国际检验医学杂志
國際檢驗醫學雜誌
국제검험의학잡지
INTERNATIONAL JOURNAL OF LABORATORY MEDICINE
2014年
23期
3153-3155
,共3页
日本血吸虫%聚合酶链反应%大肠埃希菌%表达效率
日本血吸蟲%聚閤酶鏈反應%大腸埃希菌%錶達效率
일본혈흡충%취합매련반응%대장애희균%표체효솔
Schistosoma japonicum%polymerase chain reaction%Escherichia coli%expression efficiency
目的：构建日本血吸虫（Sj）重组质粒 pGEX-Sj32并研究其在大肠埃希菌 BL21中的表达效率。方法从该实验室保存的 BL21（pET28α-Sj32）重组菌中抽提质粒 pET28α-Sj32，PCR 扩增 Sj32抗原编码基因，定向克隆入穿梭载体 pGEX-1λT，构建重组质粒 pGEX-Sj32。将重组质粒转化大肠埃希菌 BL21（DE3），经异丙基硫代-β-D-半乳糖苷（IPTG）诱导表达；表达产物用SDS-PAGE 和 Western blot 进行鉴定。结果 PCR 成功扩增出 Sj32编码基因，并成功构建了重组质粒 pGEX-Sj32；SDS-PAGE显示相对分子质量约为58×103的重组蛋白，薄层扫描分析显示表达蛋白约占菌体总蛋白的21％；Western blot 显示重组蛋白可被日本血吸虫感染兔血清识别。结论成功构建日本血吸虫重组质粒 pGEX-Sj32，该重组质粒在大肠埃希菌 BL21中得到了高效表达，且表达蛋白具有特异的抗原性。
目的：構建日本血吸蟲（Sj）重組質粒 pGEX-Sj32併研究其在大腸埃希菌 BL21中的錶達效率。方法從該實驗室保存的 BL21（pET28α-Sj32）重組菌中抽提質粒 pET28α-Sj32，PCR 擴增 Sj32抗原編碼基因，定嚮剋隆入穿梭載體 pGEX-1λT，構建重組質粒 pGEX-Sj32。將重組質粒轉化大腸埃希菌 BL21（DE3），經異丙基硫代-β-D-半乳糖苷（IPTG）誘導錶達；錶達產物用SDS-PAGE 和 Western blot 進行鑒定。結果 PCR 成功擴增齣 Sj32編碼基因，併成功構建瞭重組質粒 pGEX-Sj32；SDS-PAGE顯示相對分子質量約為58×103的重組蛋白，薄層掃描分析顯示錶達蛋白約佔菌體總蛋白的21％；Western blot 顯示重組蛋白可被日本血吸蟲感染兔血清識彆。結論成功構建日本血吸蟲重組質粒 pGEX-Sj32，該重組質粒在大腸埃希菌 BL21中得到瞭高效錶達，且錶達蛋白具有特異的抗原性。
목적：구건일본혈흡충（Sj）중조질립 pGEX-Sj32병연구기재대장애희균 BL21중적표체효솔。방법종해실험실보존적 BL21（pET28α-Sj32）중조균중추제질립 pET28α-Sj32，PCR 확증 Sj32항원편마기인，정향극륭입천사재체 pGEX-1λT，구건중조질립 pGEX-Sj32。장중조질립전화대장애희균 BL21（DE3），경이병기류대-β-D-반유당감（IPTG）유도표체；표체산물용SDS-PAGE 화 Western blot 진행감정。결과 PCR 성공확증출 Sj32편마기인，병성공구건료중조질립 pGEX-Sj32；SDS-PAGE현시상대분자질량약위58×103적중조단백，박층소묘분석현시표체단백약점균체총단백적21％；Western blot 현시중조단백가피일본혈흡충감염토혈청식별。결론성공구건일본혈흡충중조질립 pGEX-Sj32，해중조질립재대장애희균 BL21중득도료고효표체，차표체단백구유특이적항원성。
Objective To construct the recombinant plasmid pGEX-Sj32 of Schistosoma japonicum(Sj )and to research its ex-pression in Escherichia coli (E.coli)BL21.Methods Sj32 gene was amplified by PCR from template of plasmid pET28α-Sj32 ex-tracted from recombinant bacterium BL21 (pET28α-Sj32 )stored by our laboratory,and then cloned into the vector pGEX-1λT to construct pGEX-Sj32.The recombinant plasmid pGEX-Sj32 was transformed into E.coli BL21(DE3).The recombinant strains were induced by isopropyl-β-d-thiogalactoside(IPTG),and the expressed products were identified by SDS-PAGE and Western blot.Re-sults Sj32 coding gene was successfully amplified by PCR and cloned into the vector pGEX-1λT,and the recombinant plasmid pGEX-Sj32 was constructed successfully.The molecular mass of the expressed recombinant protein was proximately 58 000 as de-tected by SDS-PAGE.The amount of the expressed protein was about 21% of the total bacterial protein.Western blot confirmed that the expressed protein could be recognized by the immune sera from rabbit infected with Schistosoma japonicum.Conclusion The recombinant plasmid pGEX-Sj32 is successfully constructed.The Sj32 protein was highly expressed in E.coli and the expressed recombinant protein possesses the specific antigenicity.
