Cloning,Expression And Functional Analysis Of Transgelin Gene From Angiostrongylus Cantonensis

Angiostrongylus cantonensis is a food-borne parasite that causes angiostrongylosis,a disease characterized by eosinophilia meningoencephalitis.Human get infection via eating food or water containing the third stage larvae(L3)of A.cantonensis.Humans are not natural definitive hosts of A.cantonensis,the larvae will not develop into adult worms but migrate and remain in the central nervous system(CNS),in a few cases,the larva will migrate to the eyes.Accompanying acute severe headache or meningoencephalitis,as well as neck stiffness and other symptoms,it even can lead to coma,fuzzy consciousness,and death.Onset of disease is quick and the incubation period is as short as 3 days,which indicated that the worm can trigger a strong inflammatory response.However,the current research on the inflammatory pathway caused by A.cantonensis infection is not understanding,and it is not clear about the interaction between parasite and host.Therefore,further study on the inflammatory pathway will help us to explore the pathogenic mechanism of the disease.Transgelin protein(TLP)exists widely in vertebrates and invertebrates,and it is an important factor to stabilize cytoskeleton and maintain cell life activities.It binds to F-actin to stabilize the cytoskeleton,and it also mediates the process of deconstruction to maintain the dynamic equilibrium of the cytoskeleton.Studies showed that TLP is involved in a variety of life activities and is highly expressed in organs and tissues with more life activities.It can also mediate downstream inflammatory pathways and promote the occurrence of inflammatory response.For example,TLP can bind to MAPK proteins and effectively promote the expression of the signal complex of MAPK pathway,which would activate the MAPK pathway and mediate the inflammatory response.In addition,in bacterial infection,it regular the expression of NF-κB pathway and stimulates the activation of macrophages to defend pathogen invasion.Based on the relationship between transgelin proteins and inflammatory pathways.we will conduct a preliminary study on the structure and function of Ac-TLP.We expect that study will further reveal the pathogenic mechanism of A.cantonensis and lay the foundation for prevention and treatment.ObjectiveThe TLP gene sequence was identified from the cDNA library of A.cantonensis.The full-length gene was cloned and prokaryotic expression was performed to obtain the recombinant protein.At the same time,the immunoreactivity of Ac-TLP gene was analyzed and the expression of Ac-TLP was detected in different stage of A.cantonensis.The effect of rAc-TLP in macrophage inflammation was studied.Therefore,the effect of Ac-TLP on angiostrongylosis and its mechanism are further explored and a theoretical basis is provided for pathogenic mechanism of angiostrongylosis.Methods1.Bioinformatics Analysis of Ac-TLP Gene Using bioinformatics software and structure analysis website,the structure analysis and function prediction of Ac-TLP gene were predicted.2.Ac-TLP Gene Cloning and Construction of Recombinant Plasmid The designed primers were used to amplify the Ac-TLP gene by PCR,and then it was linked with the prokaryotic expression vector p ET-32a(+).The double digestion and sequencing were carried out.3.Induction and Purification of rAc-TLP The recombinant pET-32a(+)-Ac-TLP plasmid was transformed into E.coli(BL21)for induction and expression.The recombinant protein was induced by IPTG,and the optimal induction conditions were determined to obtain the recombinant protein,and then the soluble analysis was conducted.The protein was purified by Ni-NTA affinity chromatography.After identified by SDS-PAGE,the protein concentration was measured by BCA method.4.The recombinant protein rAc-TLP immunoreactivity analysis Used the serum of A.cantonensis infected mice as the positive control,and the serum of normal mice as the negative control.The immunoreactivity of rAc-TLP was analyzed via western blotting.5.Real-time quantitative fluorescence PCR(qRT-PCR)Collecting the parasites in different development stages,and then RNA was extracted and cDNA was reverse transcript.Primers were designed to detect the relative expression levels of Ac-TLP in different stages for analysis.6.To detect the NF-κB pathway in macrophage Divided into five groups by different intervention methods: macrophage + Elution Buffer(Elution Buffer group),macrophage + Trx protein(Trx group),macrophage + rAc-TLP(rAc-TLP group).Western blotting was used to detect the relative expression level of p-p65,using p65 as internal reference,to detect the NF-κB pathway.7.The NF-κB pathway was detected in the brain tissues of mice.Four groups were set: uninfected group,7D,14 D and 21 D.Total protein was extracted from the brain tissues of mice in each group,and the expression level of p-p65 in the brain tissues was detected by western blotting,so as to detect the NF-κB pathway.Results1.The full length of Ac-TLP gene was 471 bp,and the ORF encoded 156 amino acids(aa).The isoelectric point of the encoded protein was predicted to be 7.6,and the molecular weight was 17.71 k Da,indicating that it was a hydrophilic protein.It does not contain transmembrane structure and signal peptide.A complete Calpnion-Homology(CH)domain was found in this sequence.It has multiple phosphorylation sites and may be regulated by multiple pathways.2.The gene was successfully cloned by PCR.The results of double digestion and sequencing showed that the recombinant plasmid p ET-32a(+)-Ac-TLP was successfully constructed.3.The recombinant protein was successfully induced.After SDS-PAGE analysis,the recombinant protein was soluble protein,and the concentration of the recombinant protein rAc-TLP was 200mg/ m L after purification by affinity chromatography.4.Western blotting showed that rAc-TLP could be recognized by the serum of the mice infected with A.cantonensis,but not by the serum of the negative mice.The Trx protein did not react with either serum.5.The qRT-PCR results showed that Ac-TLP was expressed in all inflection stages of parasite,and the highest expression level was detected in the female stage,suggesting that the gene might be related to the life activity of the parasite.6.Western blotting results showed that rAc-TLP can activate the NF-κB pathway in macrophages.The expression of p-p65 in rAc-TLP group was significantly higher than that the control group and the blank group,and there was no statistical difference between the blank group and the control group.7.Western blotting showed that the expression of p-p65 at 7,14 and 21 D was significantly higher than uninfected group.NF-κB pathway was activated during the infection of A.cantonensis.ConclusionThe prokaryotic expression plasmid p ET-32a(+)-Ac-TLP was successfully constructed,and the soluble recombinant protein was purified.Western blotting showed that the recombinant protein had immune reactivity.QRT-PCR analysis showed that Ac-TLP was expressed in different developmental stages of A.cantonensis,and the expression level was the highest in female stage which suggest that its high expression level might be related to the developmental stages of A.cantonensis.And in vitro,rAc-TLP could induce macrophages to activate the NF-κB pathway and promote inflammatory response in vitro.At the same time,western blotting result showed that NF-κB pathway was activated during the infection of A.cantonensis.