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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。
Objective To clone the fragment of Toxoplasma gondii surface antigen P22 and to determine its sequence. Methods One pair of primers was designed and synthesized. The target gene fragment of P22 was amplified by PCR. The fragment was purified by low melting point agarose and double digested with restriction endonucleases BamHI and KpnI. The recombinant plasmid p5.6 The recombinants p5.6 / P22 were constructed and transformed into E. coli DH5α. The positive recombinant plasmids were screened by rapid acid method. The positive clones were further identified by PCR and restriction analysis. The identified recombinants were sequenced by the dideoxy chain termination method. Results The DNA fragment of about 593bp was amplified from the Toxoplasma gondii nucleic acid extract, which was consistent with the expected size of the amplified fragment. The positive control plasmid of p5.6 / P22 was double-digested PCR identification was consistent with the expected results; the results of the sequencing confirmed the correctness of the insert. Conclusion The fragment of P22 gene encoding Toxoplasma gondii surface antigen was successfully cloned in vitro and verified by sequence analysis. The expression of P22 antigen on Toxoplasma gondii and the preparation of Toxoplasma gondii vaccine were well prepared.