微小隐孢子虫糖蛋白GP900对HCT-8细胞Akt和MAPK通路的影响  

The recombinant glycoprotein GP900 of Cryptosporidium parvuminduced activation of Akt and MAPK pathways in HCT-8 cells

作　　者：张梦鸽 宫鹏涛 张西臣 李建华 ZHANG Meng-ge;GONG Peng-tao;ZHANG Xi-chen;LI Jian-hua(College of Veterinary Medicine, Jilin University, Changchun130062, China)

机构地区：[1]吉林大学动物医学学院,吉林长春130062

出　　处：《中国病原生物学杂志》2018年第5期457-461,467,共6页Journal of Pathogen Biology

基　　金：国家重点研发计划项目(No.2017YFD0501300)

摘　　要：目的探讨微小隐孢子虫糖蛋白GP900对HCT-8细胞Akt和MAPK信号通路的影响,为进一步研究微小隐孢子虫与宿主细胞的相互作用提供理论依据。方法以微小隐孢子虫卵囊cDNA为模板,PCR扩增GP900基因片段,回收后连接到pMD-18T载体,构建重组克隆质粒pMD-18T-GP900。双酶切pMD-18T-GP900和pET-32a载体并进行连接,构建pET-32a-GP900重组质粒,双酶切及测序鉴定正确后转化入大肠埃希菌BL21(DE3),重组菌用IPTG诱导,通过SDS-PAGE分析GP900的表达。采用Ni-NTA亲和层析富集和超滤纯化GP900重组蛋白用Triton-114去除内毒素,直至重组蛋白内毒素含量在0.01~0.1Eu/mg为止,然后用Hydrophobic beads吸附残留的Triton-114,收集重组蛋白。用GP900重组蛋白刺激HCT-8细胞,收集不同刺激时间的细胞,提取全蛋白,采用Western blot检测Akt、p38和ERK的磷酸化;用不同浓度GP900刺激后检测细胞中上述通路蛋白的磷酸化与GP900剂量的关系。结果 pET-32a-GP900重组质粒经双酶切及测序鉴定构建正确,表达的重组GP900蛋白分子质量与理论值相符。纯化的重组蛋白刺激HCT-8细胞后,Akt在60min发生磷酸化且随着GP900浓度增加磷酸化程度呈剂量依赖,p38在60min发生磷酸化但不呈剂量依赖,ERK在90min发生磷酸化但不呈剂量依赖。结论成功构建了GP900重组质粒,表达的GP900重组蛋白可诱导HCT-8细胞Akt、p38和ERK信号通路活化。Objectives To understand the effect of Cryptosporidium parvum GP900 on Akt and MAPK signaling pathways in HCT-8 cells,which may help us to clarify the mechanism of the interactions between C.parvumand host cells. Methods The coding sequences of GP900 were amplified with PCR using cDNA fromC.parvumas a template.After gel extraction,GP900 was inserted into a pMD-18 Tvector.The vectors pMD-18T-GP900 and pET-32awere then digested with EcoRI and XhoI,and a GP900 fragment was cloned into the pET-32avector to construct a pET-32a-GP900 plasmid.All recombinant plasmids were verified with double restriction enzyme digestion and sequencing.The plasmid pET-32a-GP900 was transformed into E.coli B L21（DE3）,and its expression was induced with IPTG.The expressed recombinant protein GP900 was analyzed using SDS-PAGE and purified using chelating Ni-NTA and filtered with an ultrafiltration membrane.Endotoxin in the recombinant protein was removed using Triton X-114 until endotoxin was in the range of 0.01-0.1 Eu/mg.Remaining Triton X-114 was removed by adsorption to hydrophobic beads and then the recombinant protein was collected.The total lysate of HCT-8 cells was collected after co-incubation with recombinant protein GP900 for different times,and Western blotting was used to detect the phosphorylation of Akt,p38,and ERK.After stimulation with different concentrations of GP900,the relationship between the phosphorylation of signaling pathway proteins and the dose of GP900 was determined using Western blotting. Results The recombinant plasmid pET-32a-GP900 was correctly constructed according to restriction enzyme digestion and sequencing.The molecular mass of the expressed recombinant protein GP900 was consistent with the theoretical value.After stimulation with purified recombinant protein GP900,the phosphorylation of Akt in HCT-8 cells was induced at 60 min,and the amount of phosphorylation increased in a dose-dependent manner as the GP900 concentration increased.The phosphorylation of p38 was induced at 60 min but not in a d

关 键 词：微小隐孢子虫 GP900重组蛋白 AKT P38 ERK 

分 类 号：R382.3[医药卫生—医学寄生虫学]