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Development of Monoclonal Antibody Against Nucleoprotein of TGEV SC-H Isolate

Author: LiNa
Tutor: WenXinTian；CaoSanJie
School: Sichuan Agricultural University
Course: Preventive Veterinary Medicine
Keywords: TGEV recombinant nucleoprotein purification monoclonal antibody development
CLC: S852.65
Type: Master's thesis
Year: 2008
Downloads: 156
Quote: 1
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Abstract


Porcine transmissible gastroenteritis (TGE) is a highly contagious, enteric disease of swine caused by porcine transmissible gastroenteritis virus. The main clinical symptoms of infected pigs is vomiting、diarrhea and dehydration. TGE have lead to enormous loss to pig breed .In this study, monoclonal antibodies against recombinant nucleoprotein of TGEV SC-H isolate was development which be significant to diagnosis、prevention and control of TGE.1. Preparation, purification and identification of recombinant nucleoprotein of TGEV.In this experiment, recombinant plasmid pET-32a of N gene of TGEV named pET-32a-N was transfected into E.coli BL21 and the bacteria was induced with IPTG at 37℃. SDS-PAGE electropherogram shows that the molecular weight of the target protein was about 67kD. The recombination N protein was found in the inclusion body fraction, so the purification of it was under denaturing conditions at room temperature. It was demonstrated by SDS-PAGE that recombinant N protein was observed as a single limpid strap after purification, and its concentration was about 1500μg/ml. The result of Western-blotting presented that the purified recombinant N protein could identify the specific antisera against TGEV corpuscle certainly. It’s showed that the recombinant N had good reactionogenicity.2. Development of monoclonal antibodies against recombinant nucleoprotein of TGEV.The monoclonal antibodies(McAbs) against transmissible gastroenteritis virus(TGEV) recombinant nucleoprotein was developed by fusing SP2/0 cells with splenocytes of BALB/c mice immunized with the recombinant N protein.The fusing rate was 85%.And The positive rate was 65% detected by indirect ELISA.Three hybridoma cells, JY(4)-1, JY(4)-2, JY(4)-3,were examinated by indirect ELISA and be subcloned for four times by the limited dilution. The ELISA titers of 3 hybridoma cells’culture supernatants ,ascite and serum all were 1:512,1:10~5,1; 10~5 .No cross-reaction was observed between the McAbs and PPV、PRRSV and PRV. The McAbs also showed strong reactivity in IFA on the ST cells which be infected with TGEV.In this study, monoclonal antibody against TGEV SC-H isolate recombinant nucleoprotein has been prepared. The experiment confirmd the McAbs had satisfactory specificity which laid a good foundation for further study of nucleoprotein and establish diagnostic method of TGEV.

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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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