| Avian Pasteurella multocida disease, also known as fowl cholera, is an acute, contact lost blood infectious disease caused by Pasteurella multocida, and has a serious impact on the development of the poultry industry. Bacause of Pasteurella multocida including many serotypes. It is substantially important to develope new type of safe and efficient Pasteurella multocida subunit vaccine. Glucose 6- phosphate dehydrogenase(6-PGD) is the key enzyme of pentose-phophate pathway in sugar metabolism of bacteria. In the process of bacterial metabolism regulate the growth and reproduction of bacteria by regulating 5-ribose and reduced coenzyme Ⅱ phosphate(NADPH). We prokaryoticly expressed the glucose 6- phosphate dehydrogenase of C48-1 strain, finally establish indirect ELISA detection method and the method of researching recombinant protein immunoprotective.1. In reference to KEGG 6-PGD gene sequences of Pm70 strains(registration number:T00046)has published, we design primers, then successfully amplified out of the 6-PGD gene sequences, the segment length of 1453 bp, with PCR by using whole genome of C48-1 strain as a template. Insert the purpose gene into the prokaryotic expression vector PET-28 a, constructing prokaryotic expression plasmid PET-28a-6PGD successfully.The sequencing results shows that: Amplification sequences with Gen Bank reported Pm70 strains, 3480 strains, HN06 strains, ATCC 43137 strains, HB03 strain homology is above 99%.2. We transform recombinant plasmid to E.coli BL21(DE3) and expressed purpose proteins under IPTG induction.Obtained target protein, approximately 50 k D, is as the same size as expected results. After that, further optimization of induction time was performed, and we detected the target protein soluble in 37℃ and 16℃. Western-blot results showed that the recombinant protein has good reactionogenicity which laid a solid foundation of indirect preliminary ELISA method to study the immune protection function.3. This experiment establishes indirect ELISA detection method using purified 6-PGD recombinant protein as envelope antigen. The study optimized reaction conditions of indirect ELISA which namely placed 37℃ 2 h and 4℃ for the night with concentration of 10ug/m L of package antigen, and the best sealing fluid is 5% skimmed milk powder for 30 min, then serum concentration of 1:200 and goat-anti-chicken secondary antibody diluted 1:60000 incubated for 30 min and 60 min respectively. The coefficient of variation number of repeat and repeat inside batch experiment were from 0.433% to 7.23%, and blocking test block rate were greater than 50%.4. We immune chicken using purification of recombinant proteins, C48-1 inactivated bacteria with white oil adjuvant and physiological saline as the negative control group for twice which interval time of two weeks. Then by the indirect ELISA method for detection of serum antibody leves using separation of serum. The results showed that the antibody level of recombinant protein group was obviously higher than that of total bacteria inactivated seedlings group and the negative control group. After first immune, strong strain C48-1 of 10LD50 affectd chicken, and the results showed that the bacteria inactivated seedlings group, protection against recombinant protein group and the physiological saline group can provide 100%,60%,0% protection respectively.To sum up, indirect ELISA method can be used to establish the clinical sample testing and Pasteurella multocida disease epidemiology investigation. The recombinant protein can induce chicken produce higher levels of specific antibodies, and have certain immune protection and laid the foundation of 6-PGD for further research of pathogenesis and subunit vaccine research. |
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