[Expression, purification and antigenic characterization of the Epitope-G1 gene of bovine ephemeral fever virus in Escherichia coli].
The epitope-G1 gene, cloned from the pMD-G plasmid including G protein gene of bovine ephemeral fever virus (BEFV), was subcloned into expression vector pGEX-4T-1 to construct pGEX-G1 recombinant plasmid successfully. The pGEX-G1 was transformed into E. coli BL21(DE3) to be induced with IPTG. The optimal expression conditions for G1 gene were obtained, which included reaction temperature 16 degrees C, induction time 18h and IPTG concentration 0.1 mmol/L. The soluble target protein was purified with Glutathione Sepharose TM(4B) and the purity reached 80%. The inclusion body washed with 2% deoxycholic acid sodium salt and dissolved with 0.5% N-lauroyl sarcosine sodium was recovered by the way of dialysis, then the protein was purified with Glutathione Sepharose TM(4B) and its purity was above 85%. The protein purified had nicer reaction activity by analysis of Western blot. The target protein was used as coating antigen to detect the sera against BEFV by an indirect ELISA. The 12 positive sera to BEFV were detected and the average of OD490 was 1.813 +/- 0.231, while the average of OD490 from 12 negative sera was 0.359 +/- 0.032, and the distinction was very remarkable (P < 0.01). All the rabbits inoculated with the target protein had produced high titer of antibodies, which indicated that the target protein had immunological activity. The average of OD49, detecting the 8 positive sera to rabies virus (RV) with the target protein purified was 0.324 +/- 0.031 which closed the datum obtained from the negative sera to BEFV, and it showed no cross-reaction between the sera to RV and BEFV. All the results above indicated that the target protein expressed had nicer biological activity and specificity, so the protein could be used as coating antigen to develop ELISA Kit for diagnosing BEF.
Zheng
Fu-ying
FY
Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China. zfycaas@126.com
Lin
Guo-zhen
GZ
Qiu
Chang-qing
CQ
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D002417:Cattle; D004810:Ephemeral Fever / Q000276:immunology* / Q000821:virology; D018115:Ephemeral Fever Virus, Bovine / Q000235:genetics / Q000276:immunology*; D000939:Epitopes / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D011817:Rabbits; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*
D000914:Antibodies, Viral; D000939:Epitopes; D014759:Viral Envelope Proteins