Differentiation Between Vaccinated And Infected Animals By Food And Mouth Disease Virus On The Basis Of Antibody Against 3AB By ELISA

Foot and Mouth Disease (FMD) virus by the foot and mouth disease (FMDV) from cloven-hoofed animals were suffering from the reach of sexually transmitted diseases. There are seven FMDV serotypes, more than 70 kinds of subtypes, high variability, and to prevent the virus have caused great problems. It can lead to the import and export of animal products trade restricted, causing huge economic losses and political influence between countries. Establish an accurate and fast distinction between infected live virus vaccine and injected animals in the detection and diagnosis of FMD control the incidence of livestock quarantine areas are very urgently needed.The ELISA of using antigen of Non-structural protein 3AB in Foot-and-mouth disease virus can be used for differentiation of infection from vaccination. The 3AB gene fragment amplified by PCR from the template of 3ABC gene fragment was inserted into pET32а(+). The recombinant plasmid pET3AB was transferred into BL21(DE3) plysS and the Target protein was induced by IPTG with 0.8OD550. The SDS-PAGE and Western Blot results showed the recombinant plasmid pET3AB was constructed successfully and the NS-3AB was expressed strictly. The NS-3AB is unsoluble .The MW of NS-3AB is about 50Kda. the ELISA result indicated that the NSP-3AB can be used as antigen to differentiation FMDV-infected from vaccinated pig and cattle.NSP-3AB ELISA were developed using the recombinant Non-structural protein 3AB as detecting antigen. It can diagnose if the detected animal infected FMDV. Detecting antigen based on the non-structural protein 3AB was coated onto reaction microplate wells, recombined protein A/G marked with horseradish peroxide enzyme was used as the second antibody, the anti-non-structural protein antibody detecting method was established. Specific of NSP-3AB ELISA was 96.68% in 1,746 immunity swine serum; it was 97.96% in 1,077 health swine serum; its positive rate was 100% for 36 swine serum infected FMDV by man. However, the specific of bovine serum was respectively 94.04%, 97.96%, 100%.Further improve the 3AB-ELISA detection method, using skim milk and E. coli lysate as the method of blocking agent, effectively reducing the background value and eliminate non-specific response. TMB used for the color of the testing environment than the requirements of the OPD wider to enable the detection of practicality better.The result of Comparison between 3AB-ELISA and similar foreign kit, the detection rate of positive bovine serum, 3AB-ELISA the positive rate was 91.7%, UBI kit was 79.2%, higher than the UBI kit 12.5 percentage points. On the negative bovine serum testing, they are almost unanimous.