弓形虫表面抗原 P22编码基因片段的亚克隆与表达

SUBCLONING AND EXPRESSION OF THE GENE ENCODING OF THE P22 SURFACE ANTIGEN OF TOXOPLASMA GONDII

目的 亚克隆弓形虫 RH株表面抗原 P2 2编码基因 ,构建表达质粒 p BK/ P2 2 ,并对其在大肠杆菌 (E.coli)中的表达作初步研究。 方法 以限制性内切酶 Bam H 和 Kpn 双酶切质粒 p BCG5 .6 / P2 2 ,获得弓形虫表面抗原 P2 2编码基因目的片段 ,在以低熔点琼脂糖回收纯化后 ,插入表达质粒载体 p BK- CMV的多克隆位点 ,构建重组体 p BK/ P2 2 ,并转化大肠杆菌 DH5 α,快速酚法初筛阳性重组子 ,阳性克隆以 PCR法与限制性酶切分析鉴定后 ,以 IPTG进行诱导在 E.coli DH5 α中表达 ,表达产物以 SDS- PAGE与免疫印迹分析。 结果 双酶切质粒 p BCG5 .6 / P2 2 ,获得约 5 93bp的 P2 2编码基因片段 ,与预期片段大小相符 ;所构建 p BK/ P2 2重组体阳性克隆经双酶切和 PCR鉴定与预期结果一致 ;SDS-PAGE与免疫印迹显示 ,表达产物的大小约 2 8ku。 结论 成功亚克隆并构建了弓形虫表面抗原 P2 2编码基因 p BK/P2 2表达质粒 ,诱导表达了弓形虫 P2 2表面抗原蛋白 。

Objective To Subclone and express the gene encoding of P22 surface antigen of Toxoplasma gondii RH strain. Methods The plasmid pBCG5.6/P22, which containing the P22 encoding gene of T. gondii , was digested by the restriction enzyme of BamHⅠ and KpnⅠ for obtaining the P22 gene fragment, about 593 bp in length. After purification, the P22 gene fragment was inserted into the polylinker of the plasmid pBK CMV, and the recombinant plasmid pBK p22 was identified by PCR and restriction digestion methods. The recombinant plasmid pBK P22 was transferred into E.coli DH5α, and induced by IPTG to express P22 fusion protein. The expressed product was analyzed by SDS PAGE and Western blot. Results The recombinant plasmid pBK/P22 was constructed, and expressed in the E.coli DH5α. The results of the SDS PAGE and Western blot indicated that the expressed P22 fusion protein was about 28 ku. Conclusion The gene encoding of the T. gondii P22 surface protein was successfully sub cloned into the plasmid pBK CMV and expressed in the E.coli DH5α.