Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (23): 4429-4436.doi: 10.3864/j.issn.0578-1752.2019.23.024

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• SPECIAL FOCUS: PREVENTION AND CONTROL OF WATERFOWL INFECTIOUS DISEASE • Previous Articles    

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

BI KeRan1,2,LI Yin1(),HAN KaiKai1,ZHAO DongMin1,LIU QingTao1,LIU YuZhuo1,HUANG XinMei1,YANG Jing1   

  1. 1 Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Nanjing 210014
    2 College of Marine Life and Fisheries, Jiangsu Ocean University, Lianyungang 222005, Jiangsu
  • Received:2019-04-23 Accepted:2019-07-22 Online:2019-12-01 Published:2019-12-01
  • Contact: Yin LI E-mail:muziyin08@163.com

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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

Fig.1

The OAS-RNaseL antiviral pathway"

Fig. 2

OASL-mediated enhancement of RIG-I signaling"

Fig. 3

Agrose gel electrophoresis patterns of duck OASL gene by RT-PCR and colony PCR"

Fig. 4

The result of SDS-PAGE and Western-blotting from recombinant protein M: Protein marker; 1: Before IPTG induction; 2: All bacteria induced by IPTG; 3: Precipitation induced by IPTG; 4: Supernatant induced by IPTG"

Fig. 5

The result of SDS-PAGE from the purified recombinant protein M: Protein marker; 1: Unpurified recombinant protein; 2: Effluent; 3: Eluent"

Fig. 6

The result of SDS-PAGE from antibody"

Fig.7

Domain organizations of OAS family protein"

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