国际遗传学杂志
國際遺傳學雜誌
국제유전학잡지
INTERNATIONAL JOURNAL OF GENETICS
2013年
6期
231-238
,共8页
王英明%陈帅%张清仪%李德彬%闫林慧%廖远平%王丁力%卢大儒%朱化星
王英明%陳帥%張清儀%李德彬%閆林慧%廖遠平%王丁力%盧大儒%硃化星
왕영명%진수%장청의%리덕빈%염림혜%료원평%왕정력%로대유%주화성
蛋白质片段互补检测技术%Split-TEV GPCR激活检测系统%IL-8%不同亚型%活性检测
蛋白質片段互補檢測技術%Split-TEV GPCR激活檢測繫統%IL-8%不同亞型%活性檢測
단백질편단호보검측기술%Split-TEV GPCR격활검측계통%IL-8%불동아형%활성검측
Protein fragment complementation assay%Split-TEV GPCR activation assay%IL-8%Different isoforms%Activity analyses
目的 通过蛋白质片段互补检测(protein fragment complementation assay,PCA)技术检测重组人源性IL-8的生物学活性.方法 利用PCA方法构建了Split-TEV GPCR激活检测系统,为了验证其有效性,在不同的宿主系统中表达和纯化了各种亚型的人源性IL-8重组蛋白用于进行活性检测.蛋白样品包括:①IL-8亚型Ⅰ(残基21-99),通过在哺乳动物细胞HEK 293中分泌表达前体IL-8基因获得,其N端信号肽(残基1-20)被切除;②IL-8亚型Ⅱ(残基23-99)和亚型Ⅲ(残基28-99),通过在大肠杆菌BL21(DE3)中表达获得.结果 Split-TEV GPCR激活检测系统证明纯化后的重组人源性IL-8样品对天然受体IL8 RB都具有明显的激活活性,其EC50值分别为:12.32 ±0.89 ng/mL(亚型Ⅰ)、15.14±1.84 ng/mL(亚型Ⅱ)和2.85±0.50 ng/mL(亚型Ⅲ).结论 成功建立了一种新型的重组人IL-8活性检测方法,为进一步的理论研究和IL-8中和抗体的高通量筛选奠定了基础.
目的 通過蛋白質片段互補檢測(protein fragment complementation assay,PCA)技術檢測重組人源性IL-8的生物學活性.方法 利用PCA方法構建瞭Split-TEV GPCR激活檢測繫統,為瞭驗證其有效性,在不同的宿主繫統中錶達和純化瞭各種亞型的人源性IL-8重組蛋白用于進行活性檢測.蛋白樣品包括:①IL-8亞型Ⅰ(殘基21-99),通過在哺乳動物細胞HEK 293中分泌錶達前體IL-8基因穫得,其N耑信號肽(殘基1-20)被切除;②IL-8亞型Ⅱ(殘基23-99)和亞型Ⅲ(殘基28-99),通過在大腸桿菌BL21(DE3)中錶達穫得.結果 Split-TEV GPCR激活檢測繫統證明純化後的重組人源性IL-8樣品對天然受體IL8 RB都具有明顯的激活活性,其EC50值分彆為:12.32 ±0.89 ng/mL(亞型Ⅰ)、15.14±1.84 ng/mL(亞型Ⅱ)和2.85±0.50 ng/mL(亞型Ⅲ).結論 成功建立瞭一種新型的重組人IL-8活性檢測方法,為進一步的理論研究和IL-8中和抗體的高通量篩選奠定瞭基礎.
목적 통과단백질편단호보검측(protein fragment complementation assay,PCA)기술검측중조인원성IL-8적생물학활성.방법 이용PCA방법구건료Split-TEV GPCR격활검측계통,위료험증기유효성,재불동적숙주계통중표체화순화료각충아형적인원성IL-8중조단백용우진행활성검측.단백양품포괄:①IL-8아형Ⅰ(잔기21-99),통과재포유동물세포HEK 293중분비표체전체IL-8기인획득,기N단신호태(잔기1-20)피절제;②IL-8아형Ⅱ(잔기23-99)화아형Ⅲ(잔기28-99),통과재대장간균BL21(DE3)중표체획득.결과 Split-TEV GPCR격활검측계통증명순화후적중조인원성IL-8양품대천연수체IL8 RB도구유명현적격활활성,기EC50치분별위:12.32 ±0.89 ng/mL(아형Ⅰ)、15.14±1.84 ng/mL(아형Ⅱ)화2.85±0.50 ng/mL(아형Ⅲ).결론 성공건립료일충신형적중조인IL-8활성검측방법,위진일보적이론연구화IL-8중화항체적고통량사선전정료기출.
Objective To measure the biological activity of recombinant human IL-8 (rhIL-8) using protein fragment complementation assay (PCA).Methods A Split-TEV GPCR activation assay was established based on the PCA method and its validity was tested by biological activity analyses of three rhIL-8 isoforms,which were expressed and purified from different expression systems.The IL-8 samples include:1) Form Ⅰ of rhIL-8 (residues 21-99),which was obtained by expression of the pre-IL-8 residues 1-99) gene in mammalian cells HEK 293.The recombinant protein was secreted by the cells into growth medium after removal its N-terminal signal peptide (residues 1-20) ; 2) Form Ⅱ (residues 23-99) and form Ⅲ residues 28-99) ofrhIL-8,which were expressed in E.coli BL21 (DE3)cells.Results The Split-TEV GPCR activation assay has indicated that all three isoforms of purified rhIL-8s can activate the receptor IL8RB with the EC50 values of 12.32 ± 0.89,15.14 ± 1.84 and 2.85 ± 0.50 ng/ml respectively.Conclusion A novel method for biological activity determination of rhIL-8 has been established successfully,which will be valuable for facilitation of theoretical research and high-throughput screening of neutralizing antibodies to IL-8.
