Preparation of Smo Protein Polyclonal Antibody and Its Immunolocalization in Apis mellifera

doi:10.7668/hbnxb.20195605

Abstract

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

CLC Number:

Q511抗体蛋白与干扰素蛋白

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.

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Figures/Tables 7

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

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

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.

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

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

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

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

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