The Effect Of Cold Stress On The Expression Of TsHSP70 Gene And Its Study On The Antioxidant Capacity In Trichinella Spiralis

Trichinosis is a food-borne zoonosis caused by Trichinella spiralis,which is prevalent worldwide.It not only affects animal husbandry,but also causes harm to human and animal health.In severe cases,it can lead to death.Usually,the host is infected by eating meat containing T.spiralis muscle larval cysts,which causes serious damage to many organs of the body and leads to various complications.Freezing method is often used to treat meat,in order to kill the parasitic T.spiralis in meat.However,the biological characteristics of T.spiralis have the ability of low temperature resistance,which has a certain influence on the harmless treatment of meat.In this study,the changes of heat shock protein 70(TsHSP70)in muscle larvae of T.spiralis under cold stress were studied to explore the protective mechanism of TsHSP70 against cold stress of T.spiralis.Firstly,in this experiment,the transcription levels of enolase(Ts ENO1),cold shock DNA binding domain proteins(Ts CDSPs),heat shock protein 70(TsHSP70)and heat shock protein 90(TsHSP90)genes in T.spiralis muscle larvae were detected by real-time quantitative PCR(q PCR)under cold,heat and salinity stress.After cold stress,the m RNA transcription levels of TsHSP70 and Ts CDSPs increased and reached the peak at-4℃ for 2 h,while the changes of Ts ENO1 and TsHSP90 were not obvious and did not have obvious regularity.The m RNA levels of TsHSP70 and TsHSP90 increased after heat stress,but Ts ENO1 and Ts CDSPs did not change.Ts CSDPs m RNA transcription level increased after salinity stress,and the increase was most significant at 20‰salinity culture for 3 h.TsHSP90 m RNA transcription level increased and reached the peak at 30‰salinity culture for 3 h,while Ts ENO1 and TsHSP70 did not change.It is speculated that TsHSP70 and Ts CDSPs can protect T.spiralis muscle larvae during cold stress,TsHSP70 and TsHSP90 during heat stress,and Ts CSDPs and TsHSP90 during salinity stress.Under cold and heat stress,the activity and survival rate of T.spiralis muscle larvae were decreased with the decrease of temperature at the same incubation time.Under the same temperature condition,with the increase of culture time,the viability and survival rate of T.spiralis muscle larvae in each group decreased,and the survival rate was only 20% at-10℃ for 4 h,indicating that continuous cold and heat stress was not conducive to the survival of T.spiralis muscle larvae in vitro.The results of the activity and survival rate of T.spiralis muscle larvae under salinity stress showed that the in vitro survival of T.spiralis muscle larvae was not affected by the salinity of 15～30‰ in a short period of time.According to the above experimental results,since the transcription level of TsHSP70 m RNA changed significantly with cold stress,subsequent experimental studies were focused on cold stress and TsHSP70 gene.Based on the heat shock protein 70 gene of T.spiralis published by NCBI,the TsHSP70 gene was cloned from T.spiralis muscle larvae by molecular biology technique,and the cloned gene fragment was linked with p MD-18 T vector and transferred into the capable cell DH5α.The sequencing results showed that the homology with the original sequence reached 99.23%.The cloned plasmid was linked with the expression vector p ET-32a(+)after double digestion,and then transferred into competent cell BL21.The strains with correct sequencing results of bacterial fluid were expanded and induced for protein expresson.The induction time and concentration of the recombinant protein inducer IPTG were optimized,and the expression of the recombinant protein was the highest when induced by 1.0 m M IPTG at 37℃ for 6 h.The recombinant protein was expressed in the form of inclusion body.The recombinant TsHSP70 protein was purified and injected into rabbits to prepare polyclonal antibody serum of TsHSP70.The antigenicity of the prepared polyclonal antibodies was detected by ELISA and Western blot,and immunofluorescence was detected by laser confocal microscopy.The antibody titer reached 1:51,200.The prepared polyclonal antibody showed good reactivity and specificity with purified TsHSP70 protein and HSP70 protein from T.spiralis muscle larvae.Fluorescence localization showed that TsHSP70 protein was mainly distributed in the epidermal tissue of