Construction And Study On Its Antigenicity Of The Heat-labile Enterotoxin Type ? Mutant Of Escherichia Coli

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.The main virulence factors of calf diarrhea caused by Escherichia coli are the adhesin,enterotoxin,Shiga toxin.Type II heat-labile enterotoxin(LT-II)is the pathogeny of calf diarrhea.The research of LT-II is still not extensive.So in-depth studies of the virulence mutation of LT-II have great significance on the prevention and control of calf diarrhea caused by Enterotoxigenic Escherichia coli,and being used in the researches on the foundations of the attenuated vaccines of E.coli diarrhea.Basde on consulting literature,refer to the study of LT-II a and LT-II b this experiment speculated that the 13,14 amino acid site of the r LT-IIc1 B subunitis is the key site for binding to the specific receptor.Using SOE-PCR to mutated.Before being translated into Rossatta competent cells,cloned the gene fragment into the p ET30 a expression vector,and this can induce the expressing the r LT-IIc1(T13I-T14I)mutant protein.Mixed the no mutation recombinant protein of the rLT-IIc1(T13I-T14I)and the recombinant protein of the r LT-IIc1 with the white oil adjuvants at the rate of 1:1.The specific Ig G levels were detected by indirect ELISA method.the.Y-1cells were used to detect the r LT-IIc1(T13I-T14I),rLT-IIc1 recombinant protein,wild toxin lowest lethal dose of cells.In vitro neutralization experiments detection of neutralization titers and cross-neutralization titers.SDS-PAGE and Western blot results showed that,r LT-IIc1(T13I-T14I)was highly expressed,Indirect ELISA showed the antibody titers of each group after antigen immunization were different.Antibody levels rose to the highest at 35 days and there was significant difference compared with the control group.The titer of r LT-IIc1(T13I-T14I)was 1:5000,the titer of wild toxin was 1:6400,rLT-IIc1 was 1:6400,higher than other immune group.The cytotoxicity test showed that Y-1 mouse adrenal cortex cells exhibited significant lesions after treatment with rLT-IIc1(T13I-T14I),r LT-IIc1 recombinant protein,wild toxins..The minimum lethal dose of rLT-IIc1(T13I-T14I)was 3.52×10-6mg / m L.The minimum lethal dose of rLT-IIc1was1.78×10-8mg / mL,the minimum lethal dose of wild toxin was4.87×10-11 mg / mL.Results showed that the neutralization titer of rLT-IIc1 serum to rLT-IIc1 protein was 1:89,the neutralization titer of rLT-IIc1 serum to rLT-IIc1(T13I-T14I)protein was 1:62,the neutralization titer of rLT-IIc1(T13I-T14I)serum to r LT-IIc1 protein was 1:45,the neutralization titer of rLT-IIc1(T13I-T14I)serum to r LT-IIc1(T13I-T14I)protein was 1:35.The results shows that LT-?c1(T13I-T14I)is immunocompetent,its can neutralized the Unmutated combinant toxin.The minimum lethal dose for Y-1 cells was 3.52×10-6 mg / m L.