Cloning, Expression Of VP₂ Gene In Foot-and-mouth Disease And Establishment Of Antibodies Detection Methods

Foot-and-mouth disease(FMD) is a highly contagious and acute disease of cattle, goats,pigs and sheep which is caused by Foot-and-mouth disease virus(FMDV).In developed country,killing all infected and exposed animal could be the first choice to control and eliminate the disease.But in developing country,vaccination is more acceptable.In China,vaccination is the main measures to prevent and control the disease.So, evaluation of antibody level after vaccination is important.In the past,some researches indicated that structual protein VP₂ of FMDV could induce specific antibodies against FMDV.Otherwise,the four structural protein's mutation probability is VP₁＞VP₃＞VP₂＞VP₄.VP₁ is more variable and VP₂ is relatively conserved.So,VP₂ protein has many advantages as a tool to detect antibodies against FMDV.Monoclonal antibody has high sensitivity and specificity,so it is widely used in biomedicine.In this study,VP₂ gene of O and Asia-1 sreotype FMDV were cloned and expressed in E.coli respectively.monoclonal antibodies against VP₂ protein of Asia-1 type FMDV were prepared.Based on the protein expressed and monoclonal antibodies,competitive ELISA for the detection of VP₂ antibodies was established.clinical tests indicated that the ELISA method had high sensitivity and specificity.Research contents are as follows:1.cloning and expression of VP₂ genes of o type FMDV and Asia 1 type FMDVAccording to the gene sequences pulicated in Genbank,primers were designed and synthesizd.660bp VP₂ genes of O type FMDV and 693bp Asia-1 type FMDV were amplified by PCR.Sequence analysis showed the gene fragments were VP₂ genes of FMDV.Then,the VP₂ genes were subcloned into vector and expressed in E.coli.2.establishment of indirect ELISA using VP₂ protein of o type and asial type FMDVIndirect ELISA was established by coating VP₂ protein as detection antigen.All impact factors to reaction were optimizated,including consentration of VP₂ protein,temperature,reaction time and silling buffer,et al.,these work would contribute to the application of the ELISA kit.3.Generation of monoclonal antibodies against AsiaI type foot-and-mouth disease virus:Balb/c mice were immunized with the E.coli expressed fusion protein.The splenocytes from immunized mice were fused with myeloma cells SP2/0.The hybridism cells were screened by indirect ELlSA and limited dilution method.Two hybndoma cell Iines secreting mAbs against Asia I type foot-and-mouth disease were obtained.The ELISA titers of the ascites induced by the two hybridism cells were above 100×2⁹. Specificity of mAbs was analyzed by Western-blot.All the monoclonal antibodies were proved to be specific.No cross-reactions with nontarget proteins were found.4.Establishment and prime application of the competitive ELISA for the detection of asial FMDV antibodyThe competitive ELISA method used to detect the antibody against FMDV was established by using VP₂ protein as antigen and the HRP-labled monoclonal antibody 5F5 as detection antibody.All the items which may influence the reaction were perfected,including temperature in each steps and final concentration of each solution.At last the best way of this method was found,which provide the foundation for the next step-use of the ELISA kit.