Expression of genes for orthopoxviral TNF-binding proteins in insect cells and investigation of the recombinant TNF-binding proteins
Expression of genes for orthopoxviral TNF-binding proteins in insect cells and investigation of the recombinant TNF-binding proteins
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DOI：
10.1007/s11008-005-0032-x
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发表时间：
2005-03-01
影响因子：
1.2
通讯作者：
Shchelkunov, SN
中科院分区：
文献类型：
--
作者：
Gileva, IP;Ryazankin, IA;Shchelkunov, SN
通过PCR从病毒基因组中分离天花病毒（VARV）、猴痘病毒（MPXV）或牛痘病毒（CPXV）的TNF结合蛋白（CrmB）基因，并在Sf 21昆虫细胞系中的杆状病毒系统中表达。通过各种理化和免疫学方法研究纯化的重组蛋白的性质。固相酶联免疫吸附试验表明，病毒蛋白抑制hTNF与hTNF多克隆抗体的结合，其抑制效率依次为VARV-CrmB > CPXV-CrmB > MPXV-CrmB。重组蛋白制剂的生物活性通过其中和TNF对L929鼠成纤维细胞系的细胞毒性的能力来评估。CrmB显示以物种特异性方式中和人、小鼠和兔TNF的细胞毒性。还显示VARV-CrmB抑制hTNF细胞毒性的效率超过多克隆抗hTNF抗体的效率。正痘病毒CrmB蛋白可以为开发新的抗TNF药物提供基础。
Genes for TNF-binding proteins (CrmBs) of the variola virus (VARV), monkeypox virus (MPXV) or cowpox virus (CPXV) were isolated by PCR from viral genomes and expressed in a baculovirus system in the Sf21 insect cell line. Properties of the purified recombinant proteins were studied by various physicochemical and immunological methods. Using solid-phase enzyme-linked immunosorbent assay, it was shown that viral proteins inhibited hTNF binding with polyclonal anti-hTNF antibodies, with the efficiency of inhibition decreasing in the series VARV-CrmB > CPXV-CrmB > MPXV-CrmB. Biological activity of the recombinant protein preparations was assessed by their ability to neutralize TNF cytotoxicity on the L929 murine fibroblast cells line. CrmBs were shown to neutralize cytotoxicity of human, mouse, and rabbit TNF in a species-specific manner. It was also shown that the efficiency of VARV-CrmB in inhibiting hTNF cytotoxicity exceeded that of polyclonal anti-hTNF antibodies. Orthopoxviral CrmB proteins can provide a basis for development of new, anti-TNF drugs.