Cloning, Monoclonal Antibody Preparation and Expression Analysis of Actin Gene in Castor

doi:10.7668/hbnxb.20190221

Abstract

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

CLC Number:

Q786
S563.03

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.

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