鸭寡腺苷酸合成酶样蛋白原核表达及多克隆抗体制备

doi:10.3864/j.issn.0578-1752.2019.23.024

Abstract

Abstract:

【Objective】In previous study, we have obtained the full-length gene of duck oligoadenylate synthase-like protein (duOASL) and confirmed that duOASL could inhibit DTMUV replication. To further investigate the inhibitory effect of duOASL on DTMUV replication, the recombinant protein (pGEX-OASL) with GST tag and polyclonal antibody of duOASL protein was obtained by prokaryotic expression and polyclonal antibody preparation technology. These results would lay a foundation for performing further studies to determine mechanism(s) underlying the antiviral activity duOASL at the molecular level. 【Method】Primers were designed based on the ORF sequence of duOASL gene from healthy Cherry Valley ducks. Total RNA was extracted from the spleen tissues of duckling by using HP Total RNA Kits. The cDNA was synthesized using random primers and M-MLV reverse transcriptase. Full-length of duOASL gene was obtained by synthesising pGEX-OAS-F and pGEX-OAS-R primers by using the cDNA. The PCR results were detected by 1.5% agarose electrophoresis. The sequences of the same size as the target band were purified and cloned to pEASY-T1 vector. The cloned gene ORF was inserted into the expression vector pGEX-4t-1 with GST tag and by BamH I and Xho I digestion. The recombinant plasmid was transformed into E. coli BL21 and expressed under the induction with 0.5 mmol·L ^-1 IPTG for 4 h. The expression of the fusion protein was detected by SDS-PAGE and Western-blotting. Then the polyclonal antibody was obtained from rabbits which had immunized by the purified duOASL recombinant protein. The purity and titer of the polyclonal antibody were determined by SDS-PAGE and ELISA.【Result】The recombinant protein was a soluble protein and the molecular weight was 84 kD. The purity was 0.5 mg·mL ^-1. The antibody titer of polyclonal antibodies was 1:512 000.【Conclusion】Above results would lay a foundation for further studies of duOASL inhibiting DTMUV replication.

Key words: OASL, prokaryotic expression, polyclonal antibody

BI KeRan,LI Yin,HAN KaiKai,ZHAO DongMin,LIU QingTao,LIU YuZhuo,HUANG XinMei,YANG Jing. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Oligoadenylate Synthase-Like Protein[J].Scientia Agricultura Sinica, 2019, 52(23): 4429-4436.

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References 29

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Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Oligoadenylate Synthase-Like Protein

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