T.spiralis muscle larvae,and the protein was abundant in the head,indicating that TsHSP70 protein should be excreted protein.In order to further verify the correlation between cold stress and TsHSP70 expression,q PCR and Western blot were used to detect TsHSP70.When T.spiralis muscle larvae were cultured at the same time and different temperatures,the gene transcription level and protein translation level of muscle larvae cultured at-4℃ were increased by 117.13% and 61.83%,respectively,compared with the control group,and the expression level was significantly increased.The changes of gene transcription level and protein translation level of TsHSP70 were detected in muscle larvae cultured at-4℃ for different time.Compared with the control group,the muscle larvae cultured at2 h increased by 130.86% and 158.64%,respectively,with the most significant changes.These results further demonstrated that the transcription level and protein content of TsHSP70 gene increased under cold stress,and TsHSP70 is highly likely to be involved in the protective process of T.spiralis muscle larvae under cold stress.The changes in the transcription level of TsHSP70 gene were detected after co-culture with T.spiralis muscle larvae using heat shock protein inhibitors and inducers.The concentration and time of the inhibitors and inducers were optimized,and the worms were treated with the inhibitors and inducers after optimized conditions.The effect of inhibitors and inducers on the expression of TsHSP70 in gene transcription level and protein translation level was analyzed.The results showed that muscle larvae cultured at 37℃ with 30 μg/m L quercetin solution for 6 h decreased by 32.99%and 55.38% compared with the control group.The muscle larvae cultured at 37℃ with 20μM 2-phenylethyne sulfo namide(PES)solution for 48 h decreased by 58.45% and 76.84% compared with the control group.The muscle larvae cultured in 8 m M glutamine solution for 24 h increased by 41.68% and 19.78% compared with the control group.The above experimental results showed that the inhibitor quercetin and PES could reduce the expression of TsHSP70,while the inducer glutamine could increase the expression of TsHSP70.The optimal action conditions of PES and glutamine were selected to study the subsequent antioxidant capacity of T.spiralis muscle larvae under cold stress.The T.spiralis muscle larvae were cultured in PBS solution and PES at 20 μM concentration for 48 h and in 8 m M glutamine solution for 24 h,respectively.After cold stress at-4℃ for 0,2,4,6,8 and 10 h,the larvae were collected and homogenized into tissues.The SOD activity,MDA content and GSH content were determined by the kit.To study the regulation of TsHSP70 on antioxidant capacity of T.spiralis muscle larvae.The results showed that the activity of SOD in T.spiralis muscle larvae was firstly increased and then decreased with the extension of cold stress time,and the activity of SOD in glutamine culture group was significantly increased compared with the control group,while the activity of SOD in PES culture group was firstly decreased and then increased with the increase of time.The MDA content of T.spiralis increased with the increase of cold stress time.The MDA content of T.spiralis muscle larvae treated with inhibitors and inducers changed slightly but not significantly.GSH content increased firstly and then decreased with the extension of cold stress time.GSH content in glutamine culture group was higher than that in control group,and GSH content in PES culture group was relatively stable.The experimental results showed that the weaker cold stress could increase the antioxidant capacity of T.spiralis muscle larvae.When the intensity of cold stress is beyond the scope for adjusting worm antioxidation ability,the muscle larvae will suffer some degree of oxidative damage.The TsHSP70 can protect the T.spiralis muscle larvae from oxidative damage by enhancing the antioxidant ability of T.spiralis.In summary,this study confirmed that cold stress significantly increased the transcription level and protein translation level of TsHSP70.TsHSP70 can protect the body under cold stress by enhancing the antioxidant capacity of T.spiralis muscle larvae,laying a foundation for the further study of the protective mechanism of TsHSP70 in the stress process of T.spiralis,and providing a new research idea for the prevention and treatment of Trichinosis.