hsTR55/TR75-preS1融合蛋白的表达与应用

Expression and Application of hsTR55/TR75 preS1 fusion receptors

目的简化测定hTNFα及其突变体的受体竞争结合活性的方法。方法将乙型肝炎病毒表面抗原(HBsAg)前区(preS1)肽段(2～50)编码区,分别融合于hsTR55和hsTR75基因的C末端组成融合受体分子基因,并利用细小RNA病毒属脑炎心肌炎病毒EMCV的内核糖体进入位点(internalribosomeentrysite,IRES),构建了hsTR55/hsTR75以及它们与preS1组成的融合受体分子hsTR55hsTR75-preS1基因和新霉素抗性(neoR)基因的双顺反子表达载体,转染BHK-21细胞后用G-418持续筛选得到稳定表达目的蛋白的细胞株。结果RT-PCR及ELISA均证实,两种融合受体分子表达,并且表达上清对hTNFα的活性均具有一定的中和作用。在对表达上清进行ELISA定量后,我们还用Biosensor方法测定了这些可溶性受体分子与hTNFα的结合常数。结果表明,在融合preS1肽段前后,hTNFα与受体分子的解离常数变化不大。结论利用表达的两种融合受体分子,成功地建立了一种检测hTNFα及其突变体的受体竞争结合活性的ELISA法,并利用该法测定了野生型hTNFα及其4种突变体的受体竞争结合活性,所获结果与利用125I-hTNFα测定的结果具有相当的可比性。

Aim To simplify the method about the receptor competitive binding assay of hTNFα and its mutants. Methods The gene encoding the N terminal 2 50 amino acids of HBsAg preS1 was amplified by PCR and fused to the 3′ end of two human soluble TNF receptor genes respectively to form the hsTR55 preS1/hsTR75 preS1 fusion genes. The recombinant bicistronic expression vectors were further constructed, which contained one of the human soluble TNF receptor fusion genes and the encephalomyocarditis virus(EMCV) internal ribosome entry site (IRES), followed by the neomycin phosphotransferases as the selectable marker. BHK 21 cells transfected with these vectors by electroporation were selected with G 418, and the positive colonies expressing the protein of interest were obtained. Results Supernatant of all transfectants could fairly neutralize hTNFα induced cytotoxicity to L929 cells. The expression of hsTR55 preS1/hsTR75 preS1 in those cells respectively has been further demonstrated by RT PCR and indirect ELISA at RNA transcription and protein translation levels. Their Kd(dissociation constant) value has also been assayed by Biosensor method. The results showed that the fused HBsAg preS1 peptide did not affect the Kd of hsTR55 or hsTR75 with hTNFα and its muteins. Conclusion A novel ELISA method was developed for studing the interaction between hTNFα and its two receptors using those expressed fusion receptors.