Paratuberculosis Mycobacterium 34KD Protein In The Diagnosis Of Bovine Paratuberculosis ELISA

Bovine paratuberculosis is caused by Mycobacterium paratuberculosis(MP) and it is a kind of chronic wasting disease in ruminants globally, which is called Paratuberculosis enteritis.This kind of disease was first discovered and described by Johne in 1891 and so it was also called Johne’s disease.OIE listed as a Class B disease. This disease can cause proliferative enteritis, and can lead to intractable diarrhea, the incubation period of this disease ranging from several months to a year or two. Stool is thin, and accompanied by the smell of stench, diarrhea often has jet out with feces and mucus bubbles. And at last it result in death. This disease can be transmit from sheep and other animals to humans, this disease are widely popular on a global scale,and there is no effective cure methtod, prevention is the mainly way we can do, and the prevention can be done from the transmission ways of the disease. And we need to reinforce the management of daily feeding to increase the animal ’s own resistance. The most important is to do a thorough quarantine with PPD assay and do a second test for cows which were detected out of this disease, once the we can make a definite diagnosis of disease the animal must be isolated and make appropriate phase-out and killing. In order to control the spread of paratuberculosis the herd will be vaccinated to reduce the incidence of the disease in some countries, this approach also has a big drawbacks that can affect serology and allergy testing, vaccination can make healthy disease-free animal serum become positive, and it can also produce a granuloms in the injection site, which would affect allergy test results of bovine paratuberculosis. Since after vaccination the normal quarantine of cattle is affected and for worrying about impeding the import and export trade of livestock, so some countries have regulations that do not allow animals to be vaccinated. Paratuberculosis is considered to be the most important factor in economic losses in cattle industry, especially caused serious economic losses for the cattle industry and the dairy industry. So the development of new and safe and high performance vaccine and sensitive and specific diagnostic antigen agentia have become extremely urgent.In this study, E.coli expression system was used to obtain vice 34 kD mycobacterial antigens,the 34 kD protein of this bacterial strain expressed was used as bovine paratuberculosis diagnostic antigen or subunit vaccines for paratuberculosis. Using the chromosomal DNA as a template, with a specific primers for PCR amplification. Gel extraction kit was used to purify PCR products, and then cloned into JM109 to construct the recombinant cloning vector pMD-18T-34. By blue-white and plasmid size and restriction analysis and PCR screening to identify recombinant plasmid and do the sequence analysis.The recombinant plasmid pMD-18T-34 and the expression vector pET28 a are recycled by gel extraction and purified by BamHⅠand EcoR Ⅰdouble digestion, and then cutting to T4 DNA Lingase connected under 16 ℃2 h, 4 ℃overnight, construct recombinant plasmid pET28a-34, and transformed into competent E.coli DH5α to do the screening and identification of recombinant plasmids. The recombinant plasmid was transformed into E.coli BL-21, and after IPTG induction, SDS-PAGE and Western blotting analysis was used to examine teh target gene expression in E.coli BL-21.Using the purified 34 kD gene to do the test of tuberculosis detection in collected blood samples, and the first is to determine the optimum concentration of antigen coating and the serum diluted multiples, the bigest value measured by the method of indirect ELISA experiment OD492 P/N ratio is 11.888, means the the optimum coating concentration for recombinant protein is 0.6 ug/mL, the best dilution ratio is 1:400 for the serum samples and we used it for testing, using 34 kD antigen to test the 359 bovine serum, PPD got 28 positive detection serums, and 34 kD antigen got 23 positive detection serums, 34 kD got 326 negative detection serums and PPD got 321 negative detection serums, and the positive coincidence rate was 82.14%, the negative coincidence rate was 98.46%, So this research results not only has important application value in TB diagnosis but also has great significance in developing new type of vaccine.