微小扇头蜱P0基因的克隆及其编码蛋白的生物信息学分析

doi:10.3969/j.issn.1004-1524.20231117

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (8): 1800-1810.DOI: 10.3969/j.issn.1004-1524.20231117

微小扇头蜱P0基因的克隆及其编码蛋白的生物信息学分析

李中波¹^,²^,³(), 杨甜¹, 罗世民¹^,^*(), 黄翠琴²^,³^,^*()

1.怀化职业技术学院动物科技学院,湖南怀化 418000
2.龙岩学院生命科学学院,福建龙岩 364012
3.动物源性人兽共患病防控福建省高校工程研究中心,福建龙岩 364012

收稿日期:2023-09-21 出版日期:2024-08-25 发布日期:2024-09-06
作者简介:黄翠琴,E-mail: 13605919659@163.com
*罗世民,E-mail: 15807451608@163.com;
李中波(1990—),男,湖南新宁人,硕士,助理研究员,研究方向为病原分子生物学诊断与寄生虫学。E-mail: 18390913065@163.com
通讯作者: 罗世民,黄翠琴
基金资助:
怀化市重点实验室项目(2023R2208);湖南省教育厅优秀青年项目(23B1072);湖南省自然科学基金(2022JJ50319);动物源性人兽共患病防控福建省高校工程研究中心项目(2022K004)

Cloning of P0 gene in Rhipicephalus microplus and bioinformatics analysis of its encoded protein

LI Zhongbo¹^,²^,³(), YANG Tian¹, LUO Shimin¹^,^*(), HUANG Cuiqin²^,³^,^*()

1. College of Animal Science and Technology, Huaihua Vocational and Technical College, Huaihua 418000, Hunan, China
2. College of Life Science, Longyan University, Longyan 364012, Fujian, China
3. Engineering Research Center for the Prevention and Control of Animal Original Zoonosis, Fujian Province University, Longyan 364012, Fujian, China

Received:2023-09-21 Online:2024-08-25 Published:2024-09-06
Contact: LUO Shimin,HUANG Cuiqin

摘要/Abstract

摘要：

为探究微小扇头蜱P0基因序列特征,预测P0蛋白的理化性质和二、三级结构,筛选出P0蛋白的B、T优势抗原表位,本研究克隆了微小扇头蜱P0基因,运用Clustal X软件分析P0基因序列特征,用在线软件EXPASY、PRABI和SWISS-MODEL预测P0蛋白的理化性质和二、三级结构,用在线软件ABCpred Prediction、Scratch、IEDB和NetCTL筛选P0蛋白的B、T优势抗原表位。试验结果显示:微小扇头蜱P0基因全长957 bp,碱基A含量为24.0%,T含量为20.3%,G含量为27.5%,C含量为28.2%,A+T含量为44.3%,G+C含量为55.7%,共编码318个氨基酸;P0蛋白分子量为34 ku,理论等电点(pI)为5.86,平均亲水系数为-0.153,不稳定指数为38.15;P0蛋白的二级结构含163个α螺旋(占比51.25%),130个无规卷曲(占比40.88%),25个延伸链(占比7.86%),其中以α螺旋为主要结构;P0蛋白的三级结构以α螺旋的含量最高,该蛋白的全局模型质量评估(global model quality estimation, GMQE)、定性模型能量分析(qualitative model energy analysis, QMEAN)值分别为0.49和0.52±0.05,无信号肽和跨膜结构域,但存在40个磷酸化位点和1个糖基化位点;P0蛋白有13个B淋巴细胞优势抗原表位和6个T淋巴细胞优势抗原表位。综上所述,微小扇头蜱P0基因序列呈GC偏好,P0蛋白是以α螺旋为主要结构成分的亲水性酸蛋白,具有B、T淋巴细胞优势抗原表位,是今后研制防控微小扇头蜱疫苗的理想靶标。

关键词: 微小扇头蜱, P0基因, P0蛋白, 抗原表位

Abstract:

