弓形虫棒状体蛋白2和膜表面蛋白1融合基因的克隆与表达

Cloning and Expression of the Fused Gene of Rhoptry Protein ROP2 and Major Surface Protein P30 from Toxoplasma gondii

目的进行弓形虫棒状体蛋白2(ROP2)和膜表面蛋白1(P30)融合基因的克隆与表达,为弓形虫ROP2-P30基因工程复合抗原的制备做准备。方法半套式PCR扩增编码弓形虫P30的基因片段,克隆至已构建成功的重组质粒pUC119/ROP2中,经PCR和酶切鉴定正确的重组质粒pUC119/ROP2-P30再以SacⅠ/HindⅢ双酶切克隆至表达载体pET28b上,鉴定正确的重组质粒pET28b/ROP2-P30转化大肠埃希菌表达菌株BL21-Codon Plus(DE3)-RIL,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。结果从弓形虫RH株基因组DNA中扩增出700 bp P30基因片段,成功构建重组质粒pET28b/ROP2-P30,该质粒经PCR和酶切鉴定,与预期结果一致,并在大肠埃希菌中高效表达,产生相对分子质量(Mr)约为69 000的重组目的蛋白。结论弓形虫ROP2和P301融合基因克隆成功,并表达出预期的复合重组蛋白ROP2-P30。

Objective To clone and express the fused gene fragment coding rhoptry protein ROP2 and major surface protein P30 from Toxoplasma gondii as a preparation for the construction of the complex ROP2-P30 antigen by gene engineering. Methods The gene fragment encoding P30 was amplified by PCR from T.gondii RH strain and subeloned into the recombinant plasmid pUC119/ROP2 already constructed. The recombinant plasmid pUC119/ROP2-P30 was digested by Sac Ⅰ/HindⅢ and inserted into the same site of expression vector pET28b. The recombinant plasmid of pET28b/ROP2-P30 was transformed to E.coli and expressed under the induction of IPTG. Results The gene fragment 700 bp encoding P30 was obtained from the total DNA of T.gondii by PCR. The recombinant plasmid pET28b/ROP2-P30 was successfully constructed, which was highly expressed in E.coli, a fusion protein with molecular weight of 69 000. Conclusion The fusion geue encoding the rhoptry protein ROP2 and the major surface protein P30 of T. gondii has been successfully cloned and expressed to be an expected recombinant fusion protein ROP2-P30 with molecular weight 69 000.