| Trichinella spiralis is a zoonotic parasite with worldwide distribution,which can infect humans and a variety of mammals and cause Trichinosis.Trichinella has caused serious harm to human health and animal husbandry,and is one of the food-borne zoonotic parasitic diseases.The adult larvae are mainly parasitic in the duodenum and jejunum of the host,while the newborn larvae are in the intestinal mucosa and reach the skeletal muscle cells with the blood circulation of the whole body,and then they can form cysts through re-development.On the 14 th day after infection,under the action of gastric acid,infectious muscle larvae in tomont are releases.After a few hours,the muscle larvae drill into the small intestine and produce the newborn larvae to continue the next stage of life history.Since T.spiralis larvae need to go through gastric acid environment,so the study was carried out with reference to the acid fast system of E.coli.This study is based on ornithine dependent acid fast system to study the effect of acidic conditions on the transcription and expression of T.spiralis ornithine decarboxylase(Ts ODC)gene.Then the role of ornithine-dependent acid-fast system in the acid-fast mechanism of T.spiralis was studied.In order to study the changes of gene expression in muscle larvae of T.spiralis under acidic conditions,mouse muscles were ground and collected.According to the gene sequences of Ts ODC and T.spiralis heat shock protein 60(Ts HSP60),T.spiralis heat shock protein 70(Ts HSP70)and T.spiralis heat shock protein 90(Ts HSP90)published in Gen Bank.q PCR primers were synthesized and q PCR method was used to detect the changes in the expression level of the selected genes.Four gradients were set from low to high culture p H,i.e.,p H 1.5,p H 2.5,p H 4.5 and p H 6.6,and three gradients were set for culture time,i.e.,0.5 h,1 h and 2 h.The results showed that Ts HSP60 had no change trend under acidic conditions.Ts HSP90 increased with the increase of culture time in weak acid environment.The m RNA expression level of Ts HSP70 was significantly higher than that of PBS group when cultured at p H 2.5 for 1h(P<0.01),suggesting that Ts HSP70 could be increased under strong acidic conditions and may be involved in the acid resistance of T.spiralis.The general trend of Ts ODC study was as follows:in the same culture time,Ts ODC m RNA expression showed an upward trend in the range of p H 1.5-2.5,and a downward trend in the range of p H 2.5-6.6;Under the same p H condition,Ts ODC m RNA expression increased with the increase of culture time.Compared with PBS group,Ts ODC m RNA expression increased by 160%(P<0.01)when cultured at p H 2.5 for 2 h,and showed a significant regularity.There was no significant difference at p H 6.6.As there are many influencing factors of heat shock protein,the interference of other external factors cannot be excluded.Therefore,ornithine decarboxylase was selected for the follow-up study.These results suggested that the expression of ornithine decarboxylase gene in muscle larvae of T.spiralis was significantly induced by acidic conditions,which was involved in the acid resistance process of T.spiralis.In this study,we designed and synthesized specific primers with Ts ODC restriction site based on the published m RNA sequences of Trichinalis ornithine decarboxylase gene using molecular biology techniques.The PCR fragment was linked with p MD-18 T vector and transformed into the clone competent cell DH5α.After bacterial solution sequencing,the cloned sequence was analyzed with the sequence published by Gen Bank.After enzyme digestion,it was linked with the expression vector p Cold I and transformed into the competent BL21 cell.After sequencing verification,IPTG was used as inducer for expression.The maximum expression level was obtained after 1 mmol/L IPTG induction at 16℃ for 20 h by optimizing the inductive expression conditions.SDS-PAGE analysis showed that the size of the target band was in line with the predicted theoretical value,the molecular weight was about 43 k Da,and the target protein mainly existed in the form of inclusion body.The purified protein was mixed with adjuvant and immunized New Zealand white rabbits subcutaneously at multiple points to prepare ornithine decarboxylase polyclonal antibody serum.The prepared serum was used as the primary antibody to detect the whole protein of T.spiralis muscle larvae and the purified recombinant protein by Western-Blot,and the specific bands were obtained,indicating that the prepared serum could bind the recombinant protein.The titer of serum antibody was detected by ELISA method.The results showed that the titer of multiple antibodies was 1:204800,with good immunogenicity and reactivity,and high specificity.Immunofluorescence localization showed that ornithine decarboxylase was mainly distributed on the surface of the muscle larvae of T.spiralis,and the expression level was high in the head and tail parts.The muscle larvae of T.spiralis treated with curcumin and rapamycin,as well as arginine and ornithine decarboxylase polyantisera,respectivively,were cultured in p H 2.5 for 2h with PBS treatment as control.Real-time fluorescent quantitative PCR(q PCR)technology was used to detect the influence of different preparations on the expression of Ts ODC gene,and the optimal culture conditions for in vitro preparations were determined as follows: Arginine 10 μg/ m L,24 h,survival rate is up to 78%;Polyclonal antibody 1mg/m L,48 h,survival rate is as low as to 38%;Curcumin20 μM,48 h,survival rate is as low as to 33%;Rapamycin concentration was 2 μM for 48 h,survival rate is as low as to 25%.The expression level of Ts ODC protein detected by Western-Blot was consistent with the q PCR results.Compared with PBS control group,arginine group,polyantiserum group,curcumin group and rapamycin group were increased by 68%(P<0.01),decreased by 11.6%,25.8%(P<0.05)and 47.8%(P<0.01),respectively.After acid treatment in the rapamycin treatment group and the PBS control group,p H of the medium was detected,and the results were consistent with the previous experimental results.Rapamycin treatment did not increase significantly in the PBS group,indicating that it inhibited the activity of ornithine decarboxylase and affected the acid resistance of T.spiralis.These results indicated that inhibitors and promoters could affect the expression of ornithine decarboxylase gene under acidic conditions,and proved that ornithine decarboxylase could regulate the acid resistance of T.spiralis.Above all,this experiment by means of cultivating in different acidity condition of T.spiralis ornithine decarboxylase gene,ornithine decarboxylase in the distribution of T.spiralis body surface,and the inhibitor and promoter of ornithine decarboxylase gene expression and its research on the influence of protein content,proved the ornithine decarboxylase participate in the process of T.spiralis acid,has a regulatory role.The results laid a foundation for the further study of the acid resisting mechanism of T.spiralis and provided a theoretical basis for the diagnosis and prevention of Trichinosis. |
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