蓖麻肌动蛋白基因的克隆、抗体制备和表达分析

doi:10.7668/hbnxb.20190221

摘要/Abstract

摘要： 为探讨蓖麻肌动蛋白（Actin）基因（RcActin）是否可作为蓖麻基因表达研究中的内参基因。以蓖麻生长根为试验材料，根据GenBank中公布的RcActin全长cDNA序列（登录号：XM_002522148.3）设计合成特异性引物，经PCR扩增获得RcActin的编码序列（CDS），并将其插入原核表达载体pET32a（+）中。重组表达载体pET32a（+）-RcActin转化大肠杆菌BL21（DE3），IPTG诱导表达融合6个组氨酸标签的目的蛋白，表达蛋白经钴离子螯合磁珠纯化后，采用Western Blot验证，结果表明，pET32a（+）-RcActin可诱导表达分子量约为61.46 ku的融合蛋白His-RcActin。用纯化的His-RcActin免疫BALB/c小鼠，经细胞融合及筛选获得9株能分泌抗蓖麻RcActin蛋白质单克隆抗体（Monoclonal antibody，McAb）的杂交瘤细胞株。利用阳性杂交瘤细胞株刺激小鼠，取腹水，采用间接ELISA方法测定，结果表明，5株阳性单克隆细胞株腹水效价达到1：1 000 000及以上。用蛋白A/G亲和层析法纯化His-RcActin McAb；采用间接ELISA方法对纯化McAb的特异性进行测定，3株阳性细胞株能分泌针对RcActin的特异性McAb；通过抗体亚类鉴定试剂盒鉴定纯化McAb的亚型，结果表明，3株阳性细胞株分泌的McAb亚类均为IgG1。采用间接ELISA方法对3株阳性细胞株分泌McAb的稳定性进行鉴定，获得1株在体外传19代或液氮冻存4个月后、能稳定分泌McAb的阳性细胞株。采用Western Blot和间接ELISA方法对从该细胞株中纯化的McAb的特异性、效价进行验证，结果表明，获得的McAb具有针对RcActin的特异性且效价为1：512 000。以制备的McAb为一抗，利用Western Blot方法分析RcActin在正常水肥管理且时空一致条件下的蓖麻8个组织中的表达量，结果表明，蓖麻不同组织中RcActin的含量存在显著性差异（P<0.05）。RT-qPCR分析结果表明，蓖麻不同组织中RcActin mRNA也存在显著性差异（P<0.05）。通过Western Blot方法分析RcActin在不同浓度NaCl胁迫下盆栽蓖麻根系中的表达量；通过RT-qPCR对不同组织中RcActin mRNA表达量进行分析，结果表明，蓖麻不同组织中RcActin蛋白质和RcActin mRNA的表达量均存在显著性差异（P<0.05）。RcActin表达研究为蓖麻功能基因表达分析中内参基因的筛选提供了一定的理论依据。

关键词: 蓖麻, 肌动蛋白, 基因克隆, 单克隆抗体, 组织表达

Abstract: In order to know whether the actin gene can be used as the internal reference gene in castor gene expression, the expression patterns of RcActin were investigated in transcriptional and post-transcriptional levels. Specific primers were designed and synthesized according to the full-length cDNA of RcActin published in GenBank (Accession number:XM_002522148.3). The coding sequence (CDS) sequence of RcActin was isolated by RT-PCR using the growing roots of castor as the experimental material. The obtained gene was inserted into prokaryotic expression vector pET32a(+) with His-tag, then the recombinant expression vector pET32a(+)-RcActin was established. pET32a(+)-RcActin was transformed into E.coli BL21(DE3) cells, and the target protein by fusing 6 histidine tag was expressed induced by IPTG in the bacteria. The fusion protein purified with cobalt-coated magnetic agarose beads were verified by Western Blot analysis. The results showed that a molecular mass of fusion protein His-RcActin close to 61.46 ku was produced. BALB/c mice were immunized with purified His-RcActin, and 9 hybridoma cell strains with the ability to secrete monoclonal antibody (McAb) against His-RcActin (anti-His-RcActin monoclonal antibody, anti-His-RcActin McAb) was obtained after cell fusion and being screened. The titers of anti-His-RcActin McAb from ascitics of mice were tested with indirect ELISA, which were stimulated separately with positive hybridoma cells. The results showed that the antibody titer of ascites stimulated with 5 positive hybridoma cell strains were higher than 1:1 000 000. Anti-His-RcActin McAbs were purified by protein A/G affinity chromatography from 5 ascitics and the specificity of purified McAbs was verified with indirect ELISA. The results showed that 3 positive hybridoma cell strains of them which could secrete specific McAbs against RcActin (anti-RcActin McAbs) were prepared. The Ig subtype of 3 specific anti-RcActin McAbs was identified by mouse monoclonal antibody isotyping kit. The results showed that 3 specific McAbs were classified as IgG1. Indirect ELISA results of the stability of 3 positive strains with secreting anti-RcActin McAbs showed that 1 positive cell strain, which were in vitro after 19 generations or 4 months of liquid nitrogen cryopreservation, could secret anti-RcActin McAbs as normal. Western Blot and indirect ELISA were used to verify the specificit and titer of anti-RcActin McAbs from this stable strain. The results showed that the secreted could specifically react with RcActin protein from castor and its titer was 1:512 000. Taking anti-RcActin McAbs as the primary antibody, Western Blot was used to analyzed the expression levels of RcActin protein in eight tissues of the castor plants at the same spatio-temporal scales growing normally under normal water and nitrogen management. The results showed that there were significant differences(P<0.05) in the expression of RcActin protein in different tissues of castor. Real-time quantitative RT-PCR (RT-qPCR) was used to detect the expression of RcActin mRNA in the above-mentioned tissues of the castor plants. The results showed that there were significant differences(P<0.05)in the expression of RcActin mRNA in the detected castor tissues. Western Blot was used to analyzed the expression levels of RcActin protein in the roots of the potted castor plants under NaCl stress at different concentrations. The relative expression levels of RcActin mRNA in different tissues were analyzed by RT-qPCR. The results showed that there were significant differences(P<0.05)in the expression of RcActin protein and RcActin mRNA in the detected castor tissue. The study on RcActin expression provides reference for the screening of internal marker genes in the analysis of functional genes expression from castor.

Key words: Castor, Actin, Gene cloning, Monoclonal antibody, Tissue expression

中图分类号:

Q786
S563.03

王芳, 汤璧蔚, 董乐, 刘宝, 黄慧, 黄苹苹, 张立群, 栾芙蓉. 蓖麻肌动蛋白基因的克隆、抗体制备和表达分析[J]. 华北农学报, 2020, 35(3): 12-23. doi: 10.7668/hbnxb.20190221.

WANG Fang, TANG Biwei, DONG Le, LIU Bao, HUANG Hui, HUANG Pingping, ZHANG Liqun, LUAN Furong. Cloning, Monoclonal Antibody Preparation and Expression Analysis of Actin Gene in Castor[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(3): 12-23. doi: 10.7668/hbnxb.20190221.

http://www.hbnxb.net/CN/Y2020/V35/I3/12

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