具有抗肿瘤和抗菌活性的长效改性蜂毒肽(GPG)设计及其生物活性评价

doi:10.11924/j.issn.1000-6850.casb20200200116

摘要/Abstract

摘要：

为获得低毒、具有抗肿瘤和抗菌活性的长效改性蜂毒肽,提高蜂毒肽的临床应用价值,本试验以多肽构效关系为指导,选择两亲性α-螺旋型阳离子多肽分子结构为改性蜂毒肽的设计模板,用杂合肽法设计了一系列多肽序列,用生物信息学工具比较它们的生物活性并进行初选,再用固相法合成初选所得少量序列的多肽,比对合成多肽的生物活性,精选获得生物活性高的多肽。实验表明精选所得多肽具有长效性,确定其为设计目标肽(GPG),当GPG浓度为150 μmol/L时,其溶血率为零;浓度为33.3 μmol/L时,其对小鼠肝癌细胞(H₂₂)、人肝癌细胞(SMMC-7721)和人结肠癌细胞(SW-1116)的抑癌率分别为(78.0±0.6)%、(74.0±1.2)%、(74.0±0.5)%;浓度为100 μmol/L时,其对大肠杆菌的抑菌圈直径为(15.18±0.30) mm;当注射GPG（100 μmol/只）时,能延长致死剂量大肠杆菌（5×10⁷个/只）攻毒的小鼠寿命63.2 h。该设计省时省力地优选得溶血活性低、抗癌抗菌活性强、半衰期长的全新结构目标肽GPG,其有临床应用前景;以GRGDSP肽链为多肽氮端端基,可提高其生物活性及半衰期。

关键词: 改性蜂毒肽, 长效肽, 分子设计, 抗肿瘤活性, 抗菌活性

Abstract:

The purpose is to improve the clinical application value of melittin. A series of polypeptide sequences were designed by hybridpeptide method. Based on the structure-activity relationship,we used the molecular structure of amphiphilic α-helix cationic peptide as the design template, to get the long-effective modified melittin with low toxicity, antitumor and antibacterial activity. Their bioactivities were compared and screened first by bioinformatics tools, then chemical synthesis was performed, and the bioactivities of the synthetic peptides were compared to obtain the designed target peptide (GPG). The experiment showed that GPG had long-term efficacy. When the concentration of GPG was 150 μmol/L, its hemolytic ratio was still zero, when the concentration was 33.3 μmol/L, the tumor inhibition ratio of H₂₂, SMMC-7721 and SW-1116 tumor cells was (78.0±0.6)%, (74.0±1.2)% and (74.0±0.5)%, respectively. When the concentration was 100 μmol/L, the diameter of inhibition zone on Escherichia coli was (15.18±0.30) mm. When GPG (100 μmol per mouse) was injected, the life prolongation period of the lethal dose of Escherichia coli was 63.2 h. The above design can be time-saving and labor-saving, so that the target peptide (GPG) can be obtained, with low hemolyticity, strong anticancer and antibacterial activities, and long half-life. The result indicates that GPG is a whole new polypeptide series with clinical application prospect, and GRGDSP chain could be used as N terminal domain of modified melittin and its biological activities and half-life are improved.

Key words: modified melittin, long-effective peptide, molecular design, antitumor activity, antibacterial activity

中图分类号:

S896.5

叶若柏, 吴珍红, 缪晓青. 具有抗肿瘤和抗菌活性的长效改性蜂毒肽(GPG)设计及其生物活性评价[J]. 中国农学通报, 2020, 36(16): 34-41.

Ye Ruobai, Wu Zhenhong, Miao Xiaoqing. Design and Bioactivity Evaluation of Long-effective Modified Melittin (GPG) with Antitumor and Antibacterial Function[J]. Chinese Agricultural Science Bulletin, 2020, 36(16): 34-41.

