边缘革蜱Dm 86基因表达及免疫原性分析

doi:10.6048/j.issn.1001-4330.2022.09.029

摘要/Abstract

摘要：

【目的】 研究Bm86同系物Dm86基因及其编码蛋白，为抗边缘革蜱疫苗候选抗原提供基础。【方法】 以边缘革蜱饱血雌蜱cDNA为模板扩增克隆Dm86基因，分析生物学特性及生活史各阶段表达水平，截短基因构建重组质粒，经IPTG诱导后SDS-PAGE鉴定，纯化蛋白，制备多克隆抗体进行Western Blotting 鉴定。【结果】 PCR扩增获得了1 773 bp目的片段，Dm86蛋白由591个氨基酸组成，分子量为64 857.81，理论等电点为6.78，酸性，具有不稳定性、亲水性和多个B细胞抗原表位；Dm86基因在边缘革蜱不同阶段均有不同程度的表达，饱血蜱阶段的表达量较高；重组蛋白大小为39kDa，以包涵体形式表达；抗体效价可达1∶25 600，能够识别重组蛋白。【结论】 Dm86重组蛋白具有一定的反应原性和免疫原性。

关键词: 边缘革蜱; Dm86基因; 生物信息学分析; 荧光定量PCR; 原核表达; Western blot

Abstract:

【Objective】 To understand Dm86 gene and its coding protein of Bm86 homologue，provide the basis for anti-Dermacenter marginatus vaccine candidate antigen.【Method】 this experiment amplified the Dm86 gene using the cDNA of fulminated female ticks，truncated genes to construct recombinant plasmids，SDS-PAGE identified after IPTG induction，purified protein and prepared polyclonal antibodies for Western Blotting identification. 【Result】 The target fragment of 1，773 bp was obtained by PCR amplification. According to the analysis of biological characteristics，Dm86 protein was presumed to be composed of 591 amino acids，with molecular weight of 64857.81 and theoretical isoelectric point of 6.78. It was acidic，had unstable hydrophilicity and multiple B cell epitopes. Fluorescence quantitative PCR results showed that Dm86 gene was expressed to different degrees in different stages of ticks，and the expression level was higher in ticks at satiety stage. High antigenicity truncated gene building proteins. SDS-PAGE results showed that specific bands appeared around 39 kDa and expressed as an inclusion body. The titer of polyclonal antibody was 1∶25600，Western blot results show that，the recombinant protein can react with polyclonal antibody to produce specific bands. 【Conclusion】 Recombinant protein Dm86 has certain reactivity and immunogenicity，which can lay the foundation for the study of candidate antigens of vaccine against D. marginatus.

Key words: D. marginatus; Dm86; bioinformatics analysis; fluorescence quantitative PCR; prokaryotic expression; western blot

中图分类号:

S188

李敏, 马英, 呼尔查, 何文文, 史倩云, 阿力木江·加帕尔, 蒋倩, 巴音查汗. 边缘革蜱Dm 86基因表达及免疫原性分析[J]. 新疆农业科学, 2022, 59(9): 2324-2332.

LI Min, MA Ying, Huercha , HE Wenwen, SHI Qianyun, Alimujiang Jiapaer, JIANG Qian, Bayinchahan . Expression of Dm86 Gene of Dermacenter Marginatus and Analysis of Immunogenicity[J]. Xinjiang Agricultural Sciences, 2022, 59(9): 2324-2332.

图/表 10

表 1 Dm86基因PCR引物

Table 1 Dm86 gene PCR primer

引物名称 Prime name	引物序列（5'→3'） Primer sequence（5'→3'）	片段长度 Fragment length（bp）
Dm86-out-F	5′-TAAGGTCAAGGATTTATTCTG-3′	1 773
Dm86-out-R	5′-TTAAGCACTTGATGGTCCAGGATC-3′	1 773
Dm86-q-F	5′-ACCAGCAACAAACGAGG-3′	165
Dm86-q-R	5′-GGTCCAGGATCTGAACAAC-3′	165
Dm86-ep-F	5′-CCGGAATTCATGGAAAACGAAGAATCAAAGTG-3′	345
Dm86-ep-R	5′-CCGCTCGAGTTAAGCACTTGATGGTCCAGGATC-3′	345

图 1 边缘革蜱Dm86基因PCR扩增注：M：DNA 标准DL2000；1-4：克隆引物扩增结果；5：阴性对照

Fig. 1 Result of PCR amplification of Dm86 gene of D.marginatus Note：M：DNA standard DL 2000；1：PCR products of Dm86；2：Negative control

图 2 边缘革蜱Dm86基因的系统发育

Fig. 2 Phylogenetic analysis of Dm86 gene of D.marginatus

图 3 生物信息学注：A：亲水性； B：信号肽； C：二级结构；D：B细胞抗原表位；E：N端糖基化位点； F：三级结构

Fig. 3 Analysis of Protein Biology Note：A： hydrophilism； B： signal peptide； C： Secondary structure； D：B cell antigen epitopes；E： N glycosylation site ； F：Ttertiary structure

图 4 各发育周期相对表达量注：ns，差异不显著（P>0.05）；*：差异显著（P<0.05）

Fig. 4 Relative expression levels of each developmental period Note:ns: No significant difference（P>0.05）；*：Significant difference（P<0.05）

图 5 吸血期相对表达量注：ns，差异不显著（P>0.05）；***:差异极显著

Fig. 5 Relative expression level in the bloodsucking stage Note:ns: No significant difference（P>0.05）；***：very significantly different（P<0.001）

图 6 边缘革蜱截短Dm86基因PCR扩增结果注：M：DNA 标准DL2000； 1: 阴性对照；2：Dm86基因PCR产物

Fig. 6 Result of PCR amplification of Dm86 gene of D.marginatus Note：M：DNA standard DL 2000； 1: Negative control； 2：PCR products of Dm86 gene

图 7 Dm86重组质粒酶切验证注：M：DNA 标准DL8 000；1：Dm86-pGEX-4T-1 双酶切产物

Fig. 7 Dm86 recombinant plasmid enzyme digestion validation Note：M：DNA standard DL 8000；1：Products of Dm86-pGEX-4T-1 by restriction enzymes digestion

图 8 重组蛋白SDS-PAGE 注：M：120 kDa Ladder ；1：pGEX-4T-1；2：诱导前；3：诱导后沉淀；4：诱导后上清；5：纯化后上清

Fig. 8 SDS-PAGE of protein Note：M：120 kDa Ladder；1：pGEX-4T-1；2：before induction；3：precipitation after induction；4：supernatant after induction；5：supernatant after purification

图 9 蛋白多克隆抗体的Westernblotting分析注：M：120 kDa Ladder； 1：兔抗血清； 2：阴性对照

Fig. 9 Western blotting analysis of polyclonalantibody against Dm86 protein Note：M：120 kDa Ladder； 1：Rabbit antiserum； 2：negative control

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