摘要
为了揭示鸡重组粒细胞巨噬细胞集落刺激因子(rchGM-CSF)在免疫中的作用,本试验对海兰灰蛋鸡GM-CSF基因进行预测并通过PCR去掉信号肽,改造蛋白质编码区(CDS)3′端的终止密码子,使C末端带有c-myc和6His标签,构建毕赤酵母表达质粒pPICZαA/chGM-CSF,电转化感受态毕赤酵母GS115,甲醇诱导表达,经SDS-PAGE和Western blot鉴定后进行大规模诱导表达,收集上清,用His Bind Ni-NTA层析柱进行纯化,并通过淋巴细胞增殖试验鉴定rchGM-CSF的生物活性。结果显示,rchGM-CSF成功表达,在第3~4天时分泌表达达到高峰;rchGM-CSF按1∶5倍稀释时能极显著促进淋巴细胞分裂增生(P<0.01),按1∶25倍稀释时能显著促进淋巴细胞增殖(P<0.05),表明rchGM-CSF具有一定的免疫促进功能。
This study aimed to express chicken recombinant granulocyte macrophage colony stimulating factor(rchGM-CSF)in Pichia Pastoris and examine its bioactivity.The chGM-CSF gene was cloned and its signal peptide segment was predicted and knocked out by PCR.The stop codon at 3'end of the protein-coding region(CDS)was modified to label with a C-terminal c-myc and 6His.The expression vector pPICZαA/chGM-CSF was constructed and electrotransformed into P.pastoris GS115.The rchGM-CSF expression was induced by methanol and identified by SDS-PAGE and Western blot.After large-scale expression,the supernatant was collected and purified through His Bind Ni-NTA chromatography.The rchGM-CSF bioactivity was assessed by MTT technique.The results showed that the rchGM-CSF was successfully expressed and secreted into culture medium,peaking between 3-4 days,and rchGM-CSF at 1∶5 dilution could significantly promote lymphocyte proliferation(P<0.01),and at 1∶25 dilution could promote lymphocyte proliferation(P<0.05).Thus,the rchGM-CSF expressed in P.pastoris may potentially enhance immune function.
作者
王黎霞
张鹏
张建军
安健
WANG Li-xia;ZHANG Peng;ZHANG Jian-jun;AN Jian(College of Animal Science and Veterinary,Beijing Vocational College of Agriculture,Beijing 102442,China;College of Animal Science and Technology,Beijing University of Agriculture,Beijing 102206,China)
出处
《中国兽医杂志》
CAS
北大核心
2022年第10期35-39,43,共6页
Chinese Journal of Veterinary Medicine
基金
打造高水平专业群动物医学专业群技术平台与社会服务建设项目(11000022T000000444734)。
