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There are currently only one FDA-approved drug and no vaccines for cryptosporidiosis, largely due to the limited knowledge of the molecular mechanisms involved in the invasion of the pathogens. Previous studies have shown that GP60, which is cleaved into GP40 and GP15 after expression, is an immunodominant mucin protein involved in the invasion of <jats:italic>Cryptosporidium<\/jats:italic>. The protein is highly O-glycosylated, and recombinant proteins expressed in prokaryotic systems are non-functional. Therefore, few studies have investigated the function of GP40 and GP15.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods<\/jats:title>\n                <jats:p>To obtain recombinant GP40 with correct post-translational modifications, we used CRISPR\/Cas9 technology to insert <jats:italic>GP40<\/jats:italic> and <jats:italic>GP15<\/jats:italic> into the <jats:italic>UPRT<\/jats:italic> locus of <jats:italic>Toxoplasma gondii<\/jats:italic>, allowing heterologous expression of <jats:italic>Cryptosporidium<\/jats:italic> proteins. In addition, the Twin-Strep tag was inserted after <jats:italic>GP40<\/jats:italic> for efficient purification of GP40.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>Western blotting and immunofluorescent microscopic analyses both indicated that GP40 and GP15 were stably expressed in <jats:italic>T. gondii<\/jats:italic> mutants. GP40 localized not only in the cytoplasm of tachyzoites but also in the parasitophorous vacuoles, while GP15 without the GPI anchor was expressed only in the cytoplasm. In addition, a large amount of rec<jats:italic>Tg<\/jats:italic>GP40 was purified using Strep-TactinXT supported by a visible band of\u2009~\u200950\u00a0kDa in SDS-PAGE.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>The establishment of a robust and efficient heterologous expression system of GP40 in <jats:italic>T. gondii<\/jats:italic> represents a novel approach and concept for investigating <jats:italic>Cryptosporidium<\/jats:italic> mucins, overcoming the limitations of previous studies that relied on unstable transient transfection, which involved complex steps and high costs.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Graphical Abstract<\/jats:title>\n                \n              <\/jats:sec>","DOI":"10.1186\/s13071-024-06159-y","type":"journal-article","created":{"date-parts":[[2024,2,15]],"date-time":"2024-02-15T16:02:39Z","timestamp":1708012959000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Stable expression of mucin glycoproteins GP40 and GP15 of Cryptosporidium parvum in Toxoplasma gondii"],"prefix":"10.1186","volume":"17","author":[{"given":"Muxiao","family":"Li","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaohua","family":"Sun","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Haoyu","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Na","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yaoyu","family":"Feng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lihua","family":"Xiao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yaqiong","family":"Guo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2024,2,15]]},"reference":[{"key":"6159_CR1","doi-asserted-by":"publisher","first-page":"85","DOI":"10.1016\/S1473-3099(14)70772-8","volume":"15","author":"W Checkley","year":"2015","unstructured":"Checkley W, White AC Jr, Jaganath D, Arrowood MJ, Chalmers RM, Chen XM, et al. 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All experiments were conducted under the approval of the Laboratory Animals Research Center of Guangdong Province and South China Agricultural University (approval number: 2021C005).","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare that they have no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"65"}}