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目的筛选与甲型副伤寒杆菌(副甲)CMCC50973噬菌体LSPA1早期蛋白GP23相互作用的宿主蛋白。方法 Sau3AⅠ酶切副甲基因组,回收250~1 500 bp的条带与p AT质粒连接,构建p AT-副甲基因文库;扩增噬菌体早期基因gp23,与p BT构建p BT-gp23作为诱饵质粒;将p AT-副甲基因文库和p BT-gp23共转KS宿主菌,利用细菌双杂交技术筛选蓝色单菌。检测筛选出阳性单菌的Lac Z活性,并提质粒测序,分析其编码蛋白。结果成功构建副甲基因文库,其库容量满足实验的要求,基因文库阳性率接近100%;筛选出的插入基因为编码嘌呤透性酶。结论宿主蛋白(嘌呤透性酶)可能与副甲噬菌体LSPA1早期蛋白GP23有相互作用。
Objective To screen the host proteins that interact with GP23, an early stage protein of LSPA1 of Paragonimus paratyphi A (CMCC50973). Methods The para-methionine was digested with Sau3AⅠ and ligated with plasmid p AT-A from 250-1 500 bp to construct the p AT-paramyxovirus library. The early phage gp23 was amplified and p BT-gp23 was constructed with p BT as the bait plasmid The p AT-para-methionine library and p BT-gp23 were co-transformed into KS host bacteria, and the blue single bacteria were screened by bacterial two-hybrid technique. The positive single bacterium was tested for LacZ activity, and the plasmid was sequenced to analyze the encoded protein. Results The parathyroid library was successfully constructed. The library capacity of the library met the requirement of the experiment. The positive rate of the gene library was close to 100%. The inserted gene was identified as purine permease. Conclusion The host protein (purine permease) may interact with GP2, an early protein of paraphathiophage LSPA1.