CAJ | 학술논문

目的：构建人类磷脂酰肌醇蛋白聚糖3（Glypican3, GPC3）重组原核表达载体。方法以人类肝脏cDNA文库为模板扩增出GPC3基因产物，与pMD-18 simple T 载体进行连接构建出T克隆载体；从T载体上获取基因片段，并对pET-32a（+）空载体进行双酶切，用T4连接酶连接，构建出pET-32a（+）-GPC3原核表达载体；对构建好的载体再次测序，并进行诱导表达。结果 PCR产物长度为837 bp，即扩增的GPC3 C末端长度，与NCBI网站的相比较，序列完全一致，说明pET-32a （+）载体中正确插入了目的片段，并且诱导表达出蛋白。结论成功构建了融合表达Pet-GPC3载体，蛋白诱导表达成功。
목적：구건인류린지선기순단백취당3（Glypican3, GPC3）중조원핵표체재체。방법이인류간장cDNA문고위모판확증출GPC3기인산물，여pMD-18 simple T 재체진행련접구건출T극륭재체；종T재체상획취기인편단，병대pET-32a（+）공재체진행쌍매절，용T4련접매련접，구건출pET-32a（+）-GPC3원핵표체재체；대구건호적재체재차측서，병진행유도표체。결과 PCR산물장도위837 bp，즉확증적GPC3 C말단장도，여NCBI망참적상비교，서렬완전일치，설명pET-32a （+）재체중정학삽입료목적편단，병차유도표체출단백。결론성공구건료융합표체Pet-GPC3재체，단백유도표체성공。
Objective To construct the prokaryotic expression vector of the human GPC3 gene for further study. Methods The human GPC3 gene from human liver cDNA library were obtained, and then inserted the fragment of GPC3 into pMD -18 simple T vector. Gene part from T vector and pET-32a (+) vector were digested with restrictive enzymes. Then the amplified product was linked to digested pET-32a (+) vector using T4 ligase. Finally, the reconstructed product was sequenced and was expressed. Results The 837 bp length of GPC3 C terminal was amplified and its sequence was the same as shown in the NCBI web, which demonstrated that the desired gene fragment had been inserted into the pET-32a (+) vector and induced protein expression. Conclusion The recombinant fusion expression vector Pet-GPC3 has been constructed and proteins are expressed successfully.