CAJ | 학술논문

目的构建肝癌相关新基因BC047440原核表达载体,表达并纯化出其重组蛋白.方法从HepG2细胞中提取总RNA,经逆转录-聚合酶链式反应(RT-PCR)扩增BC047440cDNA,克隆进质粒pMD19-T,转化E.coliDH5α,测序正确后,酶切目的基因片段,插入质粒pET-28a(+),转化E.coliBL21,IPTG诱导蛋白表达,Ni-NTA柱亲和层析纯化重组蛋白.结果电泳证实RT-PCR扩增产物与预期目的基因BC047440长度一致.RT-PCR纯化产物与载体pMD19-T连接后,经蓝白斑筛选,挑出5个白色菌落做酶切鉴定,其中2个菌落被证实为阳性克隆.测定其基因序列,结果显示与Genbank公布的BC047440基因序列完全一致.将BC047440cDNA插入质粒pET-28a(+),转化E.coliBL21,培养过夜后,挑选7个白色菌落做酶切鉴定,证实均为阳性克隆.IPTG诱导其中一个克隆表达蛋白,SDS-PAGE电泳分析表明,在相对分子质量23000左右出现新的蛋白表达条带,诱导4h后表达蛋白量最多,占菌体总蛋白的22.3%.将诱导3h的菌体超声破碎后,Ni柱亲和层析,50和125mmol/L咪唑洗脱液SDS-PAGE电泳显示出清晰的单一条带.结论成功构建BC047440基因原核表达载体,表达并纯化出重组蛋白,为深入研究其临床应用打下基础.
목적 구건간암상관신기인BC047440원핵표체재체,표체병순화출기중조단백.방법 종HepG2세포중제취총RNA,경역전록-취합매련식반응(RT-PCR)확증BC047440cDNA,극륭진질립pMD19-T,전화E.coliDH5α,측서정학후,매절목적기인편단,삽입질립pET-28a(+),전화E.coliBL21,IPTG유도단백표체,Ni-NTA주친화층석순화중조단백.결과 전영증실RT-PCR확증산물여예기목적기인BC047440장도일치.RT-PCR순화산물여재체pMD19-T련접후,경람백반사선,도출5개백색균락주매절감정,기중2개균락피증실위양성극륭.측정기기인서렬,결과현시여Genbank공포적BC047440기인서렬완전일치.장BC047440cDNA삽입질립pET-28a(+),전화E.coliBL21,배양과야후,도선7개백색균락주매절감정,증실균위양성극륭.IPTG유도기중일개극륭표체단백,SDS-PAGE전영분석표명,재상대분자질량23000좌우출현신적단백표체조대,유도4h후표체단백량최다,점균체총단백적22.3%.장유도3h적균체초성파쇄후,Ni주친화층석,50화125mmol/L미서세탈액SDS-PAGE전영현시출청석적단일조대.결론 성공구건BC047440기인원핵표체재체,표체병순화출중조단백,위심입연구기림상응용타하기출.
Objective To construct prokaryotic expression vector of BC047440 gene to express and purify its recombinant protein. Methods The total RNA was extracted from HepG2 cells. The BC047440 gene fragment was amplified from the total RNA by RT-PCR. The resulting product was cloned into pMD19-T vector and sequenced. Then the confirmed BC047440 cDNA was cloned into plasmid pET-28a( + ) and transformed into E. coli BL21 where it was induced to express proteins by i-sopropyl-1-thio-b-Dgalactopyranoside (IPTG). The expression of protein was analyzed through SDS-PAGE cells induced for 3 h by IPTG harvesting. Then it was sonicated briefly and the proteins were purified through affinity chromatography. Results The target sequences were specifically amplified through RT-PCR, cloned into pMD19-T vector, and then transformed into E. coli DH5a. Five white colonies were selected and cut by BamHI and EcoRl separately. The plasmids of two white colonies showed positive results and was sequenced, demonstrating to be the same as that of BC047440 gene in GenBank. Then BC047440 cDNA was cut down from pMD19-T, ligated into the vector pET-28a( + ) and then transformed into E. coli BL21. After an overnight incubation, seven white colonies were picked and showed positive results after being digested by both BamHI and EcoRI. After IPTG induction of one positive colony, a new protein band about Mr 23000 showed on SDS-PAGE. The percentage of expressed product over total bacterial proteins after 4 hours of induction was 22. 3%. After affinity chromatography, SDS-PAGE showed only one clear band existed in 50 mmol/L or 125 mmol/L imidizone elution. Conclusion The prokaryotic expression vector of BC047440 gene has been successfully constructed. Meanwhile, purified recombinant proteins are obtained through affinity chromatography, laying foundation for further study of its clinical use.