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
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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(45.652 ± 5.975) pg/ml, P < 0.05 ]. Conclusion The recombinant plasmid pcold Ⅲ-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.
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(45.652 ± 5.975) pg/ml, P < 0.05 ]. Conclusion The recombinant plasmid pcold Ⅲ-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.
引文
[1]李雨春,胡锡敏,童重锦,等.海南省人群广州管圆线虫病血清学、危险因素及知晓率调查[J].中国寄生虫学与寄生虫病杂志,2011,29(1):74-75.
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[22]黎志财,周静,张倩,等.IL-10对细菌脂蛋白诱导乳鼠心肌细胞凋亡的抑制作用[J].中国老年学杂志,2012,32(19):4203-4205.
[23]姚君,王立生,王建尧,等.重组人白介素10对低氧条件下人单核细胞凋亡变化规律的影响[J].广东医学,2011,32(1):40-42.
[24]Zhang Y,Zhang GQ,Hendrix LR,et al.Coxiella burnetii induces apoptosis during early stage infection via a caspaseindependent pathway in human monocytic THP鄄1 cells[J].PLo S One,2012,7(1):e30841.
[25]Cohen SB,Crawley JB,Kahan MC,et al.Interleukin鄄10rescues T cells from apoptotic cell death:association with an upregulation of Bcl鄄2[J].Immunology,1997,92(1):1鄄5.
[2]张仪,吕山,杨坤,等.我国广州管圆线虫自然疫源地分布首次调查[J].中国寄生虫学与寄生虫病杂志,2009,27(6):508-512.
[3]Huang HC,Yao LL,Song ZM,et al.Development-specific differences in the proteomics of Angiostrongylus cantonensis[J].PLo S One,2013,8(10):e76982.
[4]王晓燕,林岚,刘江.广州管圆线虫病患者中枢神经系统MRI的影像学表现[J].中国寄生虫学与寄生虫病杂志,2012,30(1):49-51.
[5]张榕燕,李莉莎,林金祥.幼儿重症广州管圆线虫病1例[J].中国寄生虫学与寄生虫病杂志,2010,28(1):71,74.
[6]Yan BL,Sun WW,Yan LZ,et al.Structural and functional characterisation of FOXO/Acan-DAF-16 from the parasitic nematode Angiostrongylus cantonensis[J].Acta Trop,2016,164:125-136.
[7]Feng Y,Zeng X,Li WH,et al.The pathogenesis of optic neuritis caused by Angiostrongylus cantonensis in BALB/c mice[J].Parasit Vectors,2014,7:339.
[8]Song ZM,Huang HC,Tan F,et al.Differential proteomics analysis of female and male adults of Angiostrongylus cantonensis[J].Exp Parasitol,2012,131(2):169-174.
[9]姚丽丽.广州管圆线虫V期幼虫特异性抗原的筛选及免疫鉴定[D].温州:温州医科大学,2014.
[10]Huang W,Zhou Q,Yuan X,et al.Proteasome inhibitor YSY01A enhances cisplatin cytotoxicity in cisplatin-resistant human ovarian cancer cells[J].J Cancer,2016,7(9):1133-1141.
[11]Wang X,Mazurkiewicz M,Hillert EK,et al.The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitinspecific protease-14 and induces apoptosis of multiple myeloma cells[J].Sci Rep,2016,6:26979.
[12]Papaevgeniou N,Chondrogianni N.The ubiquitin proteasome system in Caenorhabditis elegans and its regulation[J].Redox Biol,2014,2:333-347.
[13]孙鹏,刘淼,冯利兴,等.蛋白酶体结构和活性调节机制的研究进展[J].生物化学与生物物理进展,2015,42(12):1084-1093.
[14]Stout GJ,Stigter EC,Essers PB,et al.Insulin/IGF-1-mediated longevity is marked by reduced protein metabolism[J].Mol Syst Biol,2013,9:679.
[15]Chen J,Li L,Su J,et al.Proteomic analysis of G2/M arrest triggered by natural borneol/curcumin in Hep G2 cells,the importance of the reactive oxygen species-p53 pathway[J].J Agric Food Chem,2015,63(28):6440-6449.
[16]Ames E,Hallett WH,Murphy WJ.Sensitization of human breast cancer cells to natural killer cell-mediated cytotoxicity by proteasome inhibition[J].Clin Exp Immunol,2009,155(3):504-513.
[17]陈建华,汤正好.泛素-蛋白酶体系统与免疫系统的关系[J].国际免疫学杂志,2010,33(4):286-289.
[18]陈佳,吴松明,朱兴全,等.寄生虫蛋白酶体的研究进展[J].畜牧兽医学报,2012,43(1):7-13.
[19]曹弈堃,刘风英,华冰,等.慢性实验性变态反应性脑脊髓炎小鼠模型不同阶段抗原提呈细胞免疫应答作用分析[J].宁夏医学杂志,2017,39(2):100-103.
[20]Hasan Z,Ashraf M,Tayyebi A,et al.M.leprae inhibits apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 gene expression[J].BMC Microbi鄄ol,2006,6:78.
[21]王庆敏,雷呈祥,肖安,等.结核分枝杆菌MPT64抗原对巨噬细胞凋亡的抑制作用[J].第二军医大学学报,2013,34(5):502-506.
[22]黎志财,周静,张倩,等.IL-10对细菌脂蛋白诱导乳鼠心肌细胞凋亡的抑制作用[J].中国老年学杂志,2012,32(19):4203-4205.
[23]姚君,王立生,王建尧,等.重组人白介素10对低氧条件下人单核细胞凋亡变化规律的影响[J].广东医学,2011,32(1):40-42.
[24]Zhang Y,Zhang GQ,Hendrix LR,et al.Coxiella burnetii induces apoptosis during early stage infection via a caspaseindependent pathway in human monocytic THP鄄1 cells[J].PLo S One,2012,7(1):e30841.
[25]Cohen SB,Crawley JB,Kahan MC,et al.Interleukin鄄10rescues T cells from apoptotic cell death:association with an upregulation of Bcl鄄2[J].Immunology,1997,92(1):1鄄5.