广州管圆线虫蛋白酶体α5基因的克隆、表达及重组蛋白对人THP-1巨噬细胞凋亡的影响

摘要/Abstract

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（RPMI 1640）。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。

关键词: 广州管圆线虫, 蛋白酶体, 重组蛋白, 人THP-1巨噬细胞, 凋亡

Abstract:

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 human THP-1 macrophages. Methods The pas-5 gene was cloned from the cDNA of A. cantonensis, inserted into the prokaryotic expression vector pcoldⅢ to construct the recombinant plasmid pcoldⅢ-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 expressed 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 cytokine 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 pcoldⅢ-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(45.652 ± 5.975) pg/ml, P < 0.05］. Conclusion The recombinant plasmid pcoldⅢ-pas-5 was constructed, and the recombinant protein PAS-5 can inhibit the apoptosis of THP-1 macrophages, which may be related to the increased level of IL-10.

Key words: Angiostrongylus cantonensis, Proteasome, Recombinant protein, Human THP-1 macrophages, Apoptosis

闫兰竹1,2，史晓萌1，祖雁文1，陈茜茜3，李星潘1，闫宝龙1，黄慧聪1*. 广州管圆线虫蛋白酶体α5基因的克隆、表达及重组蛋白对人THP-1巨噬细胞凋亡的影响[J]. 中国寄生虫学与寄生虫病杂志.

YAN Lan-zhu1,2, SHI Xiao-meng1, ZU Yan-wen1, CHEN Xi-xi3, . Cloning, expression of the gene for proteasome α5 subunit of Angiostrongylus cantonensis and the effect of the recombinant protein on human THP-1 macrophages apoptosis[J]. Chinese Journal of Parasitology and Parasitic Diseases.

[1]	黄梦宇, 陈帝坤, 陈慧儒, 范梦培, 柳俊有, 权云帆. 海口市湿地公园褐云玛瑙螺广州管圆线虫感染情况调查[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(5): 640-643.
[2]	张娟, 陶洪, 李彦忠, 王婷婷, 杨晶晶, 向以斌, 陈奕杉, 周晓梅. 2017—2022年云南省常见螺类广州管圆线虫感染情况调查[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(4): 464-469.
[3]	刘玉婷, 厉广栩. 儿童重型广州管圆线虫病1例[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(6): 813-816.
[4]	李佳铭, 王艺璇, 杨宁爱, 马慧慧, 兰敏, 刘春兰, 赵志军. 刚地弓形虫ROP16蛋白对MH-S细胞极化和凋亡的影响及其相关机制[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(5): 579-586.
[5]	潘火云, 谭丽梅, 徐翼, 李旭芳, 叶家卫, 房春晓, 杨峰霞, 陈敏霞, 杨花梅, 曾凡森. 12例儿童广州管圆线虫病临床分析[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(5): 668-672.
[6]	邵晗, 李思源, 李军. 二甲双胍对多房棘球蚴囊泡和原头节自噬及凋亡的影响[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(1): 43-49.
[7]	周文正, 孙俊刚, 赵喜滨, 曹力. 三维适形调强放疗对大鼠继发性股骨细粒棘球蚴感染的疗效研究[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(4): 443-448.
[8]	王婵, 杨松昊, 董飞, 杜先才, 杨继辉, 牛楠, 朱明星, 王浩, 王娅娜, 赵巍. LncRNA 017418及细胞因子在细粒棘球蚴rP29免疫小鼠中的表达分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(2): 201-206.
[9]	赵亚男, 洪勇, 李朝品. Der p1 T细胞表位融合蛋白对哮喘小鼠的特异性免疫治疗效果[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(1): 74-79.
[10]	柴强, 李朝品. 重组蛋白Blo t 21 T特异性免疫治疗哮喘小鼠效果研究[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(3): 286-290.
[11]	岳志远, 张仪, 郭云海, 秦志强, 黄芸, 张伟, 茅光耀. 福寿螺感染广州管圆线虫后G型溶菌酶基因差异表达分析[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(3): 326-331.
[12]	梁凯, 李艳文, 王贝贝, 傅晓茵, 刘晓泉, 何姗姗, 唐莉莉, 刘登宇, 石焕焕. 刚地弓形虫重组蛋白表面抗原1与4诱导小鼠免疫保护性的比较研究[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(2): 155-160.
[13]	白慧芳, 柯琪文, 陈韵秋, 刘芷晴, 张玲敏, 吴春云, 袁桂秀, 詹希美, 程梅. 广州管圆线虫半胱氨酸蛋白酶AcCBL1和AcCBL2基因的克隆、表达及鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(5): 469-473.
[14]	高德俊, 常爽, 单秀梅, 范巍巍, 毛佐华. 刚地弓形虫棒状体蛋白ROP16对小鼠脑神经细胞凋亡的影响[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(6): 570-574.
[15]	胡求安, 张仪, 郭云海, 吕山, 夏尚, 刘和香. 广东省南澳岛福寿螺和鼠类密度及其广州管圆线虫感染现状调查[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(3): 239-245.

广州管圆线虫蛋白酶体α5基因的克隆、表达及重组蛋白对人THP-1巨噬细胞凋亡的影响

Cloning, expression of the gene for proteasome α5 subunit of Angiostrongylus cantonensis and the effect of the recombinant protein on human THP-1 macrophages apoptosis

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价