The Establishment And Primary Application For RT-PCR Detection Assay Of FMDV And ELISA Assay Of Their Antibodies

Foot-and-mouth disease caused by foot-and-disease virus is a highly contagious disease among cloven hoofed animals and was listed first among type-A infectious disease by Office International des Epizooties. FMD seriously impair healthy development of animal husbandry and result in considerable economic losses. Therefore, all the countries pay much attention to its study and prevention of FMD. This paper is divided into three parts. In the first part, We developed a detection assay of FMDV and differential serotypes by RT-PCR. The second part is about the prokaryotic and eukaryotic expression of VP2 gene of FMD and developing I-ELISA method of detecting VP2 antibody. The third part is about the prokaryotic expression of FMDV 3ABC gene and developing I-ELISA method of detecting 3ABC antibody.In the first part, according to the 3D gene, 1D gene and 2A gene data of FMDV in Genebank, 2 pairs of primers P1/P2 and S1/S2 used for the detection and the differentiation of seven serotypes of FMDV were designed. The interested gene of FMDV was cloned by RT-PCR method and the comparative homology was analysed. In sensitiveness test, the malicious TCID₅₀ of FMDV was measured using Reed-Muench method. The viral RNA was extracted separately after ten times of gradient dilution. The result of the test indicated that the designed primers could be measured to 10TCID50. The RT-PCR results of IBRS-2 cell, HCV, VSV, BVDV, IBRV were negative. The results of infected cow's tissue (heart, tongue, the spleen and so on), the young mouse's tissue and the saliva of infected cow, were positive using this method. Through homology analysis of amplification products of primers S1/S2, the primer pair not only could detect the FMDV but also differentiate the seven serotypes. The primary application results suggested that the RT-PCR is a specific and sensitive method which can be used for detection and diagnosis of latent and subclinical FMDV O type and Asia I type.In the second part, VP2 gene of FMDV was cloned by the primer pair V1/V2 containing Pst â…  / Kpn â…  sites. The recombinant clone vector pT-VP2 was digested with Pst â…  and Kpn â… , then cloned into prokaryotic expression vector pPROEX^TMHTb and eukaryotic transfer vector pBlueBacHis2A respectively. The recombinant eukaryotic transfer vector pBL-VP2 was used to transfect into sf9 cell. After plaque scan and two times amplification of the virus stocks, the target protein was expressed in insect cell sf9, the molecular weight was 33ku. The Fusion protein antigenicity was identified by Western-blot and Dot-ELISA analysis using cattle antisera as first antibody.The recombinant prokaryotic expression vector pPR-VP2 was transformed into the E. coli...