CAJ | 학술논문

目的克隆表达牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)牙龈素中黏附素区域的Hgp44基因,并纯化目的蛋白.方法通过聚合酶链反应(PCR)和基因重组技术,克隆得到PgATCC33277的Hgp4基因,插入到克隆载体pMD18-T中并测序鉴定.经过酶切后将目的基因片段与表达载体pET22b相连,构建出表达质粒pET22b-Hgp44.将重组质粒转化到感受态细胞BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG)诱导表达融合蛋白,通过SDS聚丙烯酰胺凝胶电泳及蛋白质印迹法进行分析.用固定化金属亲和层析法对重组蛋白进行分离纯化.结果目的基因片段约为1100 bp,与预期大小相符,测序结果与GenBank中ATCC33277国际标准菌株的RgpA的等位基因序列U15282一致.IPTG诱导后的菌体经SDS聚丙烯酰胺凝胶电泳后形成一个以包涵体形式存在的44 000的融合蛋白.蛋白质印迹法检测证实其具有免疫原性.用镍离子金属螯合层析柱纯化出了目的蛋白,最后得到约3.5 mg/L的目的蛋白.结论成功克隆表达了PgHgp44基因,并纯化出了目的蛋白.
목적 극륭표체아간계람단포균(Porphyromonas gingivalis,Pg)아간소중점부소구역적Hgp44기인,병순화목적단백.방법 통과취합매련반응(PCR)화기인중조기술,극륭득도PgATCC33277적Hgp4기인,삽입도극륭재체pMD18-T중병측서감정.경과매절후장목적기인편단여표체재체pET22b상련,구건출표체질립pET22b-Hgp44.장중조질립전화도감수태세포BL21(DE3)중,용이병기-β-D-류대반유당감(isopropyl-β-D-thiogalactoside,IPTG)유도표체융합단백,통과SDS취병희선알응효전영급단백질인적법진행분석.용고정화금속친화층석법대중조단백진행분리순화.결과 목적기인편단약위1100 bp,여예기대소상부,측서결과여GenBank중ATCC33277국제표준균주적RgpA적등위기인서렬U15282일치.IPTG유도후적균체경SDS취병희선알응효전영후형성일개이포함체형식존재적44 000적융합단백.단백질인적법검측증실기구유면역원성.용얼리자금속오합층석주순화출료목적단백,최후득도약3.5 mg/L적목적단백.결론 성공극륭표체료PgHgp44기인,병순화출료목적단백.
Objective To clone and express the gene of Hgp44 in adhesin domains of gingipains from Porphyromonas gingivalis(Pg) and to purify the protein.Methods The genomic DNA of Pg was isolated from PgATCC33277.The Hgp44 gene fragment was amplified by polymerase chain reaction (PCR)and then inserted into the cloning vector pMD18-T and sequenced.The correct fragment was linked with a prokaryotic expression vector pET-22b to construct the recombinant expression plasmid pET22b-Hgp44.The pET22b-Hgp44 confirmed by enzyme digestion was transformed into competent Escherchia coli( Ec ) BL21 (DE3) cells.Expression of fusion protein was induced by isopropyl-3-D-thiogalactoside (IPTG),and purified by immobilized metal-chelating affinity chromatography ( IMAC ) using a Ni2 + matrix column.SDS-polyaerylamide gel electrophoresis(SDS-PAGE) and Western blotting analysis were used to examine the fusion protein.Results A 1 100 bp fragment was successfully amplified and verified by the agarose gel electrophoresis and sequencing.The generated recombinant expression vectors pET22b-Hgp44 were verified by enzyme digestion and agarose gel electrophoresis.The expression of fusion protein in Ec BL21 ( DE3 )cells was examined by SDS-PAGE and Western blotting analyses,and the data showed that the protein was 44 000 in size and expressed mostly in the form of inclusion body.The purification of fusion protein was achieved using Ni2+ affinity chromatography.About 3.5 mg/L fusion protein was obtained.Conclusions Hgp44 was successfully expressed in the prokaryotic expression system and purified by IMAC using a Ni2+matrix column.