摘要
目的体外分析Toll样受体4(TLR4)诱导感染弓形虫棒状体基部蛋白38(ROP38)对小鼠树突状细胞(DC)成熟的影响。方法以异硫氰酸胍方法提取刚地弓形虫RH株速殖子总RNA,采用PCR扩增ROP38,将ROP38连接至原核表达载体构建重组质粒pG EX-4T-ROP38;利用ProtS cale在线软件分析ROP38蛋白的亲水性和疏水性,利用DNAStar8.0分析ROP38蛋白的抗原表位;将重组质粒pG EX-4T-ROP38转化E.coli Rosetta感受态细胞,挑取阳性单菌落用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导重组质粒表达,Western blot法检测ROP38蛋白水平。利用0.28 mg重组ROP38蛋白刺激小鼠骨髓来源DC,同时采用TLR4抗体封闭和空白处理为对照组。采用流式细胞术分析CD11c表达水平,运用SPSS23.0软件进行单因素方差分析。结果成功扩增弓形虫ROP38基因,其大小为516 bp,测序结果显示所获基因与GenB ank中已报道的序列(XM_002366710.2)同源性高达99%;ROP38基因序列生物信息学分析表明,ROP38蛋白含有5个α螺旋,5个β折叠,5个亲水性区域和8个可预测的抗原表位;重组质粒pG EX-4T-ROP38经IPTG诱导后,表达蛋白质的相对分子质量(Mr)为45 000,主要以包涵体形式存在。ROP38抗原刺激小鼠源DC后,其CD11c+表达水平明显高于TLR4抗体封闭组和空白对照组。结论 TLR4可诱导ROP38刺激小鼠DC的成熟。
Objective To investigate the effect of rhoptry protein 38 （ROP38） from Toxoplasma gondii （ T. gondii） on the maturation of dendritic cells （DCs） by Toll-like receptor 4 （TLR4） induction in vitro. Methods The total RNA from T. gondii RH strain was extracted by guanidine thiocyanate method, and then cDNA was synthesized with reverse transcription reaction. After ROP38 gene was amplified by PCR, the recombinant pGEX-4T-ROP38 was constructed and expressed under IPTG induction. The recombinant ROP38 protein was detected by SDS-PAGE and Western blot analysis. The secondary structure and antigenicity of the ROP38 were predicted through DNAStarS. 0 and PretScale. In vitro, DCs were isolated and cultured for 6 days, then reacted with ROP38 antigen for 2 hours. The CD11c was detected by flow cytometry, and the data were analyzed with ANOVA by SPSS 23.0 software. Results The amplified gene was about 516 bp as expected. The sequence analysis showed that its homology was 99% compared with the reported sequence （ XM_002366710.2 ） from GenBank. It was found that the relative molecular mass （Mr） of recombinant ROP38 was 45 kD. The prediction of structure indicated that there were 5 co-helices, ,5 β-sheets, 5 hydrophilic regions and 8 potential epitopes in ROP38 protein. In vitro, the expression of CDllc on DCs was significantly up-regulated after stimulated with ROP38, and the expression level of CD11c in ROP38 infection group was significantly higher than that of the other groups. Conclusion ROP38 promotes the maturation of DCs mediated by TLR4.
作者
张衡
吴尚桦
施之强
汪汕
陆维
吴以振
孙裴
徐前明
ZHANG Heng;WU Shanghua;SHI Zhiqiang;WANG Shan;LU Wei;WU Yizhen;SUN Pei;XU Qianming(College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, Chin)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2018年第3期199-204,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
安徽省高校自然科学基金(KJ2016A838)
