摘要
目的获得广州管圆线虫蛋白酶体α5基因(pas-5),构建其重组质粒,探讨PAS-5重组蛋白对人THP-1巨噬细胞凋亡的影响。方法以广州管圆线虫逆转录DNA为模板,PCR扩增pas-5基因,构建pcoldⅢ-pas-5重组质粒,转化至大肠埃希菌DH5α感受态细胞,取菌液进行PCR、双酶切和测序鉴定。阳性质粒经0.1 mmol/L异丙基-β-D-硫代半乳糖苷诱导表达后,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)分析重组蛋白表达情况,采用镍离子亲和层析法纯化重组蛋白。将用佛波酯诱导的人THP-1巨噬细胞经PAS-5(实验组)孵育18 h后,采用流式细胞术观察细胞凋亡情况,采用ELISA检测细胞培养上清中细胞因子白细胞介素-10(IL-10)水平,另设阴性对照组(牛血清白蛋白)和空白对照组(RPMI1640)。结果Pas-5基因PCR扩增产物约为800 bp,与预期大小相符。经菌液PCR、双酶切和测序鉴定获得pcoldⅢ-pas-5重组质粒。SDS-PAGE分析结果显示,PAS-5重组蛋白主要以包涵体形式存在,其相对分子质量约28 000。Western blotting分析结果显示,重组蛋白能与兔抗鼠His单克隆抗体特异性结合。镍离子亲和层析纯化重组蛋白后获得单一条带。流式细胞术分析结果显示,阴性对照组和实验组人THP-1巨噬细胞凋亡率分别为(0.380±0.194)%和(0.052±0.036)%,两者差异有统计学意义(P<0.05)。ELISA检测结果显示,实验组细胞培养上清中IL-10水平为(77.606±1.766)pg/ml,高于阴性对照组的(45.652±5.975)pg/ml(P<0.05)。结论获得了pas-5基因和重组质粒pcoldⅢ-pas-5,重组蛋白PAS-5可抑制人THP-1巨噬细胞凋亡,与IL-10水平提高有一定关系。
Objective To obtain the gene for proteasome α5 subunit（pas-5） of Angiostrongylus cantonensis,construct the recombinant plasmid, and investigate the effect of PAS-5 recombinant protein on the apoptosis of hu-man THP-1 macrophages. Methods The pas-5 gene was cloned from the c DNA of A. cantonensis, inserted into the prokaryotic expression vector pcold Ⅲ to construct the recombinant plasmid pcold Ⅲ-pas-5, and transformed into Escherichia coli DH5α competent cells. The cultures underwent PCR, double digestion, and sequencing to confirm the construction of recombinant plasmid. Expression of the recombinant plasmid was induced by IPTG, and the ex-pressed recombinant proteins were analyzed by SDS-PAGE and Western blotting, and purified with Nickel affinity chromatography using Ni-NTA beads. The phorbol myristate acetate-induced THP-1 macrophages were incubated with PAS-5 for 18 h, and then flow cytometry was performed to evaluate the apoptosis of macrophages. The level of cy-tokine IL-10 in the culture supernatant was assessed by ELISA. Negative control and blank control groups were set with bovine serum albumin（BSA） and RPMI 1640, respectively, instead of PAS-5. Results The PCR amplification product of pas-5 was about 800 bp, consistent with expectation. The pcold Ⅲ-pas-5 recombinant plasmid was verified by PCR, double digestion and sequencing results. SDS-PAGE results showed that the recombinant protein PAS-5 was expressed mainly in the form of inclusion body, with a relative molecular weight of 28 000. Western blotting showed that the recombinant protein could specifically bind to rabbit anti-mouse His monoclonal antibody. A single strip was obtained from Nickel affinity chromatography. The flow cytometry showed that the THP-1 macrophages apoptosis rate was（0.380 ± 0.194）% in the BSA group, and（0.052 ± 0.036）% in the PAS-5 group（P〈0.05）.ELISA results showed a significantly higher level of supernatant IL-10 in the PAS-5 group than in the BSA group[（77.606 ± 1.766） pg/ml versus（45
作者
闫兰竹
史晓萌
祖雁文
陈茜茜
李星潘
闫宝龙
黄慧聪
YAN Lan-zhu;SHI Xiao-meng;ZU Yan-wen;CHEN Xi-xi;LI Xing-pan;YAN Bao-long;HUANG Hui-cong(Department of Parasitology, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou 325035, China;Clinical Laboratory of Bethune International Peace Hospital, Shijiazhuang 050082, China;School of the First Clinical Medical Sciences, Wenzhou Medical University, Wenzhou 325035, China)
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2018年第3期253-257,共5页
Chinese Journal of Parasitology and Parasitic Diseases
