鸡白细胞介素-18(ChIL-18)重组蛋白的生物学活性检测  

Biological Activity Assay of Purified Expression Product of Recombined Chicken Interleukin-18 Gene in Escherichia coli

作　　者：李宏梅[1] 胡敬东[1] 马凤龙 郑玉姝[1] 刘翠艳[1] 田兆菊[1] 赵宏坤[1] 

机构地区：[1]山东农业大学动物科技学院,泰安271018 [2]山东信得药业,青岛266061

出　　处：《农业生物技术学报》2007年第1期5-10,共6页Journal of Agricultural Biotechnology

基　　金：山东农业大学博士科研基金项目(No.23201);"十一五"国家科技支撑计划重大项目(No.20061122);山东省优秀中青年科学家科研奖励基金(No.2006BS06015)资助。

摘　　要：对含有鸡白细胞介素-18(ChIL-18)基因的质粒在大肠杆菌(Escherichia coli)内进行诱导表达,经亲和层析法对表达产物进行纯化,用微量细胞病变抑制法CEF-VSV系统,检测其对SPF鸡脾淋巴细胞诱导产生γ-干扰素(IFN-γ)的活性和其对病毒的抑制效果;用3H-TdR掺入法和MTT法分别检测其对T淋巴细胞诱导转化和NK细胞杀伤活性的作用。结果表明,经诱导表达出分子量44kD的目的蛋白(与GST融合表达),纯化后得到去除菌体蛋白的高纯度目的蛋白;该蛋白能够诱导脾细胞产生IFN-γ,当浓度为250ng/mL时诱导IFN-γ的活性最高,可达1×106U/mL;但该蛋白不能直接抑制水疱性口炎病毒(VSV)在鸡胚成纤维细胞(CEF)上生长;能够刺激淋巴细胞显著增殖,浓度为250ng/mL时效果最佳;适当浓度的rChIL-18蛋白能促进NK细胞的杀伤活性,浓度为150ng/mL时,作用最强。利用原核表达的重组鸡白细胞介素-18蛋白与天然鸡IL-18蛋白一样具有较广泛的生物学活性。To detect biological activity of recombined chicken interleukin-18 （IL-18）, positive vector containing chicken IL-18 gene was expressed in Escherichia coil, then the expressed product was purified by affinity chromatography. The activities of IFN-γ induced by rChIL-18 and rChIL-18 anti-virus were detected with CEF-VSV system. Both ^3H-thymidine （3H-TdR） incorporation and MTT colorimetric assays.were used to detect T lymphocytes proliferation response to rChIL-18 and the effect of rChIL-18 on cytotoxicity of NK cells, respectively. Results showed that recombinant protein with a relative molecular weight of 44 kD （including 26 kD GST） was expressed in E.coli, which could induce splenocytes to produce high activity of IFN-γ and the highest was 1 × 10^6 U/mL produced by 250 ng/mL of recombinant chicken IL-18 protein, but could not directly inhibit the growth of VSV in CEF. It was proved that purified protein could stimulate T lymphocytes proliferation and increase NK cytotoxicity at certain concentrations. It can be coneluded that chicken IL-18 protein with relatively high bioactivity can be successfully acquired by prokaryotic expressed system.

关 键 词：鸡白细胞介素-18 原核表达 纯化 生物学活性 

分 类 号：S188[农业科学—农业基础科学]