日本血吸虫新基因——蛋白酶体激活因子PA28亚单位的发现与克隆

Cloning of the proteasome activator PA28 subunit gene of Schistosoma japonicum

目的将用EST策略及同源性搜索发现的日本血吸虫新基因——蛋白酶体激活因子PA28亚单位（proteasome activatorPA28 subunit,PA28）cDNA克隆到表达质粒pET32a（+）上,为下一步对该基因进行功能研究做准备。方法将插入于pTrip1Ex2质粒上的cDNA进行测序,测序结果经BLASTx程序搜索,发现该插入cDNA序列所编码的蛋白与小鼠肌肉内的PA28亚基蛋白高度同源。根据表达质粒pET32a（+）上的克隆位点及该cDNA序列设计PCR引物,将PCR产物纯化后连接到pMD 18-T载体上,将重组T载体经EcoRI/XhoI双酶切后切下的SjPA28基因导入原核可溶性表达质粒pET32a（+）中。结果所发现的cDNA所编码的蛋白与小鼠肌肉内的PA28亚基蛋白的同一性达41％,PCR产物的片段长度与预期大小一致,重组T载体及表达质粒经EcoRI/XhoI双酶切后证明具有与目标片段长度相符的插入片段。结论本研究所发现的cDNA所编码的蛋白与小鼠肌肉内的PA28亚基蛋白高度同源,并且已成功地构建出重组表达质粒pET32a（+）-SjPA28。

Object To subclone the novel gene of Schistosoma japonicum, proteasome activator PA28 subunit (PA28) cDNA, into the expression plasmid pET32a(+), thereby preparing for further research of the function of this novel gene. Method The insert cDNA fragment was sequenced and searched with BLASTx program. It was found that the protein coded by this cDNA was highly homologous tomouse muscle PA28 subunit protein. Two PCR primers were designed according to this PA28 cDNA sequence and the cloning site in pET32a (+) plasmid. The PCR product was purified and linked with the pMD 18-T vector, and the resultant recombinant T-vector was digested with EcoR I / Xho I resulting in SjPA28 gene which was then introduced into the expression plasmid pET32a(+). Result The protein coded by this novel gene is highly homologous (41%) to mouse muscle PA28 protein, and the PCR product conformed to the expected length. The recombinant T-vector and expression plasmid were demonstrated to have the insert with the same length as that of the target fragment when digested with EcoR I/Xho I. Conclusion The novel gene codes for Schistosoma japonicum PA28 subunit protein, and the recombinant expression plasmid pET32a(+)-Sj PA28 have been successfully constructed.