HMGN2分子的分离纯化及其抗乙型肝炎病毒活性研究
摘要
高迁移率组蛋白N2(High Mobility Group Chromosal protein N2，HMGN2)是脊椎动物和非脊椎动物的细胞核中普遍存在的非组蛋白，目前对其功能还不完全清楚。在本实验室从人LAK细胞和人宫颈黏液中分离纯化寻找抗菌肽过程中，发现HMGN2具有较强的抗革兰氏阴性菌的作用，为进一步深入研究HMGN2的生物学新功能，本文重点研究其体外抗乙型肝炎病毒(Hepatitis B virus，HBV)的功能。
【目的】1．从人单核细胞株THP-1中分离与纯化HMGN2，探讨HMGN2分子的抗菌作用。2．应用HepG2.2.15细胞株作为体外抗乙型肝炎病毒的实验模型，对HMGN2分子是否抑制乙肝病毒表面抗原(HBsAg)、e抗原(HBeAg)和HBV DNA的分泌、抑制程度作初步探讨，为寻求新的抗HBV药物开辟途径。
【方法】大量培养人单核细胞株THP-1细胞，收集THP-1细胞沉淀，加入适量5％乙酸，在冰浴中电动匀浆，收集上清液，经透析除去乙酸、冷冻干燥得到THP-1细胞的酸溶性提取物。酸溶性提取物经RP-HPLC分离，用琼脂糖弥散抗菌法筛选纯化组分的抗菌活性，对活性组分进行Tricine-SDS-PAGE电泳，AU-PAGE电泳分析，得到纯化的HMGN2。
以HBV DNA转染的细胞株HepG2.2.15细胞为模型，HMGN2作用后检测细胞培养液中的HBsAg、HBeAg和HBV DNA，对HMGN2抗HBV效果进行评价。在HepG2.2.15细胞培养基中分别加入不同浓度的HMGN2蛋白，培养的第4天、第8天收集细胞培养上清液，用酶联免疫吸附测定技术(ELISA)检测上清液中HBsAg和HBeAg含量，用荧光定量PCR技术检测细胞培养上清液中HBV DNA的变化。
【结果】THP-1细胞的酸溶性粗提物通过HPLC分离，在第21分钟出峰时间(保留时间)纯化出一个分子(即HMGN2)，该分子对大肠杆菌氨苄青霉素耐药株ML-35p有较强的抑菌活性。琼脂糖弥散法检测显示HMGN2对大肠杆菌标准株ATCC25922和临床分离株54080有较强的抗菌活性，而对金黄色葡萄球菌ATCC25923及绿脓杆菌ATCC27853未检测到抗菌活性。HMGN2在本实验浓度范围内有抑制HBV增殖作用，表现为培养上清中HBsAg、HBeAg分泌减少，以及细胞上清液中HBV DNA水平降低，HMGN2抑制HBV以无血清条件下效果更明显，浓度为2μg／ml抗病毒效果达最大，可使HBV DNA拷贝数减少95.4％，继续增加剂量抗病毒效果不再增加。与人中性粒细胞α-防御素相比，HMGN2对HBV抑制作用更明显。
【结论】HMGN2分子具有抗革兰氏阴性菌作用。HMGN2在2μg／ml水平可显著抑制HBVeAg和HBVsAg的表达，可降低HBV DNA拷贝数，且存在量效关系，证明HMGN2在体外细胞培养中具有直接抗HBV活性。
The high mobility group protein N2 (HMGN2) is one of the most abundant and well characterized nonhistone nuclear proteins which are found in the cell nucleus of vertebrate and invertebrate organisms. However, the biological role of this protein has not been fully defined. Recent work from our lab has shown that the human HMGN2 purified from IL-2-stimulated peripheral blood mononuclear leukocytes had antimicrobial activity against gram negative bacteria and HMGN2 may be a novel antibacterial effector molecule. To further understand the biological function of HMGN2, in the present study we investigated the anti-HBV effect of human HMGN2.
Objective: 1. To isolate and purify antibacterial peptide HMGN2 from human monocyte THP-1 cells and determine its antimicrobial activity. 2. In order to develop new modality to treat hepatitis B infection, HepG2.2.15 cell line was used as an in vitro experimental model to study the anti-HBV activity of HMGN2. The level of HBsAg, HBeAg and HBV DNA in the supernatant of the HMGN2-treated HepG2.2.15 cells was assayed.
