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目的 选择弓形虫表面抗原P2 2、P30的有效基因片段 ,共同构建在同一克隆载体pUC19和表达载体 pGE MEXTM- 1上 ,并保证其连接方向及开放读码框的正确。方法 根据已发表P30基因序列 ,用PCR技术调取所需基因片段 ,P2 2基因片段来自重组质粒pGEX - 2T -v2 2 ,将两片段定向克隆于克隆载体 pUC19及表达质粒pGEMEXTM- 1上 ,用酶切及测序的方法对重组子进行鉴定。结果 酶切产物经电泳显示条带清晰 ,P2 2、P30基因片段的泳动位置分别在 4 4 6bp和 86 0bp的位置 ,与预计结果一致 ;测序结果表明插入的复合基因片段方向及序列均正确。结论 成功构建的含有P2 2、P30基因片段的质粒重组体 ,保证了两基因连接方向、序列及开放读码框的正确 ,为今后两基因的进一步复合表达研究奠定了基础。
Objective To select the effective gene fragments of Toxoplasma gondii surface antigen P2 2 and P30 to construct the same cloning vector pUC19 and expression vector pGE MEXTM- 1 and ensure the correct orientation and open reading frame. Methods According to the published sequence of P30 gene, the desired gene fragment was retrieved by PCR. The P2 2 gene fragment was derived from the recombinant plasmid pGEX - 2T - v2 2. The two fragments were cloned into the cloning vector pUC19 and the expression plasmid pGEMEXTM - 1. Recombinant was identified by digestion and sequencing. Results The bands of the digested products were clear by electrophoresis. The motifs of P2 2 and P30 gene fragments were located at 4 4 6bp and 86 0 bp, respectively, in agreement with the expected results. The sequencing results showed that the direction and sequence of inserted gene fragments were correct . Conclusion The recombinant plasmids containing P2 2 and P30 gene fragments were successfully constructed to ensure the correct orientation and sequence of the two genes and open reading frame, which laid the foundation for the further study of complex expression of the two genes in the future.