The Cloning And Polymorphism Analysis Of Ascaris Suum GST Gene And Its Expression In Different Development Stages

Ascaris suum(Goeze 1782)is one of the largest intestinal parasites in pigs,which can infect pigs and bring enormous economic losses in pig industry by causing serious visceral larva migrans(VLM)syndrome,growth disorder,even death.Swine ascariasis is prevalent in pigs worldwide.Pigs from intensively raised or free-range raised are both susceptibility to infection of this parasite and causing ascariasis.The occurrence of A.suum in pigs from China is in 10% to 80%.In addition,many studies has indicated that as a kind of important parasitic zoonosis,A.suum can not only lead to great production losses to livestock and poultry breeding,but also cause a serious risk to human health by infecting humans and other animals including chicken,cattle,sheep and apes.Therefore,control of A.suum has a zootechny and public health security significance.There is a necessity to identify more effective vaccine and drug candidate gene,and Gultathione S transferase(GST)gene is a possible target due to its detoxication in nematode.For years,a large number of researches on GST gene have been conducted,nevertheless,few of them is associated with A.suum.Hence,we cloned coding sequence(CDS)and predicted the structure and antigenic epitope of A.suum GST gene in this study to evaluate the possibility of GST as a vaccine candidate gene.Besides,in order to analyze GST gene single nucleotide polymorphism(SNP)of A.suum in northwestern China,allele and genotype frequency of SNPs in this gene was identified and calculated.Additionally,expression of GST gene in different development stage,drug-treated ova and drug-treated adults was detected,respectively,to illuminate the role of this gene during Ascaris development and drug metabolism.The results obtained are as follows:1.The result showed that GST protein-coding gene of A.suum from Shaanxi fufeng mutated in 243 and 248 nucleotides,which changed the 83 th amino acid comparing to the reference gene.Moreover,predicted results indicated that many secondary structure and antigenic epitopes exist in this region and the dominant epitopes were located in section 43~46 and 201~204 or the adjacent area.2.Three SNPs were discovered in GST gene of A.suum from northwestern China,called SNP2,SNP3,and SNP4.Predominant allele and genotype of these SNPs was A2,C3,A4 and A2A2,C3C3,A4G4,respectively.In addition,eight haplotypes were existed.Simultaneously,each SNP in A.suum from different regions is intermediate polymorphism(0.25 < PIC < 0.50),and three SNPs in Shaanxi and SNP4 in Gansu were inconsistent with Hardy-Weinberg equilibrium(P < 0.05).3.Microscope observation showed that Ascaris eggs developed in a way binary fission in vitro.Besides,expression of GST gene in different development stages of A.suum was stable and roughly equal,remarkably,changes were observed in drug-treated group,particularly in adults(P < 0.001).In conclusion,rich antigenic epitopes in GST gene suggested its possibility to be a potential vaccine candidate gene.Inconsistent with Hardy-Weinberg equilibrium illustrated that selection pressure resulted in the occurrence of SNPs.Expression of GST gene significantly increased in drug-treated adults indicated that the important role of this gene during drug metabolism and the onset of resistance.