利用毕赤酵母表达系统对成熟鸡白细胞介素-18蛋白突变体进行表达及生物活性测定

[Expression and bioactivity assay of mature chicken interleukin-18 protein mutant using Pichia pastoris expression system].

作者信息

Wang Xihui, Yue Shousong, Hu Jingdong, Huang Yijun, Chen Jinlong, Wang Tingting, Fan Zhongling, Zhao Hongkun

机构信息

College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Shandong Taian 271018, China.

出版信息

Wei Sheng Wu Xue Bao. 2010 Sep;50(9):1258-63.

PMID:21090268

Abstract

OBJECTIVE

We expressed mature chicken interleukin-18 (mChIL-18) in Pichia pastoris.

METHODS

The mChIL-18 gen was reconstructed by using site-specific mutagenesis based on the Pichia pastoris-preferred codons. The recombinant plasmid pPIC9K/mChIL-18 was constructed and transformed to Pichia pastoris GS115 by electroperation. Multi-copy recombinant strains were screened by Geneticin (G418). The expression of mChIL-18 protein was induced by methanol. SDS-PAGE and Western-blot were used to analyze the expressed products. The bioactivity of mChIL-18 was measured by methyl thiazolyl tetrazolium assays and chicken embryo fibroblasts-vesicular stomatitis virus (CEF-VSV) system.

RESULTS

The protein of mChIL-18 could be secreted by GS115. The optimum expression conditions, a rate of 480 mg/L,were obtained as follows:temperature 28 degrees C, pH 6.5, methanol concentration 2% and expression time 120 h. The obtained mChIL-18 protein could stimulate T lymphocytes proliferation. IFN-gamma induced by mChIL-18 could directly inhibit the growth of VSV in CEF,and its antiviral activity was about 1.7 x 10(4) U/mL which was produced by 400 microg/L of mChIL-18.

CONCLUSION

The high expression of bioactive recombinant mature chicken interleukin-18 (mChiL-18) in Pichia pastoris had been achieved.

摘要

目的

在毕赤酵母中表达成熟鸡白细胞介素-18（mChIL-18）。

方法

基于毕赤酵母偏好密码子，采用定点诱变技术对mChIL-18基因进行改造。构建重组质粒pPIC9K/mChIL-18，并通过电穿孔法将其转化至毕赤酵母GS115中。利用遗传霉素（G418）筛选多拷贝重组菌株。用甲醇诱导mChIL-18蛋白表达。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western-blot）分析表达产物。通过噻唑蓝比色法（MTT法）和鸡胚成纤维细胞-水泡性口炎病毒（CEF-VSV）系统检测mChIL-18的生物活性。

结果

mChIL-18蛋白可由GS115分泌。获得最佳表达条件，表达量为480 mg/L，具体如下：温度28℃，pH 6.5，甲醇浓度2%，表达时间120 h。所获得的mChIL-18蛋白能够刺激T淋巴细胞增殖。mChIL-18诱导产生的γ干扰素可直接抑制VSV在CEF中的生长，其抗病毒活性约为1.7×10⁴ U/mL，由400 μg/L的mChIL-18产生。