| Calf diarrhea was an acute infectious gastrointestinal infectious disease of newborn baby calves caused by enterotoxigenic Escherichia coli, its features mainly characterized was diarrhea.Severe cases died of failure, dehydration and even death, which had brought serious economic losses to the calf industry. Virulence factors of calf diarrhea caused by E.coli has contain enterotoxin(ST,LT), Shiga toxin and adhesion.The type II heat-labile enterotoxin can lead calf severe diarrhea, which have strong immunogenicity and immune adjuvant function.For LT-II related research at home and abroad is less, therefore, further study of biological activity of LT-II and immunogenicity of prevention and treatment of E.coli can be calf diarrhea has important practical significance, and also can be used for laid the foundation of E.coli diarrhea vaccine research.In order to improve the understanding of the epidemiological situation of LT-II toxin in Northeaet of China, a total of 138 E.coli isolates from 2010 to 2012 were detected for LT-II gene by PCR, and ten of these were sequenced.The results showed that eight samples were LT-IIc1 producing E.coli, one was LT-IIc4 producing E.coli and one was LT-IIc6 producing E.coli of ten LT-II positive.The polycistron ORF of LT-IIc1 was cloned into the p ET30 a expression vector,translated into Rossatta competent cells, and induced for expressing the recombinant LT-II protein.At the same time, also constructed the BL21/p HUE-LT-IIc1-A(r LT-IIc1-A), BL21/p HUE-LT-IIc1-B(r LT-IIc1-B) and BL21/p GEX-6p-1-LT-IIc1-B(rp LT-IIc1-B) fusion coding gene to Explore the LT-II immunogenicity. Mix the LT-IIc1, r LT-IIc1-A and r LT-IIc1-B with white oil adjuvants was 1:1 via intraperitoneal intraperitoneally and intranasally. after the immune,regular mice tail vein blood tests for detecting specific s Ig G and s Ig A antibody level in serum. At the same time, select August old hyline Brown laying hens, Chest point injection immunogens for preparation of egg yolk antibody, and Extraction of yolk antibody with chloroform, use of indirect ELISA for detection of antibody titer.The results of SDS-PAGE and Western Blot showed that four recombinant protein was expreeed successfully, and the recombinant toxin had induceded the apparent cytopathic effect for mouse Y1 adrenal cells. With the minimum lethal dose of 17.8pg. Meanwhile, the control group showed no obvious change.ELISA result show that for the I.P group, the LT-IIc1 recombinant toxin of mice immune serum has the highest levels of specific sIg G titer, the titer of A-subunit was1:10610, B-subunit titer was 1:14400, higher than other immune group; for the I.N group, theLT-IIc1 toxins mice serum specificity s Ig A titer was also the highest, A-subunit was 1:3400,B-subunit was 1:3600, after serial dilution LT-IIc1 toxin of Y-1 mouse cells in vitro and experimental results show that the neutralizing test showed that neutralizing titers of I.P group was1:52. After Immuned laying hens,recombinant protein LT- IIc1 group of yolk antibody level is higher, among them, the titer of A-subunit was 1:6120, B-subunit was 1:8630, r LT-IIc1-B group of yolk antibody titer was 1:9670, r LT-IIc1-A group of yolk antibody titer was only 1:3600; after dilution of yolk antibody LT-IIc1 toxin of Y-1 cells in vitro, according to the results of in vitro experimental results show that the recombinant protein LT-IIc1 group of yolk antibody neutralizing titers was 1:24; Recombinant protein r LT-IIc1-A groups of neutralization titer of 1:7.Neutralization titer of recombinant protein r LT-IIc1-B group was 1:19.The results showed that the LT-II toxin has immunogenicity and mucosal adjuvants effect,which can lay a preliminary foundation for further study of calf diarrhea. This research confirms Ig Y which are generated through recombinant protein infected hens can neutralize LT-II.Furthermore, the study provides high effective prevention agents for calf diarrhea’s prevention and cure, also supply detection antibody for the further research of LT-II. |
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