青杄PwHAP5基因原核表达及多克隆抗体制备

doi:10.13560/j.cnki.biotech.bull.1985.2021-1404

摘要/Abstract

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。

关键词: PwHAP5基因, 原核表达, 蛋白纯化, 多克隆抗体

Abstract:

Specific polyclonal antibodies against PwHAP5 gene of Picea wilsonii were prepared to provide the experimental data for follow-up studies of PwHAP5 protein localization,expression and function. The PwHAP5 gene fragment from P. wilsonii pollen was amplified by PCR and then was inserted into vector pET-48b for constructing the recombinant plasmid pET-48b-PwHAP5. The recombinant protein of PwHAP5 was induced and was purified by His-NTA nickel column. The expressed recombinant protein was tested by SDS-PAGE and injected into New Zealand rabbits as an antigen to obtain antiserum. Antiserum was purified by ProteinA,the purity and specificity of the antibody was identified by SDS-PAGE and Western blot method. As results,the prokaryotic expression vector pET-48b-PwHAP5 was successfully constructed,and the PwHAP5 protein with molecular weight of 45 kD was prepared. The titer of PwHAP5 rabbit antiserum was more than 1∶729 000 via indirect ELISA. The prepared PwHAP5 protein of rabbit polyclonal antibody has a high titer and specifically recognize antigen,which can meet the requirements of subsequent studies on the functions of PwHAP5 in the growth and development of P. wilsonii.

Key words: PwHAP5 gene, prokaryotic expression, protein purification, polyclonal antibody

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.

图/表 8

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4 间接ELISA法检测效价

Fig.4 Detected titer of antibody detected by indirect ELISA

图5 纯化后抗体纯度检测 1：蛋白分子质量标准;2：抗体

Fig.5 Purity detection of purified antibody 1：Protein marker;2：the antibody

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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