图/表 6

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设计序列号	设计多肽的氨基酸序列	简记	整体结构参数
设计序列号	设计多肽的氨基酸序列	简记	净正电荷数	疏水力矩	疏水性	α-螺旋度	两亲性
1	RGDSFLHLAKKFGKAFPAVLKVLTTG		+4	0.420	0.320	50.0	是
2	GRGDSPFLHLAKKFGKAFPAVLKVLTTG		+4	0.557	0.596	66.7	是
3	GRGDSPSFLHLAKKPGKAFPAVLKVLTTG		+4	0.572	0.526	61.1	是
4	RGDFLHLAKKFGKAFPAVLKVLTTG		+4	0.557	0.596	66.7	是
5	GRGDSPKFLHSAKKFGKAFPAVLKVLTTG	GPG	+5	0.565	0.377	58.0	是
6	GRGDSPKFLHLAKKFGKAFIGIAVLKVLTTG		+5	0.451	0.594	38.9	是
7	GRGDSPKFLHSAKKFGKAFIGIAVLKVLTTG		+5	0.439	0.497	44.4	是
8	RGDKFLHLAKKFGKAFIGIAVLKVLTTG		+5	0.451	0.594	38.9	是
9	RGDKFLHSAKKFGKAFIGIAVLKVLTTG		+5	0.439	0.497	55.6	是
10	RGDKFLHLAKKFGKAFPAVLKVLTTG		+5	0.589	0.473	38.9	是
11	GRGDSPKFLHLAKKFGKAFPAVLKVLTTG		+5	0.589	0.473	38.9	是
12	GRGDSPKWKLFKKIPAVLKVLTTG		+5	0.395	0.554	44.4	是
13	RGDKWKLFKKIEKVGQPAVLKVLTTGL		+5	0.283	0.328	50.0	是
14	GRGDSPKWKLFKKIEKVGQPAVLKVLTTGL		+5	0.283	0.328	50.0	是
15	GRGDSPKFLHSAKKFKAFMMEAVLKVLTT		+5	0.239	0.528	61.1	是
16	RGDKFLHSAKKFKAFMMEAVLKVLTT		+5	0.214	0.432	44.4	是
17	RGDKWKLFKKIPKFLHLAKKF	RP18	+7	0.333	0.332	52.4	是
18	RGDKWKLFKKIIGIKFLHSAKKF		+7	0.202	0.505	50.0	是
19	GRGDSPKWKLFKKIIGIKFLHLAKKF		+7	0.298	0.602	44.4	是
20	RGDKFLHSAKKFGKAFPAVLKVLTTG	RPG	+5	0.392	0.448	50.0	是
21	RGDKWKLFKKIPAVLKVLTTG	RCAG	+5	0.395	0.554	44.4	是