Methods: Human monocyte cell line THP-1 cells were cultured in RPMI-1640 medium with 20% fetal bovine serum and collected by centrifugation. Acid-soluble components from THP-1 cells were prepared by homogenizing the cell pellet in 5% acetic acid. The supernatant was collected and dialyzed against water and lyophilized.
The Acid-soluble components were subjected to RP-HPLC fractionation. The antimicrobial activities of the RP-HPLC fractions were determined by the agar radial diffusion assay and AU-PAGE and Tricine-SDS-PAGE were used to characterize the HMGN2 molecule.
To investigate the in vitro anti-HBV effect of HMGN2, the HepG2.2.15 cells were treated with HMGN2 at a variety of concentrations. After inoculation for 4 days or 8 days, Supernatant of HMGN2-treated HepG2.2.15 cells were collected. The concentrations of HBsAg and HBeAg in the supernatant were measured by ELISA and the replication level of HBV DNA was detected with real-time quantitative PCR.
Results: The agar radial diffusion assay indicated that the RP-HPLC fraction eluted at 21 min (fraction 21) had potent antimicrobial activity against E.coli ML-35p, and to some extent against E. Coli ATCC 25922 and clinical isolate 54080. However, this fraction was inactive against S. aureus ATCC25923 and E aeruginosa ATCC 27853 in this system. Analyzed by Tricine-SDS-PAGE, there was only one protein band in the fraction 21 which had an apparent molecular weight of approximately 20kDa. The biological characteristics of the protein in the fraction 21(RP-HPLC retention time, antimicrobial activities and apparent molecular weight) were identical to the previously reported characteristics of HMGN2.
After incubation with HMGN2 at a dose of 2ug/ml for 4 days, the concentrations of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells decreased by 30% and 40.8% respectively. HMGN2 also inhibited the replication of hepatitis B virus in HepG2.2.15 cells. After exposed to 2ug/ml of HMGN2 for 8 days, the level of extracellular HBV DNA in the supernatant of HepG2.2.15 cells decreased by 95.4%. The anti-HBV effect of HMGN2 was dose-dependent and the presence of serum weakened the anti-HBV activity. Although human neutrophilα-defensins showed anti-HBV activity, the potency of HMGN2 against HBV was more effective thanα-defensins.
Conclusion: HMGN2 had potent antimicrobial activity against Gram-negative bacteria. Besides, in the presence of 2ug/ml of HMGN2, the level of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells decreased significantly and the replication of hepatitis B virus in HepG2.2.15 cells is also inhibited. These results suggest that HMGN2 has direct anti-HBV effect in vitro.
【目的】1．从人单核细胞株THP-1中分离与纯化HMGN2，探讨HMGN2分子的抗菌作用。2．应用HepG2.2.15细胞株作为体外抗乙型肝炎病毒的实验模型，对HMGN2分子是否抑制乙肝病毒表面抗原(HBsAg)、e抗原(HBeAg)和HBV DNA的分泌、抑制程度作初步探讨，为寻求新的抗HBV药物开辟途径。
【方法】大量培养人单核细胞株THP-1细胞，收集THP-1细胞沉淀，加入适量5％乙酸，在冰浴中电动匀浆，收集上清液，经透析除去乙酸、冷冻干燥得到THP-1细胞的酸溶性提取物。酸溶性提取物经RP-HPLC分离，用琼脂糖弥散抗菌法筛选纯化组分的抗菌活性，对活性组分进行Tricine-SDS-PAGE电泳，AU-PAGE电泳分析，得到纯化的HMGN2。
以HBV DNA转染的细胞株HepG2.2.15细胞为模型，HMGN2作用后检测细胞培养液中的HBsAg、HBeAg和HBV DNA，对HMGN2抗HBV效果进行评价。在HepG2.2.15细胞培养基中分别加入不同浓度的HMGN2蛋白，培养的第4天、第8天收集细胞培养上清液，用酶联免疫吸附测定技术(ELISA)检测上清液中HBsAg和HBeAg含量，用荧光定量PCR技术检测细胞培养上清液中HBV DNA的变化。
【结果】THP-1细胞的酸溶性粗提物通过HPLC分离，在第21分钟出峰时间(保留时间)纯化出一个分子(即HMGN2)，该分子对大肠杆菌氨苄青霉素耐药株ML-35p有较强的抑菌活性。琼脂糖弥散法检测显示HMGN2对大肠杆菌标准株ATCC25922和临床分离株54080有较强的抗菌活性，而对金黄色葡萄球菌ATCC25923及绿脓杆菌ATCC27853未检测到抗菌活性。HMGN2在本实验浓度范围内有抑制HBV增殖作用，表现为培养上清中HBsAg、HBeAg分泌减少，以及细胞上清液中HBV DNA水平降低，HMGN2抑制HBV以无血清条件下效果更明显，浓度为2μg／ml抗病毒效果达最大，可使HBV DNA拷贝数减少95.4％，继续增加剂量抗病毒效果不再增加。与人中性粒细胞α-防御素相比，HMGN2对HBV抑制作用更明显。
【结论】HMGN2分子具有抗革兰氏阴性菌作用。HMGN2在2μg／ml水平可显著抑制HBVeAg和HBVsAg的表达，可降低HBV DNA拷贝数，且存在量效关系，证明HMGN2在体外细胞培养中具有直接抗HBV活性。
