鸡白介素18成熟蛋白突变体在毕赤酵母中的高效表达及其生物活性检测

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

摘要:

摘要：【目的】通过目的基因克隆，表达获得高产量具有生物活性的鸡白介素18成熟蛋白(mature chicken interleukin-18, mChIL-18)。【方法】根据毕赤酵母密码子偏嗜性，通过引物设计来定点突变鸡白介素18基因，构建了重组质粒pPIC9K-mChIL-18。将线性化的重组阳性质粒电转到毕赤酵母GS115中，经筛选多拷贝阳性子后进行诱导表达。用MTT法和微量细胞病变抑制法检测其生物活性。【结果】筛选的多拷贝重组菌在诱导温度为28℃，培养液pH值为6.5，甲醇的诱导浓度为2%，诱导时

Abstract:

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 ℃, pH 6.5, methanol concentration 2% and expression time 120 h. The obtained mChIL-18 protein could stimulate T lymphocytes proliferation. IFN-γ induced by mChIL-18 could directly inhibit the growth of VSV in CEF, and its antiviral activity was about 1.7×104 U/mL which was produced by 400 ng/mL of mChIL-18. [Conclusion] The high expression of bioactive recombinant mature chicken interleukin-18(mChIL-18) in Pichia pastoris had been achieved.