In order to explore the sequence characteristics of Rhipicephalus microplus P0 gene, predict the physical and chemical properties, and secondary and tertiary structures of P0 protein, and screen the B and T dominant epitopes of P0 protein, the P0 gene of R. microplus was cloned and analyzed by Clustal X software. The physical and chemical properties, secondary and tertiary structures of P0 protein were predicted by online software EXPASY, PRABI and SWISS-MODEL, and the B and T dominant epitopes of P0 protein were screened by online software ABCpred Prediction, Scratch, IEDB and NetCTL. The results showed that the P0 gene of R. microplus was 957 bp in length, with 24.0% A, 20.3% T, 27.5% G, 28.2% C, 44.3% A+T and 55.7% G+C, encoding 318 amino acids; The molecular weight of P0 protein was 34 ku, the theoretical isoelectric point (pI) was 5.86, the average hydrophilicity coefficient was -0.153, and the instability index was 38.15; The secondary structure of P0 protein contained 163 α-helices (51.25%), 130 random coils (40.88%) and 25 extended strands (7.86%), and the α-helix was the main structure; The content of α-helix in tertiary structure of P0 protein was the highest. The value of global model quality estimation (GMQE) and qualitative model energy analysis (QMEAN) of P0 protein were 0.49 and 0.52 ± 0.05, respectively. There was no signal peptide and transmembrane domain, but there were 40 phosphorylation sites and 1 glycosylation site; There were 13 B lymphocyte dominant antigen epitopes and 6 T lymphocyte dominant antigen epitopes in P0 protein. In conclusion, the P0 gene sequence of R. microplus showed GC preference, and the P0 protein was a hydrophilic acidic protein with α-helix as the main structural component, and had the dominant antigen epitopes of B and T lymphocytes, which was an ideal target for the development of R. microplus vaccine in the future.

Key words: Rhipicephalus microplus, P0 gene, P0 protein, antigen epitope

中图分类号:

S855.91

李中波, 杨甜, 罗世民, 黄翠琴. 微小扇头蜱P0基因的克隆及其编码蛋白的生物信息学分析[J]. 浙江农业学报, 2024, 36(8): 1800-1810.

LI Zhongbo, YANG Tian, LUO Shimin, HUANG Cuiqin. Cloning of P0 gene in Rhipicephalus microplus and bioinformatics analysis of its encoded protein[J]. Acta Agriculturae Zhejiangensis, 2024, 36(8): 1800-1810.

图/表 11

表1 引物序列

Table 1 Primer sequence

基因名称 Gene name	正向引物 Forward primer (5'→3')	反向引物 Reverse primer (5'→3')	产物长度 Product length/bp
cox1	ATTTTACCGCGATGAATATACTC	TAAATGAATGTTCTGAGGGTGG	1 600
ITS-2	ACATATCAAGAGAGCCTTCGG	ACAGCNGATA CATTCGNGNTTCC	1 200
P0	CGGGATCCATGGTCAGGGAGGATAAG	CCGCTCGAGATTGGGCGGTAAAAGC	1 000

图1 微小扇头蜱形态(40×) A,雌蜱假头基;B,雌蜱腹面;C,气门板;D,雄蜱背面;E,雄蜱腹面。

Fig.1 Morphology of Rhipicephalus microplus(40×) A, Female tick false head base; B, Female tick ventral surface; C, Valve plate; D, Male ticks back surface; E, Male tick ventral surface.

图2 微小扇头蜱cox1、ITS-2基因的PCR扩增结果

Fig.2 The PCR amplification results of cox1 and ITS-2 gene from Rhipicephalus microplus M,DL 2000 DNA marker; 1, cox1; 2, ITS-2.

图3 微小扇头蜱P0基因的PCR结果

Fig.3 The PCR amplification results of the P0 gene from Rhipicephalus microplus M,DL 2000 DNA marker; 1-10, RM-1-RM-10.

图4 基于P0基因序列构建的微小扇头蜱的无根遗传进化树

Fig.4 Rootless phylogenetic tree of Rhipicephalus microplus based on P0 gene sequences

图5 P0基因原核表达产物的SDS-PAGE(A)和Western-blot(B)鉴定结果 M,蛋白质marker;1,重组P0蛋白。

Fig.5 SDS-PAGE (A) and Western-blot (B) analysis of the prokaryotic expression products of P0 gene M, Protein marker; 1, Recombinant P0 protein.

图6 微小扇头蜱P0蛋白的氨基酸序列

Fig.6 Amino acid sequence of P0 protein in Rhipicephalus microplus

图7 微小扇头蜱P0蛋白的信号肽、跨膜结构域和磷酸化位点预测结果

Fig.7 Prediction results of signal peptide, transmembrane domain and phosphorylation site of P0 protein in Rhipicephalus microplus

图8 微小扇头蜱P0蛋白二级结构预测结果蓝色线条表示α螺旋;红色线条表示β折叠;紫色线条表示无规卷曲。

Fig.8 Prediction results of secondary structure of P0 protein in Rhipicephalus microplus The blue lines indicate α-helix; Red lines indicate β-sheet; Purple lines indicate random coil.