The high mobility group protein N2 (HMGN2) is one of the most abundant and well characterized nonhistone nuclear proteins which are found in the cell nucleus of vertebrate and invertebrate organisms. However, the biological role of this protein has not been fully defined. Recent work from our lab has shown that the human HMGN2 purified from IL-2-stimulated peripheral blood mononuclear leukocytes had antimicrobial activity against gram negative bacteria and HMGN2 may be a novel antibacterial effector molecule. To further understand the biological function of HMGN2, in the present study we investigated the anti-HBV effect of human HMGN2.
Objective: 1. To isolate and purify antibacterial peptide HMGN2 from human monocyte THP-1 cells and determine its antimicrobial activity. 2. In order to develop new modality to treat hepatitis B infection, HepG2.2.15 cell line was used as an in vitro experimental model to study the anti-HBV activity of HMGN2. The level of HBsAg, HBeAg and HBV DNA in the supernatant of the HMGN2-treated HepG2.2.15 cells was assayed.
Methods: Human monocyte cell line THP-1 cells were cultured in RPMI-1640 medium with 20% fetal bovine serum and collected by centrifugation. Acid-soluble components from THP-1 cells were prepared by homogenizing the cell pellet in 5% acetic acid. The supernatant was collected and dialyzed against water and lyophilized.
The Acid-soluble components were subjected to RP-HPLC fractionation. The antimicrobial activities of the RP-HPLC fractions were determined by the agar radial diffusion assay and AU-PAGE and Tricine-SDS-PAGE were used to characterize the HMGN2 molecule.
To investigate the in vitro anti-HBV effect of HMGN2, the HepG2.2.15 cells were treated with HMGN2 at a variety of concentrations. After inoculation for 4 days or 8 days, Supernatant of HMGN2-treated HepG2.2.15 cells were collected. The concentrations of HBsAg and HBeAg in the supernatant were measured by ELISA and the replication level of HBV DNA was detected with real-time quantitative PCR.
Results: The agar radial diffusion assay indicated that the RP-HPLC fraction eluted at 21 min (fraction 21) had potent antimicrobial activity against E.coli ML-35p, and to some extent against E. Coli ATCC 25922 and clinical isolate 54080. However, this fraction was inactive against S. aureus ATCC25923 and E aeruginosa ATCC 27853 in this system. Analyzed by Tricine-SDS-PAGE, there was only one protein band in the fraction 21 which had an apparent molecular weight of approximately 20kDa. The biological characteristics of the protein in the fraction 21(RP-HPLC retention time, antimicrobial activities and apparent molecular weight) were identical to the previously reported characteristics of HMGN2.
After incubation with HMGN2 at a dose of 2ug/ml for 4 days, the concentrations of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells decreased by 30% and 40.8% respectively. HMGN2 also inhibited the replication of hepatitis B virus in HepG2.2.15 cells. After exposed to 2ug/ml of HMGN2 for 8 days, the level of extracellular HBV DNA in the supernatant of HepG2.2.15 cells decreased by 95.4%. The anti-HBV effect of HMGN2 was dose-dependent and the presence of serum weakened the anti-HBV activity. Although human neutrophilα-defensins showed anti-HBV activity, the potency of HMGN2 against HBV was more effective thanα-defensins.
Conclusion: HMGN2 had potent antimicrobial activity against Gram-negative bacteria. Besides, in the presence of 2ug/ml of HMGN2, the level of HBsAg and HBeAg in the supernatant of HepG2.2.15 cells decreased significantly and the replication of hepatitis B virus in HepG2.2.15 cells is also inhibited. These results suggest that HMGN2 has direct anti-HBV effect in vitro.
引文
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