| Toxoplasma gondii is an obligate intracellular parasite with nucleated cells that is host-independent and can infect humans and almost all warm-blooded animals.At present,toxoplasmosis is widespread in the world.Because there is no special vaccine and therapeutic drugs,human and animal husbandry have suffered enormously,and it has become an important public health problem in the world.In recent years,great progress has been made in the study of Toxoplasma vaccine.However,due to the complex life cycle and pathogenic mechanism of Toxoplasma gondii,an ideal vaccine has not yet been developed.Therefore,the development of new potential vaccine candidate antigen molecules,laying the foundation for the development of effective Toxoplasma vaccine,has become the focus of the development of human-toothed Toxoplasma vaccine.This study is based on the proteomic analysis of the combination of Toxoplasma gondii and host cell surface receptor sialic acid.Through bioinformatics prediction and analysis,TGME49?269450 protein with up-regulated sialic acid and completely unknown function was selected for cloning,expression and purification.As well as subcellular localization,a preliminary study of its function was carried out.The aim is to explore the molecular mechanism of Toxoplasma gondii invasion of host cells through the sialic acid pathway,and to lay the foundation and provide new ideas for the identification of new Toxoplasma vaccine candidate antigens.In this study,the full-length sequence of 1053 bp of TGME49?269450 gene was downloaded using the Toxo DB database dedicated to Toxoplasma gondii.Through bioinformatics software analysis,it is predicted that the protein has a signal peptide,no transmembrane region,and good hydrophilicity and antigenicity.The total RNA of the tachyzoites of Toxoplasma gondii RH strain was extracted,and the target fragment was amplified by reverse transcription PCR.The sequence alignment was consistent with the TGME49?269450 sequence.Subsequently,recombinant prokaryotic expression vectors(TGME49?269450-p ET28 a and TGME49?269450-p GEX-4T-1)were constructed.Recombinant proteins with His and GST-tagged TGME49?269450 were purified in large quantities.Rats and rabbits were immunized with His-tagged recombinant protein to prepare polyclonal antibody serum,and the protein was confirmed to exist in Toxoplasma gondii whole protein by Western-blot.Through the subcellular localization experiment of protein,it is presumed that it is distributed in the front of the worm.The prepared rabbit-derived Ig G was subjected to an antibody-inhibiting invasion test in vitro,and it was found that the invasion rate of the insect was reduced,indicating that the antibody of the protein has a certain inhibitory effect on the invading cells of the insect.At the same time,the GST tag was used to recombine the protein,which proved that the target protein could bind to sialic acid and adhere to the cells.It is speculated that the protein may bind to the surface of the host cell to mediate worm invasion.This study showed that TGME49?269450 protein can be purified in large quantities,and it is presumed that it is distributed in the front end of the worm and can adhere to cells.Under in vitro conditions,the invasion rate of the cells can be reduced,which may be involved in the process of Toxoplasma gondii invading the host cells by the liquid acid pathway. |
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