| Brucellosis is a common zoonotic disease in China.Its main symptoms are abortion,infertility and local lesions of various tissues.Brucellosis has a great impact on the health of livestock and people.The virulence factor vir B 9 of Brucella type IV secretory system was selected as the coating protein to sequence the gene of Brucella Maltese isolated from Zibo,and then the vir B 9 gene sequence in Gen Bank was compared with the sequencing results to determine the gene sequence of vir B 9,design primers,and then take the whole gene sequence of Brucella Maltese isolated from Zibo as the template,The target gene was amplified,digested by ndei and xhoi and connected with vector p ET28a（+）.The recombinant plasmid p ET28a（+）-vir B9 was obtained.After detection,IPTG induced the expression of vir B 9 in competent cells containing recombinant plasmid BL21（DE3）.The protein was purified by Ni purification column and identified by Western blot after SDS-PAGE analysis.Using the purified virus vir B 9 as the coating antigen,an indirect ieelisa method was established,and the optimal serum dilution,vir B 9 dilution,enzyme labeled secondary antibody,blocking solution,critical value,sensitivity and repeatability were determined by dot matrix titration.The false positive rate and coincidence rate of 1557 clinical samples were detected under the optimal reaction conditions.The test results are as follows: the false positive rate of virb9 iielisa is 0.41%,and the coincidence rates with tiger red,commercial iielisa,commercial CELISA and tube agglutination are 81.39%,60.95%,99.18% and 99.59% respectively.The positive sera of other bacteria are used for detection.The results show that the specificity of this method is good.In recent years,with the development of aquaculture,the incidence rate of brucellosis has risen sharply in the world,which has a serious impact on the prevention and control of brucellosis.To carry out cattle epidemiological investigation and master the current epidemic situation of brucellosis can lay a foundation for brucellosis purification.The purpose of this study is to investigate the prevalence of bovine brucellosis in Shandong Province in 2021,and to provide scientific basis for formulating brucellosis prevention and control policies and purification policies in Shandong Province at the present stage.In this experiment,50397 serum samples were collected from 1111 farms in 16 cities in four quarters of Shandong Province in 2021.After testing,the positive samples mainly come from 37 farms in 7 urban areas.The number of positive samples is 254,the Population positive rate is 3.33%,and the individual positive rate is 0.50%.According to the analysis,the positive samples are mainly distributed in central Shandong,southern Shandong and Northern Shandong.The positive field appears in the immune zone,and there is no positive field in the buffer zone and purification zone;It mostly occurs in commercial beef cattle farms and farms with less than500 stock;The peak of brucellosis is in spring and winter.In conclusion,the virb9-i ELISA method established in this test has good sensitivity,repeatability and specificity,and is highly consistent with the results of tube agglutination,with a coincidence rate of 99.59%,which can be used for the detection of Brucella serum antibody in clinic.Then the method was applied to the follow-up epidemiological investigation.Firstly,the preliminary screening was carried out by virb9 iielisa and tiger red plate agglutination method,and then the diagnosis was carried out by test tube agglutination and commercial ELISA.Fully grasp the individual positive rate,population positive rate,spatial distribution,population distribution and seasonal distribution of bovine brucellosis in2021.To provide a theoretical basis for the prevention,control and purification of brucellosis. |
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