Screening Of Key Amino Acids Affecting The Toxicity Of Heat Labile Enterotoxin Type 2 Of Escherichia Coli And Construction Of Its Mutant Strain

Enterotoxigenic Escherichia coli(ETEC)is the main pathogen causing diarrhea in calves,piglets and human beings.Heat labile enterotoxin(LT)produced by ETEC is an important virulence factor causing diarrhea,which can cause the death of newborn calves and cause huge economic losses to animal husbandry.Because the extensive use of antibiotics can induce the emergence of drug-resistant strains,other drugs or vaccines are needed to ensure the health of newborn calves.Oral vaccine can induce intestinal mucosal immunity and humoral immunity,and the production cost of oral vaccine is low,so oral vaccine can be used to control calf colibacillosis.Enterotoxins produced by ETEC can be divided into heat labile toxin(LT)and heat stable toxin(ST).Foreign scholars have isolated E.coli expressing LT2 from calf diarrhea feces and proved that it plays an important role in the process of diarrhea dehydration.Therefore,it is of great theoretical and practical significance to explore the virulence related amino acid sites of LT2 gene and obtain the attenuated LT2 mutant E.coli strains through gene editing for the study of pathogenic mechanism of LT2 producing E.coli and the development of attenuated oral vaccine.In this study,E.coli DN1502 was selected as the parent strain.The fragment of elt2 and the mutant fragment were connected to the expression vector to obtain E.coli p ET-30a-elt2,E.coli p ET-30a-E110K,E.coli p ET-30a-Y102K,E.coli p ET-30a-V95K,E.coli p ET-30a-R5K,E.coli p ET-30a-S59K,E.coli p ET-30a-H42A and E.coli p ET-30a.The results of cell test in vitro showed that all the mutations could attenuate the toxicity of the toxin,and the mr LT2-V95K mutation had the weakest toxicity.The median lethal dose of mr LT2-V95K to mouse adrenal(Y-1)cells was1.145×10^-1 TCID₅₀/0.1m L,while The median lethal dose of r LT2 to Y-1 cells was 1.477×10^-5TCID₅₀/0.1m L.Four-week-old BALB/c mice were immunized with r LT2 and mr LT2-V95K.Three mice in each group were immunized subcutaneously,once every seven days,for a total of three times.Blood samples were collected every seven days to test the level of Ig G antibody.Meanwhile,PBS was injected subcutaneously as the control group.The results showed that compared with the control group,the two groups of mice expressed higher levels of Ig G antibody,and the antibody level of mr LT2-V95K group was always higher than that of r LT2 group(p<0.05),both reached the highest antibody titer of 1:102400 on the 21st day.The results showed that the serum of the two groups could neutralize r LT2 toxin in vitro,and the neutralization titer of mr LT2-V95K group was slightly higher than that of r LT2 group.The gene encoding LT2 was mutated by CRISPR/Cas9 double plasmid gene editing system to obtain E.coli m LT2-V95K.The growth curve showed that there was no significant difference between the parent strain and the mutant strain,and the number of viable bacteria reached the peak at 20 h.No reverse mutation occurred after 20 passages,which proved that the mutant has good genetic stability.The tolerance of the mutant strains was tested by artificial gastric juice and artificial intestinal juice,and both strains were tolerant to gastrointestinal environment.The identification of fimbriae growth showed that both could express many fimbriae,and the mutation did not affect the growth of fimbriae.Then the bacterial solutions of the two strains that induced naphthyridone acid resistance were given to 4-week-old BALB/c mice by gavage,with 6 in each group,to detect the colonization ability of the two strains in the intestine.The results showed that both strains could be colonized in the intestine for at least 7 days,and the induced resistance did not transfer horizontally to other strains in the intestine.In conclusion,the recombinant mutant can effectively stimulate mice to produce high level of Ig G antibody.Gene editing had no significant effect on the main biological characteristics of the attenuated mutant.The biological characteristics of the mutant strain meet the basic conditions of oral vaccine strain.The above studies prove that the attenuated mutant strain has the potential to be used as oral vaccine,which provides experimental data and theoretical support for the research of oral vaccine of calf E.coli diarrhea.