Transgenic Expression Characterization Of ES15/ES24 From Haemonchus Contortus Using Caenorhabditis Elegans

Haemonchosis is a major parasitic disease of ruminants such as cattle and sheep, whose pathogen is Haemonchus contortus, which feed on the mucous membrane and blood of the host. Its infection may be manifested as anemia, weight lost, stunted growth or even death in some case, which cause significant losses to livestock industry. The control of haemonchosis is depandent on the use of broad-spectrum deworming drugs currently. However, alternative methods of control are required, such as vaccine, with the emergence of anthemintic resistant strains of H. contortus and drug residues in meat products. H. contortus in host body during parasitic process or in vitro secreted a variety of molecules named excretory product (ESP). As ES antigen directly exposed to the host immune system, it is most likely to stimulate host protective immunity as a target antigen. ES antigens have become a hot spot in parasite research. Therefore, the studies carried were focused on proteins released during parasitic pHase of infection-ES15/24. Caenorhabditis elegans was choosen as an alternative expression system to try to express ES antigens. My research aimed to take C. elegans as a heterologous system, analyze the transcriptional activity of ES15/24 and make sure the feasibility of expression of ES15/24 using C. elegans, based on established transgenic platform, which would provide technical supports for the vaccination against parasitic nematodes.The core promoter region of sre-49 gene and acdh-7 gene chosen as candidate genes of target promoter for the expression of ES antigen in C. elegans were amplified from the genomic DNA of C. elegans using PCR technology. The 5'flanking region of sre-49, acdh-7 and cpr-1 were cloned into the expression vector pPD-95.77. After microinjected into C. elegans' gonad, worms were picked up to detect the expression of EGFP reporter gene. The results showed that fluorescence directed by 5' flanking region of sre-49 gene was poor. The fluorescence directed by 5'flanking region of acdh-7 gene was detected in the pHarynx, anterior intestine and posterior intestine of transgenic-worms, but the expressions were segmental and there were some differences in the expression's patterns and intensities of GFP between different individuals. The transcriptional function of the 5'flanking region of cpr-1 gene was not only strong but also stable compared to the former two genes, GFP could expressed intensively in the whole intestine of worms in all stages expect embryos. So 5'flanking region of cpr-1 gene was determined to direct the expressions of ES15/24 eventually, referred to the location of GFP, the expression intensity in L4 and adult C. elegans, and also stability between different individuals.RT-PCR was carried out to amplify the cDNA of ES15/24 from total RNA of H. contortus, referred to the sequence of ES15/24 from H. contortus reported.400bp and 600bp product fragments were amplified and inserted into plasmid cprl-pPD-95.77, aimed to construct the recombinant plasmid cpr-ES15/24-pPD-95.77 which were injected into C. elegans'gonad respectively together with a marker plasmid--PRF4 by micro injection. Through ultraviolet irradiation, stably inherited transgenic C. elegans strains had been obtained. The transcripts of ES15/24 could be found in transgenic C. elegans by single-worm PCR and RT-PCR analysis. With fluorescence microscopy, green fluorescence could be found in the guts of ES15 transgenic C. elegans, while green fluorescence could not be found in the bodies of ES24 transgenic worms. Maybe ES24 gene was toxic to C. elegans, which still requires further research.In order to obtain recombinant protein of ES 15 expressed by C. elegans, some changes were made to the recombinant vector-cpr-1 5'flanking region::GFP with the method of adding His tag or ending GFP gene in the downstream of ES15. Experimental results showed that the intensity and pattern of expression of ES15 were consistent with the transformation of the former results. As for the conditions for purification of recombinant protein from transgenic worms, those still need us to explore and optimize.Overall, this experiment is the first study about transgenic expression of ES15/24 in C. elegans using microinjection techniques. We successfully achieved the expression of ES15 antigen in C. elegans taking the advantage of established transgenic platform, which laid a theoretical foundation for the expression and purification of antigens of H. contortus using C. elegans in the future.