced protein of PwHAP5;3:the control group
图7 花粉管萌发试验和间接免疫荧光检测 A:不同时长处理下花粉管的萌发情况;B:抗体间接免疫荧光检测(a,d:对照组;b,e:一抗处理的花粉管;e,f:一抗和二抗处理过的花粉管)
Fig.7 Pollen tube germination test and indirect immuno-fluorescence assay A:Germination situation of pollen tube under different time treatment;B:indirect immunofluorescence detection of antibody(a, d:the control group;b, e:the primary antibody treated pollen tube;e, f:pollen tubes treated with primary and secondary antibodies)
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