摘要
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BamHI和KpnI双酶切后，插入质粒载体p5．6的多克隆位点，构建重组体p5．6／P22，并转化大肠杆菌DH5α，快速酸法初筛阳性重组子，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p5．6／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。
Cloning and sequence determination of P22 gene fragment of Toxoplasma gondii 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 Therecombinant plasmid of P22 gene fragment of Toxoplasma gomdii was successfully constructed.That would make it convenient for P22 diagnostic antigen expression.
出处
《中国人兽共患病杂志》
CSCD
北大核心
2000年第2期62-64,共3页
Chinese Journal of Zoonoses
关键词
P22表面抗原
克隆
序列分析
弓形体病
Toxoplasma gomdii
P22 surface antigen
DNA clone
Sequence determination