图9 微小扇头蜱P0蛋白的三级结构

Fig.9 Tertiary structure of P0 protein in Rhipicephalus microplus

图10 微小扇头蜱P0蛋白抗原表位分析结果

Fig.10 Results of the epitopes of P0 protein in Rhipicephalus microplus

参考文献 38

[1]	GOMES A F, NEVES L. Rhipicephalus microplus(Acarina, Ixodidae) in Angola: evidence of its establishment and expansion[J]. Experimental & Applied Acarology, 2018, 74(1): 117-122.
[2]	邓国藩. 中国经济昆虫志: 第十五册蜱螨目蜱总科[M]. 北京: 科学出版社, 1978.
[3]	YI J N, JIN Y C, LIU J H, et al. Genetic variation in three mitochondrial genes among cattle tick Rhipicephalus microplus originating from four provinces of China[J]. Tropical Biomedicine, 2019, 36(1): 297-303.
[4]	ESTRADA-PENA A, BOUATTOUR A, CAMICAS J L, et al. The known distribution and ecological preferences of the tick subgenus Boophilus(Acari: Ixodidae) in Africa and Latin America[J]. Experimental And Applied Acarology, 2006, 38(2/3): 219-235.
[5]	ANDERSON J F, MAGNARELLI L A. Biology of ticks[J]. Infectious Diseases Clinical, 2008, 22(2): 195-215.
[6]	徐兴莉, 杨虎. 微小牛蜱Enolase基因在昆虫细胞中的表达及鉴定[J]. 中国预防兽医学报, 2021, 43(8): 890-894.
	XU X L, YANG H. Expression and identification analysis of Enolase gene from Rhipicephalus microplus in insect cell[J]. Chinese Journal of Preventive Veterinary Medicine, 2021, 43(8): 890-894. (in Chinese with English abstract)
[7]	李中波, 尧国民, 侯强红, 等. 褐黄血蜱雌蜱不同消化阶段中肠蛋白组学分析[J]. 中国畜牧兽医, 2021, 48(2): 607-618.
	LI Z B, YAO G M, HOU Q H, et al. Proteomic profiling of the midgut contents of female Haemaphysalis flava at different digestion stage[J]. China Animal Husbandry & Veterinary Medicine, 2021, 48(2): 607-618. (in Chinese with English abstract)
[8]	FENG L L, LIU L, CHENG T Y. Proteomic analysis of saliva from partially and fully engorged adult female Rhipicephalus microplus(Acari: Ixodidae)[J]. Experimental & Applied Acarology, 2019, 78(3): 443-460.
[9]	TIRLONI L, RECK J, TERRA R M S, et al. Proteomic analysis of cattle tick Rhipicephalus(Boophilus) microplus saliva: a comparison between partially and fully engorged females[J]. PLoS One, 2014, 9(4): e94831.
[10]	XU X L, YANG H. Molecular cloning and expression analysis of enolase in the Rhipicephalus microplus(Acari: Ixodidae)[J]. Journal of Medical Entomology, 2021, 58(6): 2540-2546.
[11]	TORRENTS J, MOREL N, ROSSNER M V, et al. In vitro diagnosis of resistance of the cattle tick Rhipicephalus(Boophilus) microplus to fipronil in Argentina[J]. Experimental and Applied Acarology, 2020, 82(3): 397-403.
[12]	代艳艳, 廖秋萍, 贾桂珍, 等. 新疆南疆地区微小牛蜱Bm86基因克隆及鉴定[J]. 江苏农业科学, 2015, 43(1): 24-26.
	DAI Y Y, LIAO Q P, JIA G Z, et al. Cloning and identification of Bm86 gene of Ixodes minimus in southern Xinjiang[J]. Jiangsu Agricultural Sciences, 2015, 43(1): 24-26. (in Chinese)
[13]	刘永宏, 赵丽. 新疆南疆微小牛蜱Bm86基因及其编码蛋白剖析[J]. 中国畜牧兽医, 2014, 41(8): 45-49.
	LIU Y H, ZHAO L. Analysis of Bm86 gene and its encoding protein of Rhipicephalus microplus in South Xinjiang[J]. China Animal Husbandry & Veterinary Medicine, 2014, 41(8): 45-49. (in Chinese with English abstract)
[14]	RODRÍGUEZ-MALLON A, ENCINOSA P E, MÉNDEZ-PÉREZ L, et al. High efficacy of a 20 amino acid peptide of the acidic ribosomal protein P0 against the cattle tick, Rhipicephalus microplus[J]. Ticks and Tick-Borne Diseases, 2015, 6(4): 530-537.
[15]	ZEB I, PARIZI L F, ISRAR M, et al. Cross-species immunoprotective antigens (subolesin, ferritin 2 and P0) provide protection against Rhipicephalus sanguineus sensu lato[J]. Parasites Vectors, 2024, 17(1): 3.
[16]	GONG H Y, LIAO M, ZHOU J L, et al. Gene silencing of ribosomal protein P0 is lethal to the tick Haemaphysalis longicornis[J]. Veterinary Parasitology, 2008, 151(2/3/4): 268-278.
[17]	RODRÍGUEZ-MALLON A, ENCINOSA GUZMÁN P E, BELLO Y, et al. Efficacy of the vaccine candidate based on the P0 peptide against Dermacentor nitens and Ixodes ricinus ticks[J]. Pathogens, 2023, 12(11): 1365.
[18]	WOJDA I, CYTRYŃSKA M, FRAJNT M, et al. Protein kinases CKI and CKII are implicated in modification of ribosomal proteins of the yeast Trichosporon cutaneum[J]. Acta Biochimica Polonica, 2002, 49(4): 947-957.
[19]	RODRÍGUEZ-MALLON A, FERNÁNDEZ E, ENCINOSA P E, et al. A novel tick antigen shows high vaccine efficacy against the dog tick, Rhipicephalus sanguineus[J]. Vaccine, 2012, 30(10): 1782-1789.
[20]	CHARLES R A, BERMÚDEZ S, BANOVIĆ P, et al. Ticks and tick-borne diseases in central America and the Caribbean: a one health perspective[J]. Pathogens, 2021, 10(10): 1273.