设计序列号	设计多肽的氨基酸序列	简记	整体结构参数
设计序列号	设计多肽的氨基酸序列	简记	净正电荷数	疏水力矩	疏水性	α-螺旋度	两亲性
1	RGDSFLHLAKKFGKAFPAVLKVLTTG		+4	0.420	0.320	50.0	是
2	GRGDSPFLHLAKKFGKAFPAVLKVLTTG		+4	0.557	0.596	66.7	是
3	GRGDSPSFLHLAKKPGKAFPAVLKVLTTG		+4	0.572	0.526	61.1	是
4	RGDFLHLAKKFGKAFPAVLKVLTTG		+4	0.557	0.596	66.7	是
5	GRGDSPKFLHSAKKFGKAFPAVLKVLTTG	GPG	+5	0.565	0.377	58.0	是
6	GRGDSPKFLHLAKKFGKAFIGIAVLKVLTTG		+5	0.451	0.594	38.9	是
7	GRGDSPKFLHSAKKFGKAFIGIAVLKVLTTG		+5	0.439	0.497	44.4	是
8	RGDKFLHLAKKFGKAFIGIAVLKVLTTG		+5	0.451	0.594	38.9	是
9	RGDKFLHSAKKFGKAFIGIAVLKVLTTG		+5	0.439	0.497	55.6	是
10	RGDKFLHLAKKFGKAFPAVLKVLTTG		+5	0.589	0.473	38.9	是
11	GRGDSPKFLHLAKKFGKAFPAVLKVLTTG		+5	0.589	0.473	38.9	是
12	GRGDSPKWKLFKKIPAVLKVLTTG		+5	0.395	0.554	44.4	是
13	RGDKWKLFKKIEKVGQPAVLKVLTTGL		+5	0.283	0.328	50.0	是
14	GRGDSPKWKLFKKIEKVGQPAVLKVLTTGL		+5	0.283	0.328	50.0	是
15	GRGDSPKFLHSAKKFKAFMMEAVLKVLTT		+5	0.239	0.528	61.1	是
16	RGDKFLHSAKKFKAFMMEAVLKVLTT		+5	0.214	0.432	44.4	是
17	RGDKWKLFKKIPKFLHLAKKF	RP18	+7	0.333	0.332	52.4	是
18	RGDKWKLFKKIIGIKFLHSAKKF		+7	0.202	0.505	50.0	是
19	GRGDSPKWKLFKKIIGIKFLHLAKKF		+7	0.298	0.602	44.4	是
20	RGDKFLHSAKKFGKAFPAVLKVLTTG	RPG	+5	0.392	0.448	50.0	是
21	RGDKWKLFKKIPAVLKVLTTG	RCAG	+5	0.395	0.554	44.4	是

多肽浓度	GPG	RPG	RP18	RCAG
150.00 μmol/L	0	0	4.0±0.2	0
75.00 μmol/L	0	0	1.8±0.2	0
37.50 μmol/L	0	0	0.2±0.1	0
18.75 μmol/L	0	0	0	0
9.38 μmol/L	0	0	0	0
4.69 μmol/L	0	0	0	0

多肽浓度	GPG	RPG	RP18	RCAG
150.00 μmol/L	0	0	4.0±0.2	0
75.00 μmol/L	0	0	1.8±0.2	0
37.50 μmol/L	0	0	0.2±0.1	0
18.75 μmol/L	0	0	0	0
9.38 μmol/L	0	0	0	0
4.69 μmol/L	0	0	0	0

多肽浓度	对H₂₂抑癌率				对SMMC-7721抑癌率				对SW-1116抑癌率
多肽浓度	GPG	RPG	RP18	RCAG	GPG	RPG	RP18	RCAG	GPG	RPG	RP18	RCAG
33.3 μmol/L	78.0±0.6	74.0±1.4	71.0±1.4	70.0±1.0	74.0±1.2	72.0±1.3	71.0±1.6	70.0±1.3	74.0±0.5	72.0±1.6	72.0±1.5	71.0±1.6
16.7 μmol/L	56.0±0.9	51.3±1.2	48.5±1.1	48.1±1.1	51.0±1.6	49.6±1.5	47.2±1.5	46.7±0.8	50.0±1.5	48.3±1.2	48.5±1.4	47.9±1.4
8.3 μmol/L	32.0±1.4	26.5±1.4	24.7±1.4	24.4±0.8	25.0±1.3	24.2±1.4	22.3±1.4	21.9±1.4	22.5±1.4	20.6±1.5	22.0±1.4	21.9±1.7
4.2 μmol/L	20.1±1.6	15.4±1.5	12.6±1.0	12.4±1.0	13.5±1.0	11.7±1.2	10.5±1.3	10.3±1.4	10.7±0.6	9.8±0.8	11.1±0.5	11.0±0.8
2.1 μmol/L	3.0±1.1	1.7±0.8	0.9±0.6	0.8±0.4	1.2±0.6	0.8±0.6	0.7±0.4	0.7±0.5	0.8±0.4	0.6±0.3	0.7±0.3	0.7±0.7