羊口疮病毒ORF129基因的原核表达、纯化及鉴定

doi:10.16431/j.cnki.1671-7236.2018.07.009

Abstract

Abstract:

The aim of the experiment was to clone the ORF129 gene of orf virus,express and purify its encoded protein.According to the published ORF129 gene sequence of OV-IA82 strain in GenBank (Accession No.:AY386263.1),a pair of specific primers was designed and synthesized using Primer Premier 5.0 software,and the ORF129 gene was amplified by PCR.The whole gene sequence was ligated with the plasmid pET-28a(+) by BamH Ⅰ and Hind Ⅲ,and the recombinant plasmid pET-28a(+)-ORF129 was constructed.After identified by double digestion and sequencing,the recombinant plasmids were transformed into E.coli Rosttta,and the expression of ORF129 gene was induced by IPTG.After SDS-PAGE analysis,the expression products were disrupted,washed and dissolved by ultrasound,and then urea gradient dialysis method was used.After renaturation,the target protein was purified by affinity chromatography and identified by Western blotting.Double restriction enzyme digestion and sequencing results showed that the recombinant plasmid was successfully constructed with the correct reading frame.The ORF129 inclusion body protein was obtained when the initial D_{600 nm} value was 0.4 to 0.8,37℃,220 r/min,the final concentration of IPTG was 1 mmol/L,and the induction time was 3 h.And the protein size was 58 ku.In the arginine and glycerol refolding solution,the protein refolding rate was higher,and the protein was refolded after binding to the Ni column.When the concentration of imidazole was 500 mmol/L,the protein could be eluted.The purified protein was identified by Western blotting and proved to be successfully expressed.In this study,the prokaryotic expression recombinant plasmid of ORF129 gene was successfully constructed,and the ORF129 protein was successfully expressed and purified,which laid a foundation for the establishment of the detection method for orf virus.

Key words: orf virus; ORF129 gene; prokaryotic expression

CLC Number:

S852.65

DU Guoyu, LIU Yongjie, WU Jinyan, WANG Guangxiang, SHANG Youjun, ZHANG Yong. Prokaryotic Expression, Purification and Identification of ORF129 Gene of Orf Virus[J]. , 2018, 45(7): 1798-1803.

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Prokaryotic Expression, Purification and Identification of ORF129 Gene of Orf Virus

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