[21]	FERNANDES É K K, BITTENCOURT V R E P, ROBERTS D W. Perspectives on the potential of entomopathogenic fungi in biological control of ticks[J]. Experimental Parasitology, 2012, 130(3): 300-305.
[22]	POURSEYED S H, TAVASSOLI M, BERNOUSI I, et al. Metarhizium anisopliae(Ascomycota: Hypocreales): an effective alternative to chemical acaricides against different developmental stages of fowl tick Argas persicus(Acari: Argasidae)[J]. Veterinary Parasitology, 2010, 172(3/4): 305-310.
[23]	UCHIUMI T, KOMINAMI R. Direct evidence for interaction of the conserved GTPase domain within 28 S RNA with mammalian ribosomal acidic phosphoproteins and L12[J]. The Journal of Biological Chemistry, 1992, 267(27): 19179-19185.
[24]	SAENZ-ROBLES M T, REMACHA M, VILELLA M D, et al. The acidic ribosomal proteins as regulators of the eukaryotic ribosomal activity[J]. Biochimica et Biophysica Acta, 1990, 1050(1/2/3): 51-55.
[25]	CANALES M, ENRÍQUEZ A, RAMOS E, et al. Large-scale production in Pichia pastoris of the recombinant vaccine Gavac against cattle tick[J]. Vaccine, 1997, 15(4): 414-422.
[26]	BEAUCLAIR L, RAMÉ C, ARENSBURGER P, et al. Sequence properties of certain GC rich avian genes, their origins and absence from genome assemblies: case studies[J]. BMC Genomics, 2019, 20(1): 734.
[27]	KANO-MURAKAMI Y, SUZUKI I, SUGIYAMA T, et al. Sequence-specific interactions of a maize factor with a GC-rich repeat in the phosphoenolpyruvate carboxylase gene[J]. Molecular & General Genetics, 1991, 225(2): 203-208.
[28]	张玉婷, 龚海燕, 周勇志, 等. 镰形扇头蜱P0基因的克隆表达及初步鉴定[J]. 畜牧与兽医, 2015, 47(5): 12-15.
	ZHANG Y T, GONG H Y, ZHOU Y Z, et al. Cloning, expression and preliminary identification of Rhipicephalus haemaphysaloides P0 gene[J]. Animal Husbandry & Veterinary Medicine, 2015, 47(5): 12-15. (in Chinese with English abstract)
[29]	RICH B E, STEITZ J A. Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly[J]. Molecular and Cellular Biology, 1987, 7(11): 4065-4074.
[30]	RODRÍGUEZ MALLÓN A, JAVIER GONZÁLEZ L, ENCINOSA GUZMÁN P E, et al. Functional and mass spectrometric evaluation of an anti-tick antigen based on the P0 peptide conjugated to Bm86 protein[J]. Pathogens, 2020, 9(6): 513.
[31]	ZHANG Y T, CUI J, ZHOU Y Z, et al. Liposome mediated double-stranded RNA delivery to silence ribosomal protein P0 in the tick Rhipicephalus haemaphysaloides[J]. Ticks and Tick-Borne Diseases, 2018, 9(3): 638-644.
[32]	GONZÁLEZ L J, ENCINOSA GUZMÁN P E, MACHADO W, et al. Synthesis, LC-MS/MS analysis, and biological evaluation of two vaccine candidates against ticks based on the antigenic P0 peptide from R. sanguineus linked to the p64K carrier protein from Neisseria meningitidis[J]. Analytical and Bioanalytical Chemistry, 2021, 413(23): 5885-5900.
[33]	RODRÍGUEZ-MALLON A, ENCINOSA GUZMÁN P E, BELLO SOTO Y, et al. A chemical conjugate of the tick P0 peptide is efficacious against Amblyomma mixtum[J]. Transboundary and Emerging Diseases, 2020, 67(Suppl 2): 175-177.
[34]	张来斌, 马光皇, 刘语涵, 等. 枣瘿蚊图尔病毒(DjTV-2a)衣壳蛋白生物信息学分析及抗原表位预测[J]. 江苏农业科学, 2022, 50(9): 33-36.
	ZHANG L B, MA G H, LIU Y H, et al. Bioinformatics analysis and epitopes prediction of capsid protein of jujube gall midge tour virus (DjTV-2a)[J]. Jiangsu Agricultural Sciences, 2022, 50(9): 33-36. (in Chinese with English abstract)
[35]	高瞻, 邵军军, 常艳燕, 等. 非洲猪瘟病毒p72蛋白抗原表位预测分析及多表位疫苗的构建[J]. 中国兽医杂志, 2020, 56(1): 13-17.
	GAO Z, SHAO J J, CHANG Y Y, et al. Epitope prediction analysis of African swine fever virus p72 protein and construction of multi-epitope vaccine[J]. Chinese Journal of Veterinary Medicine, 2020, 56(1): 13-17. (in Chinese with English abstract)
[36]	SIDI T, KEASAR C. Redundancy-weighting the PDB for detailed secondary structure prediction using deep-learning models[J]. Bioinformatics, 2020, 36(12): 3733-3738.
[37]	杨军, 刘妮, 张婷, 等. HBeAg的B细胞线性表位预测及鉴定[J]. 南方医科大学学报, 2013, 33(2): 253-257.
	YANG J, LIU N, ZHANG T, et al. Prediction and identification of B-cell linear epitopes of hepatitis B e antigen[J]. Journal of Southern Medical University, 2013, 33(2): 253-257. (in Chinese with English abstract)
[38]	POLYIAM K, RUENGJITCHATCHAWALYA M, MEKVICHITSAENG P, et al. Immunodominant and neutralizing linear B-cell epitopes spanning the spike and membrane proteins of porcine epidemic diarrhea virus[J]. Frontiers in Immunology, 2021, 12: 785293.

