Research Of Expression, Protein Localization And Acid Resistance Of Trichinella Spiralis

Trichinellosis is an important foodborne zoonotic disease, which is caused by the nematode parasites of Trichinella spiralis. Human can infect the pathogen by ingestion of striated muscles harbouring infective larvae, such as raw or undercooked pork meats. When ingested by the host, the T. spiralis cysts containing striated muscles is digested in the gastric acid environment in the nest few hours. Then the infective larvae of T. spiralis are released to invade small intestine celles. Now we know the pathogen has to live with the acid environment in the stomach of the host before continuing its life circle. However, whether this parasite has the similar acid fast mechanism, which has been examined exists in Escherichia coli, remains unknown. Rencent studies have shown that glutamic acid(Glu) content was high in T. spiralis, especially in muscle larvae. And the nucleotide sequence of T. spiralis glutamate decarboxylase(Ts GAD) are avalible in the Genbank, which will facilitate our elucidating if a GAD relying acid fast mechanism exists in this important zoonotic parasite.In this study, we had obtained the target gene from the total RNA of T.spiralis by the specific primer carrying restriction enzyme cutting site designed according to the Ts GAD gene in GeneBank following the method of RT-PCR.Then the PCR product was connected with p MD18-T and trans into E.coil DH5α. After identification by PCR and restriction analysis, the target gene was cut from p MD18-T and connected with expression vector p ET-30 a and trans into BL21(DE3). The IPTG was used to inducing the expression of the target gene and the inducing condition was optimized. The expression of the target gene was induced by 1M IPTG at 37°C for 5h and the target protein was turned to SDS-PAGE, the recombinant protein expressed as inclusion body was migrated at 57 ku which conform to the date from the GeneBank.The enzymatic activity of the Ts GAD recombinant protein was certified by colorimetric method and the liveness was 1.5U which illustrated the corrective folding of the recombinant protein. The immunogenicity of the Ts GAD recombinant protein was analysised by Western blot using the antiserum obtained from the New Zealand rabbit immuned by the protein, and the result show that the antiserum could recognise the recombinant protein. The titer of the antiserum was 1:65536 testing by indirect ELISA which show its nice which revealed its nice immunogenicity and reactionogenicity. In addition, the immunofluorescence localization assay was performed and the result show that the TsGAD was consisted in the surface of the T.spiralis muscle larvae.Based on the nucleotide sequence of Ts GAD and TsGAPDH in the Genbank, a pair of real-time PCR primers was designed. By using the method of real-time PCR, we detected the expression level of Ts GAD mRNA in different PH(pH 2.5, pH 4.0, pH 6.6, and p H 9.0) in T. spiralis muscle larvae. As the results showed, with the decrease of the pH value, the expression level of Ts GAD mRNA increased gradually. The expression level of Ts GAD m RNA in pH 2.5 cultivation environment was statistically higher than that in p H 4.0, p H 6.6 or p H 9.0(p < 0.05). We also detected the cultivation time(0.5h, 1h, and 3h) in affecting the expression of level of Ts GAD mRNA, with 1 hour of cultivation time significantly higher than that of half an hour and 3 hours. We concluded that acid environment can significantly affect the expression level of Ts GAD mRNA in T. spiralis muscle larvae. This study confirmed the exists of a glutamate decarboxylase relying acid resistance mechanism in T. spiralis muscle larvae and acid cultivation environment could facilitate the expression level of TsGAD m RNA, which layed foundation for further study on the acid resistance mechanism in the parasite.