弓形虫表面抗原P22编码基因片段的克隆及序列测定

摘要

Aim: Cloning and sequence determination of P22 gene fragment of Taxoplasma gondii Methods The P22 gene fragment was amplified by PCR.After purification, the gene fragment was ligated with plasmid p5.6 at polylinker. The recombinant plasmid p5.6/P22 was transformed into E. coli DH 5a. Positive clones were screened and indentified by PCR techniche and digestion with restriction enzyme. The sequence of inserted P22 gene fragment was also determined. Results and Conclusion The recombinant plasmid of P22 gene fragment of Toxoplasma gondii was successfully constructed. That would make it convenient for P22 diagnostic antigen expression.%目的　克隆弓形虫表面抗原P22编码基因片段并进行序列测定。方法　设计合成1对引物，采用PCR法扩增出P22目的　基因片段，以低熔点琼脂糖回收纯化，并以限制性内切酶BamH Ⅰ和Kpn Ⅰ双酶切后，插入质粒载体p5.6的多克隆位点，构建重组体p5./P22，并转化大肠杆菌DH5a，快速酚法初筛阳性重组子，并以PCR法与限制性酶切分析对阳性克隆进一步鉴定。被鉴定的重组子以双脱氧链终止法进行序列测定。结果　从弓形虫核酸提取物中扩增出约593bpDNA条带，与预期扩增片段大小相符，空白对照无特异性扩增条带；所构建p5./P22重组体阳性克隆经双酶切与PCR鉴定与预期结果　一致；序列测定的结果　确证了插入片段的正确性。结论　体外成功扩增、克隆了弓形虫表面抗原P22编码基因片段，并经序列分析所验证，为弓形虫P22表面抗原的表达以及弓形虫疫苗的制备作好铺垫。