微小扇头蜱P0基因的克隆及其编码蛋白的生物信息学分析

Cloning of P0 gene in Rhipicephalus microplus and bioinformatics analysis of its encoded protein

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 11

参考文献 38

相关文章 4

编辑推荐

Metrics

本文评价

[1]	郭富城, 金丽, 苏强, 粟雨芯, 李方琳, 陈士恩, 马晓霞. PEDV N蛋白的原核表达纯化及B细胞抗原表位预测分析[J]. 浙江农业学报, 2020, 32(5): 762-769.
[2]	黄书伦, 郑凯文, 胡洋, 李章程, 程方俊, 宋振辉, 周作勇, 刘国林, 林春发, 林永润, 张婷婷. 鸭源大肠杆菌Ⅰ型菌毛的鉴定及FimA、FimH基因特征分析[J]. 浙江农业学报, 2017, 29(12): 1994-1999.
[3]	张琳, 余斌, 倪征, 陈柳, 云涛, 叶伟成, 华炯钢, 崔言顺, 张存. 鸭坦布苏病毒E蛋白线性化B细胞抗原表位的鉴定[J]. 浙江农业学报, 2017, 29(12): 2009-2014.
[4]	袁朋, 朱瑞良, 崔晨晨, 杨萍萍. 禽波氏杆菌HagA蛋白基因的序列分析及抗原表位预测[J]. 浙江农业学报, 2017, 29(10): 1642-1647.