| African swine fever(ASF)is a highly contagious,hemorrhagic disease caused by the African swine fever virus(ASFV),with a fatality rate close to 100%.Since the first case of African swine fever was discovered in Shenyang,China in 2018,the current epidemic of the disease is nationwide,and its impact on China's swine industry is particularly serious.Therefore,the establishment of a simple ASFV detection method can be of great significance to control its spread.At present,there are many kinds of proteins that can be used for serological diagnosis of ASFV.Among them,p72 protein,as the main structural protein of African swine fever virus,is encoded by the gene sequence B646 L.It has good immunogenicity and strong conservation.The p72 protein is produced in the late stage of virus infection and accounts for a high proportion of virus particle protein,about 32%.It is also an important part of the icosahedral spool of virus particles.The p72 protein has the effect of inhibiting the reaction between the virus and the cell.The p72 antibody induced by the ASFV strain is highly conservative,and the antibody titre is high.Therefore,the p72 protein is now often used by researchers for serological diagnosis of African swine fever virus.This research is based on this characteristic of the protein to carry out correlation research,and the main results are as follows:1.Construction of a prokaryotic expression plasmid: The B646 L gene was entrusted to Shenggong Biological Company to synthesize it,amplified by PCR,and cloned into the pGEX6p-1prokaryotic expression vector.The sequence was correct,and the prokaryotic expression plasmid pGEX6p-1-P72 was initially constructed.Subsequently,the constructed pGEX6p-1-P72 plasmid was transformed into an expression bacteria under the action of BL21(DE3)competence.After the bacteria were disrupted by ultrasound,the protein was identified as soluble expression,which could satisfy further purification processing.Purification is carried out according to the characteristics of the protein with the GST specific binding tag protein,and the method of affinity chromatography is used to obtain the p72 target protein with high purity and concentration.Western-blot analysis of the protein has strong specificity.2.Establish a detection method: After repeated exploration and optimization of the conditions,it was finally determined that the coating concentration was 1.25 ?g/m L,the dilution concentration of the serum to be tested was 1:200 times,and the final dilution concentration of the enzyme-labeled secondary antibody HRP was 1: 60000 times.After a series of laboratory testing methods to evaluate the sensitivity,repeatability and specificity of the testing method,and at the same time to conduct a compliance test with the kit produced by Spanish INGENASA company,the results show that the serological testing method can be applied to the laboratory Testing of clinical samples.3.Preparation of monoclonal antibodies: quantify the purified protein,and finally immunize mice at a concentration of 40 ?g/mouse.After a total of 5 immunizations,the mice with higher antibody titers were taken out of the spleen and fused with S/P 20 cells.After 3 subcloning,5 strains of 1F1,4G2,5E1,7D3 and 9F7 were obtained.Cloned antibody cell line.The antibody obtained by Westernblot and indirect immunofluorescence analysis can react well with the protein,which can pave the way for subsequent laboratory establishment of blocking ELISA detection methods and vaccine development.This study is successful establish a simple,rapid,accurate and sensitive serological diagnosis method to ASFV,which can make great contributions for control of ASFV transmission,for ASFV vaccine research and development. |
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