西方蜜蜂Smo蛋白多克隆抗体的制备及免疫定位

doi:10.7668/hbnxb.20195605

摘要/Abstract

摘要：

Hedgehog(Hh)信号通路在多种生物的生长和发育过程中发挥重要作用,Smoothened(Smo)蛋白作为Hh信号通路的关键受体,是Hh信号转导的核心调控因子。为探究Smo蛋白在蜜蜂体内的功能及其调控机制,并深入解析蜜蜂嗅觉信号转导的分子机制,利用大肠杆菌BL21表达系统表达并纯化了Smo蛋白,并以纯化的Smo蛋白为抗原免疫新西兰白兔,制备了高活性和高特异性的Smo蛋白多克隆抗体。进一步采用免疫荧光技术检测了Smo蛋白在西方蜜蜂触角中的表达定位。结果显示,经大肠杆菌诱导表达后,重组Smo蛋白在83.72 ku处富集出明显目标条带,与预测的分子质量大小一致,表明诱导表达成功;SDS-PAGE分析结果显示,经富集纯化后,获得了高浓度的重组Smo蛋白洗脱液,表明Smo蛋白的纯化效果良好。ELISA检测结果显示,制备的Smo蛋白抗体效价达到1∶364 500,表现出较高的免疫活性。免疫荧光结果显示,Smo蛋白在西方蜜蜂触角中广泛表达,尤其在毛形感器中高表达。研究成功制备了高纯度、高效价且特异性强的Smo蛋白多克隆抗体,并明确了Smo蛋白在西方蜜蜂触角中的表达定位。

关键词: 西方蜜蜂, Smoothened(Smo)蛋白, 多克隆抗体, 免疫荧光, 毛形感器

Abstract:

Hedgehog(Hh)signaling pathway plays an important role in the growth and development of various organisms,and Smoothened(Smo)protein,as a key receptor of Hh signaling pathway,is a core regulator of Hh signal transduction.In order to investigate the function of Smo protein and its regulatory mechanism in honey bees,and to deeply analyze the molecular mechanism of olfactory signal transduction in honey bees,the Smo protein was expressed and purified by using the Escherichia coli BL21 expression system,and the purified Smo protein was used as the antigen to immunize New Zealand white rabbits,and a polyclonal antibody against Smo protein with high activity and specificity was prepared.The expression localization of Smo protein in the antennae of Apis mellifera was further detected by immunofluorescence technique.The results showed that the recombinant Smo protein was enriched at 83.72 ku after induced expression in E.coli,which was consistent with the predicted molecular weight size,indicating that the induced expression was successful;the results of SDS-PAGE analysis showed that,after enrichment and purification,a high concentration of recombinant Smo protein eluate was obtained,indicating that the Smo protein was purified well.The results of ELISA assay showed that the antibody potency of the prepared Smo protein reached 1∶364 500,showing high immunoreactivity.Immunofluorescence showed that Smo protein was widely expressed in the antennae of western honey bees,especially in the hairy sensilla.The polyclonal antibody to Smo protein was successfully prepared with high purity,high potency and specificity,and the localization of Smo protein in the antennae of western honey bees was clarified.

Key words: Apis mellifera, Smoothened(Smo), Polyclonal antibody, Immunofluorescence, Sensillum trichodeum

中图分类号:

Q511抗体蛋白与干扰素蛋白

宋畅, 张宇, 郭丽娜, 郭媛. 西方蜜蜂Smo蛋白多克隆抗体的制备及免疫定位[J]. 华北农学报, 2025, 40(3): 234-238. doi: 10.7668/hbnxb.20195605.

SONG Chang, ZHANG Yu, GUO Lina, GUO Yuan. Preparation of Smo Protein Polyclonal Antibody and Its Immunolocalization in Apis mellifera[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 234-238. doi: 10.7668/hbnxb.20195605.

http://www.hbnxb.net/CN/Y2025/V40/I3/234

图/表 7

表1 酶切及连接反应体系

Tab.1 The digestion and ligation reaction system

反应 Reaction	反应成分 Reactive component	使用量 Dosage
双酶切	PCR产物 / pET-30a(+)	1.0 μg
Double digestion	10×K Buffer	2.0 μL
	Nde Ⅰ	1.0 μL
	Xho Ⅰ	1.0 μL
	0.1% BSA	2.0 μL
	DNase/RNase-free water	补至20.0 μL
连接	目的片段酶切产物	0.1 μg
Ligation	pET-30a(+)酶切产物	0.1 μg
	T₄ DNA连接酶	2.0 μL
	2×Rapid Ligation Buffer	10.0 μL
	DNase/RNase-free water	补至20.0 μL

图1 重组表达质粒序列与目的蛋白序列比对结果

Fig.1 Alignment of the recombinant expression plasmid sequence and the target protein sequence

图2 Smo重组蛋白的诱导表达鉴定 M.蛋白Marker;1.pET-30a(+)诱导(载体);2.未诱导;3.诱导后;4.诱导后破碎上清;5.诱导后破碎沉淀。

Fig.2 Identification and induced expression of Smo recombinant protein M.Protein Marker;1.pET-30a(+)induced(vector);2.Uninduced;3.After induction;4.Supernatant after induction and fragmentation;5.Precitation after induction and fragmentation.

图3 Smo蛋白纯化后的SDS-PAGE电泳分析 M.蛋白质Marker;1.破碎后处理样品;2.流出样品;3—5.洗脱后样品。

Fig.3 SDS-PAGE electrophoretic analysis of Smo protein purification M.Protein Marker;1.Post-crushing treatment sample;2.Effluent;3—5.Eluent.

图4 Smo 蛋白质检SDS电泳分析 M.蛋白Marker;1.0.5 mg/mL BSA;2.纯化后的Smo重组蛋白。

Fig.4 Smo protein detection and analysis by SDS electrophoresis M.Protein Marker;1.0.5 mg/mL BSA; 2.Purified smoothened recombinant protein.

表2 抗体的效价检测结果

Tab.2 The result of antibody potency testing

编号 Number	稀释比例 Dilution ratio	Smo纯化抗体 450 nm处OD值 OD value at 450 nm of the purified antibody
1	500	4.000
2	1 500	4.000
3	4 500	3.648
4	13 500	3.648
5	40500	2.369
6	121 500	1.444
7	364 500	0.583
8	1 093 500	0.346
9	3 280 500	0.293
10	9 841 500	0.283
11	29 524 500	0.271
12	空白	0.258

图5 Smo在西方蜜蜂触角组织定位表达分析

Fig.5 Results of Smo expression and localization in the tentacle tissue of A.mellifera

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