Isolation And Culture Of Super Lactobacilli From Swine And Screening The Bacteria Impaired ThyA Gene

Lactobacillus are important members of normal bacteria community in guts of human beings and animals, so it will integrate the biological function of beneficial bacteria with the activity of expressed products of heterogeneous genes if Lactobaeillus were used as the expression vector. But the reported Lactobacillus expression vector systems used antibiotic as the selection pressure, it will cause the diffusion of drug resistance gene. So, the non- antibiotic expression vectors were studied.The chromatosome- plasmid causing death balance system used non-antibiotic as the selection pressure. It used house-keeping gene impairment strain of bacteria as the recipient bacteria and inserted the homologous or heterogeneous integrated impairment genes of the recipient bacteria, so the heterogeneous genes were expressed stably. ThyA gene was the house-keeping gene of bacteria, it can be used as selection pressure of non- antibiotic expression vectors.In this study, three strains of LAB were isolated from the intestinal mucosa of piglets. According to the features of Gram staining, and morphological and biochemical characteristics, the three strains, LI, L5 and L13, were preliminarily identified as Lactobacillus acidophilus, Lactobacillus casei, L. delbrueckii subsp. Delbrueeki and Lactobaeillus salivarius respectively. The results of growth curve showed that, the growth velocity of L1 and L13 were more rapid than L5. Effects of gastrointestinal factors (low pH, bile salt) on the survival of strain L1 and L13 were studied. The results showed that there was no obvious effect on the survival of L1 while incubating at pH over 2.5, and L1 was still able to survival well while incubating at pH1.5 for 30 min. Strain L13 exhibited a lower acid tolerance than L1, it was only able to survival at pH3.5 and 4.5. The survival of two strains was both inhibited by bile. The survival rates of L1 and L13 were 70.00% and 46.67% respectively after incubating in the medium with 0.1% of bile for 4 hours. The survival rate of L1 strains was 1.67%, but L13 strain was only 0.0167%. The survival rates of two strains both below 0.01% while the concentration of bile was 0.3%. The culture supernatants of the two strains showed different degrees of antagonistic effect against pathogenic E. coli, salmonella and Staphylococcus aureus. The above-mentioned experiments showed that, the trait of L1 was superior to L13, so it was studied further in next experiment.Forty-five TMP hardiness, thyA mutant strains of L. acidophilus, L1, were obtained through adding trimethoprim (20mg/L) and thymidine (50 mg/L) to the selective medium of MRS and they were passaged in ditissimus medium. Finally, one thyA mutant strain which growing well and stable was obtained and named L1-30. Then it was passaged in MRS medium and selective medium of MRS respectively.The culture (200μl) was spreaded on selective medium of MRS plate after every five generation and computed the reversed mutant ratio of every one hundred mutant. The result showed that, there was no reversed mutant after 100 generation. So it was stable nether in the thymidine surrounding nor in the non- thymidine surrounding.When it was as recipient bacteria, the biological function was educed completely on the one hand, on the other hand, thyA gene can be used as norientation selective marker and pressure, so the problem of the diffusion of drug resistance gene was solved. It was important in the agriculture, food and biology industry.