一种新型重组人源性白细胞介素-8活性检测方法的建立

目的通过蛋白质片段互补检测（proteinfragmentcomplementationassay，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—TEVGPCR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HEK293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—TEVGPCR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IL8RB都具有明显的激活活性，其EC50值分别为：12．32±0．89ng／mL（亚型I）、15．14±1．84ng／mL（亚型Ⅱ）和2．854-0．50ng／mL（亚型Ⅲ）。结论成功建立了-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。

A novel method for biological activity determination of recombinant human interluekin-8

Objective To measure the biological activity of recombinant human IL-8 （ rhIL-8 ） usin~ protein fragment complementation assay （PCA）. Methods A Split-TEg 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 ceils 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 ） of rhIL- 8 , which were expressed in E. eoli BL 2 1 （ DE 3 ）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.