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Prokaryotic Expression of the Main Antigenic Domains of the E2 Envelope Glycoprotein of Csfv in E.coli

Author: LiCongCong
Tutor: LiYunLong；HeChengQiang
School: Shandong Normal University
Course: Cell Biology
Keywords: Classical swine fever virus(CSFV) E2 gene pET28a(+) pET32a(+) homologous recombination prokaryotic expression
CLC: S852.65
Type: Master's thesis
Year: 2007
Downloads: 186
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Abstract


Classical swine fever is infectious diseases of world-wide economic importance. Classical swine fever virus (CSFV) is the causative agent of species in nature and it brings serious compromise to pig.CSFV genome can encode five structural proteins and seven non-structural proteins,E2 envelope glycoprotein of CSFV is an important protein which can cause immune reaction in the host cells.Classical swine fever prevails in the world and never dies out,because it is evoluting constantly. In this study, analysis of full-length sequences from CSFV strains of 23 genomes provided the opportunity to test the role that recombination may play in CSFV genetic diversity. As the same time,the mian antigenic domains of E2 envelope glycoprotein of CSFV was expressed in E.coli,and the expressed protein was purified and used as antigen to immune animals to gain the antiserum.This paper contains two chapters and the following is the embody:1.Firstly,we collected 23 full-length CSFV strains isolated all over the world, in order to detect possible recombination events. Putative recombinant sequences were identified with the use of SimPlot program. Recombination events were confirmed by bootscaning, using putative homologous recombinant sequence as a query. A mosaic virus, CSFV 39 isolated in China was found . And its two putative parental-like strains CSFV Shimen and GXWZ02 were identified.2.We designed two pairs of primers and amplified respectively the full–length of E2 and the main antigenic domain of E2 which was called e2 (601bp)by PCR and the template was pMC18-CSFV.The PCR products were inserted into the prokaryotic expression vectors pET28a(+)and pET32a(+)respectively after double enzyme digestion.3.The two recombinant prokaryotic expression vectors were transformed into E.coli BL21(DE3) and then induced adding to 1mM IPTG at 37℃.Analysis of SDS-PAGE showed pET32a(+)-e2 expressed a protein with a molocular weight of 42KD,but no expressed protein was detected about pET28a(+)-E2.4.The expressed protein was purified by affinity chromatography and detected by Western blotting.The purified protein was dialyzed by a grades of urea and then mixed with adjuvant as the antigen to immune animals.After three weeks of immunity,we gained the antiserum .5.A new expression vector pGEX-4T-1-E2 was conducted and it expressed protein after being induced.The antiserum was father detected by western blotting and these process could prove the antiserum was to E2 protein after the stimulation to the immuned animals.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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