Research Of Glutamine Dependent Acid Resistance In Trichinella Spiralis Larvas

Trichinella spiralis is a globally distributed zoonotic zombie disease.The host infected it is due to eat raw or undercooked meat with infectious cysts,people can cause death when severe infection.In the process of infecting mammals,Trichinella spiralis must experience a very acidic environment in the stomach,and therefore we speculate that the body of Trichinella spiralis may have an acid resistant mechanism like E.coli to protect against the damage of acid environment.Recent studies have shown that glutamine dependently antacid system is the most important antacid system in E.Coli,glutamine was added in the acid medium(p H 2.5)to maintain the acid resistance of bacteria,glutamine antacid system plays an important role in bacteria.Previous studies have proved that the survival rate of Trichinella spiralis was higher under acidic conditions for 3 h(p H 2.5).However,It is not known whether the glutaminase plays an important role in the anti acid process of Trichinella spiralis,and this experiment has been studied.We designed primers based on the published NCBI gene of Trichinella spiralis glutaminase,and cloned the gene of Trichinella spiralis glutaminase by molecular biology.The cloned fragment was connected to the p MD-18 T vector and transformed into the clone competent cell DH5?,and sequence analysis of the cloned sequences after sequencing of the strain,and then we found that there are rare codons in the sequence,Therefore,Rosetta(DE3)strain was selected as the expression vector.The correct sequence was connected with the expression vector p ET-32 a after double enzyme digestion,and was transformed into Rosetta(DE3)The plasmid was extracted by extended culture expression strain and then digested by double enzyme digestion,plasmid PCR and sequencing verified,and finally induced expression.By optimizing the expression conditions,the maximum expression of 5h was induced by 0.4 mMol IPTG at 37?.The expressed protein was purified by gel filtration and mixed with Freund's adjuvant,subcutaneous injection of rabbits with multiple injections,and preparation of glutaminase antibody serum.ELISA method was used to detect the titer of immune serum antibody,and the results showed that the antibody titer reached 1.024×106.The serum was used as first antibody combined with the whole protein of Trichinella spiralis muscle larvae and purified recombinant protein and performed Western blot detection,and obtained specific bands.Immunological experiments showed that the purified recombinant glutaminase protein had good immunogenicity and immunogenicity.Immunofluorescence showed that the glutaminase was mainly distributed in thesubcutaneous tissue of Trichinella spiralis larvae.In order to study the change of the expression of glutaminase in acid condition,we minced muscle of mice and selected Trichinella spiralis and then cultured under acidic conditions(p H2.5?p H 4.0?p H 6.6 and pH 9.0).After cultured under the same conditions for 0.5 h,1 h and 3 h,we extracted RNA and reverse transcribed into c DNA,and then detected the expression of mRNA by fluorescence quantitative PCR.The results showed that the relative expression level of glutaminase mRNA in each Trichinella spiralis group was increased except for p H 9.0 group.The expression of glutaminase mRNA was increased with the decrease of p H in each time period Trichinella spiralis.Under pH 2.5 condition,the expression level of glutaminase m RNA was higher than other groups in each culture period and had significant difference(p < 0.01).This indicated that acidic conditions could significantly induce the expression of glutaminase in the muscle larvae of Trichinella spiralis.Mammalian gastric juice is not only an extremely acidic environment,but also contains large amounts of pepsin.In order to clarify whether pepsin have the effect on the expression of Trichinella spiralis glutaminase,we used the artificial gastric juice(pH 2.5)treated Trichinella spiralis for 3 h and then performed fluorescent quantitative PCR detection.After comparative analysis p H 2.5 saline 3 h group and p H 6.6 saline 3 H group,we found that the expression of pH2.5 saline group was significantly increased when compared with p H 2.5 artificial gastric juice group,and all showed significant difference(p < 0.01).However,there was no significant difference in the relative expression between p H 2.5 saline 3 h group and p H 6.6 saline 3 h group(P > 0.05).In addition,we analyzed three groups of Trichinella spiralis by immunofluorometric assay,and the fluorescence images were obtained by using Image J to select the fluorescent areas of Trichinella spiralis and analyze the optical density,and then calculated the average optical density of each group.The results showed that the average optical density values of the three groups were 0.01679,0.01860 and 0.00630 pixel,respectively.The fluorescence signal of pH 2.5saline group and p H 2.5 artificial gastric juice group was significantly stronger than that of p H 6.6saline group and had significantly difference(p < 0.01),however,the p H 2.5 saline group and p H2.5 artificial gastric juice were closer,the difference was not significant(p > 0.05).The two parts of the experimental results showed that the expression of Trichinella spiralis muscle larvae glutaminase increased significantly in the extremely acidic conditions,and the presence of pepsin had no effect on the expression of the glutaminase.The acid resistance of Trichinella spiralis plays an important role in the invasion of host,this study proves the extremely acidic conditions will be significantly increases the expression of glutaminase,the presence of glutamine-dependent acid resistance systems,which provides clues to further research of acid resistance of Trichinella spiralis.