| Trematodes of the genus Fasciola infect not only ruminants such as cattle and sheep,but also humans,and causes zoonotic fasciolosis,threatening the live and health of humans and anmals.Therefore,it is necessary to establish,efficient and accurate early diagnosis methods to control the disease.Fasciola trematodes mainly include Fasciola hepatica,Fasciola gigantica and the“intermediate type”.The current related research focuses relatively less on F.gigantica and the“intermediate type”.The present study worked on F.gigantica,while the objectives of screening,identification and analysis of the F.gigantica excretory-secretory(ES)antigens(FgES)interacting with the host.The expected results should lay foundation for the establishment of techniques for the early diagnosis of F.gigantica infection.The research is divided into three parts.In the first part,the identification of F.gigantica was performed.Firstly,144 flukes were collected from buffaloes in Nanning,Guangxi Zhuang Autonomous Region,and then were identified by morphological and molecular approaches.All the 144 flukes were identified as Fasciola trematodes through morphological identification.In addition,the secquences of the second internal transcribed spacer(ITS-2)or ribosomal DNA of144 isolates were amplified and sequenced,and the results showed that the 144 ITS-2 sequences were 99.7 % similarity with the corresponding ITS-2 sequence of F.gigantica(GenBank accession AJ557569),while different from those of F.hepatica and the “ intermediate type”.These results demonstrated that all the 144 flukes represented F.gigantica.In the second part of the study,FgES was prepared and the interaction proteins in FgES antigen were screened.The eggs for F.gigantica were collected by in vitro culture,and then the miracidium were collected and used to infect freshwater snails.After proliferation and development in snails,ceecariae were released and developed into metacercariae.The metacercariae were collected about 35 d after infecting snails,and buffaloes were infected(about500 metacercariae per buffalo).The positive serum was separately prepared after 14 d,42 d,70 d,98 d after infection.Next,the ES antigen of F.gigantica was collected by culturing in vitro,and then was used to immunize rabbits to prepare the positive serum and the antibody titer was detected by indirect ELISA.The results showed that the positive serum titer against FgES antigen reached 1: 1 000,indicating that the immunogenicity of FgES antigen is quite good,andit can be used in the subsequent experiments.The ES antigens of F.gigantica reacted with buffalo sera collected at different time by Co-immunoprecipitation and LC-MS/MS,respectively.A total of 465 non-redundant proteins were identified,with 57 being known proteins,which were distributed: 13(22.8 %),5(8.8 %)and 7(12.3 %)at 42 d,70 d and 98 d by analysis using BlastP and UniProt;8(14 %),5(8.8 %)and 1(1.8 %)for 42 d and 70 d,70 d and 98 d,42 d and 98 d,respectively,and 18(31.6 %)proteins were found at 42 d,70 d and 98 d.Furthermore,using bioinformatics method,α-tubulin(AT)and Leucine aminopeptidase(LAP)of F.gigantica were screened and predicted,which they might have diagnostic value.In the third part of the study,the genes encoding α-tubulin(AT)and Leucine aminopeptidase(LAP)of F.gigantica were cloned and expressed in prokaryotic cells,and the feasibility of these recombinant proteins as early detection antigens was explored.The results showed that the fragment length of α-tubulin(AT)gene was 1 356 bp,which encodes 452 amino acids and the theoretical molecular weight of α-tubulin was 48.99 kDa.The fragment length of Leucine aminopeptidase(LAP)gene was 1 572 bp,which encodes 523 amino acids with the theoretical molecular weight of 56.38 kDa.The secondary structures of these two proteins were all predicted by random curl,and the epitopes were distributed throughout the sequences.The main functional area of the tertiary structure was at the N-terminal.The α-tubulin gene was inserted into vector pGEX-6P-1(+)and the Leucine aminopeptidase gene was inserted into vector pET-30 a for prokaryotic expression.It was found that the recombinant protein pGEX-6P-1(+)-FgAT was expressed by IPTG induction at a molecular weight of about 76 kDa(containing 26 kDa GST tag carrying the vector)and expressed as fusion protein.After purification by Glutathione-Sepharose beads,a highly purified recombinant protein pGEX-6P-1(+)-FgAT was obtained.The recombinant protein pGE-30a-FgLAP was expressed by IPTG induction,and the recombinant protein was about 56 kDa,expressed as inclusion body.The highly purified recombinant protein was obtained by using Ni-NTA His bind Resin.The Western Blot results showed that the two recombinant proteins reacted with positive serum of rabbits and buffaloes(42 d、70 d、98 d),indicating their good immunogenicity.In conclusion,the present study identified the protein components of FgES interacting with the host successfully,including 465 non-redundant proteins.Through bioinformatics analysis,57 known proteins were further identified,and the representative α-tubulin(AT)and Leucineaminopeptidase(LAP)were cloned,expressed and their immunoreactivity were assessed.These two proteins had great potential for the diagnosis of F.gigantica infection. |
