蒙古牛皮肤Qa-1b/NKG2A的表达及Qa-1b纳米抗体的制备和功能鉴定

doi:10.11843/j.issn.0366-6964.2025.01.037

摘要/Abstract

摘要：

本研究旨在通过检测蒙古牛皮肤Qa-1b/NKG2A的表达特征，分析其参与皮肤免疫的功能，及Qa-1b在抗黑素瘤发展中可能发挥的作用。通过实时定量PCR、Western blot、免疫组织化学方法以及免疫共沉淀法对Qa-1b/NKG2A在皮肤中的表达进行相对定量和定位以及互作关系分析；以Qa-1b多肽为抗原，使用噬菌体展示技术，从羊驼黑色素瘤细胞纳米抗体文库中进行淘筛，获得结合性较强的纳米抗体(Qa-1b-VHH)序列；构建pET28a-Qa-1b-VHH原核表达重组质粒，通过IPTG诱导表达和镍柱纯化，获得Qa-1b纳米抗体。经ELISA检测Qa-1b纳米抗体与抗原的特异性后，将Qa-1b纳米抗体应用于Western blot和免疫组织化学方法检测组织中Qa-1b的表达，以及添加到B16黑素瘤细胞培养基中，通过CCK8法、细胞划痕法和Western blot法检测其对B16细胞增殖及迁移的影响以及与增殖和迁移相关蛋白表达情况。结果显示：Qa-1b和NKG2A在蒙古牛皮肤组织的毛囊外根鞘和血管内皮中表达，在犊牛皮肤中的表达量显著高于成年牛(P＜0.01或P＜0.001)。且Qa-1b和NKG2A在皮肤中存在互作关系。所获得的Qa-1b纳米抗体特异性较强，可用作Western blot和免疫组织化学中的一抗检测其他组织中Qa-1b的表达；Qa-1b纳米抗体添加到B16细胞培养基中，应用CCK 8法和细胞划痕法检测该Qa-1b纳米抗体对B16细胞增殖和迁移有明显的抑制效果，细胞增殖过程中Ras、MEK1、CyclinD1、CDK4蛋白的表达显著下调(P＜0.01或P＜0.001)。本试验揭示了Qa-1b/NKG2A参与皮肤免疫以保证毛囊正常生长，尤其是犊牛；Qa-1b纳米抗体可抑制B16细胞增殖和迁移，为增强皮肤免疫及抗黑素瘤发展的抗体药物提供理论依据。

关键词: Qa-1b, NKG2A, 纳米抗体, 皮肤免疫, 黑素瘤, B16细胞, 蒙古牛

Abstract:

To detect the expression of Qa-1b/NKG2A in skins of Mongolia cattle for analyzing the potential roles in skin immunity and inhibition of development of melanoma cells of Qa-1b. The expression of Qa-1b/NKG2A in skins was quantitatively analyzed by quantitative real-time PCR, Western blot and immunohistochemistry, and by co-immunoprecipitation for interaction between Qa-1b and NKG2A. The sequence of Qa-1b nanoloody (Qa-1b-VHH) binding with Qa-1b was obtained by phage display technique from alpaca melanoma cell nanobady library using Qa-1b polypeptide as antigen, followed by the construction of pET28a-Qa-1b-VHH prokaryotic expression recombinant plasmid and expression through IPTG inducing and purification through nickel column. After the binding of Qa-1b nanobody to antigen was detected by ELISA, the Qa-1b nanobody was used as the primary antibody in immunohistochemistry and Western blot methods to detect Qa-1b expression in tissues. Qa-1b nanobody was added into B16 cell culture medium, and its effects on the proliferation and migration of B16 cells and the expression of proteins related to proliferation and migration were detected by CCK8, cell scratch and Western blot methods, respectively. The results showed that Qa-1b and NKG2A were expressed in the out root sheath of hair follicles and vascular endothelium in Mongolian cattle skins with significantly higher expression in skins of young cattle than that in skins of the adult cattle (P < 0.01 or P < 0.001). Moreover, there existed the interaction between Qa-1b and NKG2A. The Qa-1b nanobody with strong specificity was obtained, which could be used as a primary antibody for Western blot and immunohistochemistry to detect Qa-1b expression in other tissues. The supplement of Qa-1b nanobody in B16 culture medium resulted in the obvious inhibition of cell proliferation and migration, significant down-regulation of the expression of proteins such as Ras, MEK1, CyclinD1, and CDK4 during cell proliferation(P < 0.01 or P < 0.001). The results suggested that Qa-1b might play a role in the skin immunity to ensure the normal growth of hair follicles, especially in calves, and the Qa-1b nanobody could inhibit the proliferation and migration of B16 cells, which provided a theoretical basis for the strengthened skin immunity and anti-melanoma antibody drugs.

Key words: Qa-1b, NKG2A, nanobody, skin immunity, melanoma, B16 cells, Mongolian cattle

中图分类号:

S857.5

金聪俐, 贾琼, 任泓睿, 迟志端, 白瑞, 郭湘, 范瑞文, HerridMuren. 蒙古牛皮肤Qa-1b/NKG2A的表达及Qa-1b纳米抗体的制备和功能鉴定[J]. 畜牧兽医学报, 2025, 56(1): 404-416.

JIN Congli, JIA Qiong, REN Hongrui, CHI Zhiduan, BAI Rui, GUO Xiang, FAN Ruiwen, HERRID Muren. The Expression of Qa-1b/NKG2A in the Skins of Mongolia Cattle and Preparation and Functional Roles of the Qa-1b Nanobody[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 404-416.

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引物名称 Primer name	序列(5′→3′) Primer sequence	扩增产物大小/bp Size of PCR product
β-actin-F	TTGCTGACAGGATGCAGAAG	141
β-actin-R	ACATCTGCTGGAAGGTGGAC
Qa-1b-F	AGTATTGGGAGCGGGAGACT	572
Qa-1b-R	CACCACAGATGCCCACTTCT
NKG2A-F	GCCCCTGCAAAGATACCGAA	62
NKG2A-R	TCTGTGGGTTCTAGTCATTGAGG

阳性克隆编号 No. of positive clones	S/N	阳性强度 Positive strength
Qa-1b-VHH-A-6	6.041	+++
Qa-1b-VHH-A-8	4.453	+++
Qa-1b-VHH-A-9	4.706	+++
Qa-1b-VHH-C-1	4.057	+++
Qa-1b-VHH-C-7	3.414	++
Qa-1b-VHH-D-4	3.511	++
Qa-1b-VHH-E-7	3.773	+++
Qa-1b-VHH-F-10	4.082	+++

组别 Groups	Qa-1b纳米抗体稀释度 Dilution of Qa-1b nanobody
组别 Groups	原液 Stock solution	1∶2	1∶4	1∶8	1∶16	1∶32	1∶64	1∶128
试验组 Experiment group	1.320	0.608	0.603	0.299	0.325	0.160	0.097	0.071
对照组 Control group	0.034	0.031	0.040	0.037	0.037	0.035	0.034	0.027

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