鸭<i>TLR2</i>在免疫组织和血液中的表达分析及多克隆抗体制备

doi:10.7668/hbnxb.20194892

摘要/Abstract

摘要：

为研究Toll样受体2(TLR2)在水禽如鸭先天免疫中的作用,利用荧光定量PCR分析dTLR2 mRNA在1~10周龄鸭免疫器官中表达水平以及大肠杆菌、沙门氏菌感染后血液中dTLR2 mRNA表达变化,初步解析dTLR2在鸭抗感染中的可能作用;通过体外表达鸭dTLR2胞外段,并免疫家兔获得特异性抗体以期为鸭dTLR2免疫应答研究提供生物素材。结果表明,dTLR2 mRNA在不同周龄鸭免疫器官中都有表达,脾脏和胸腺组织中,表达量在不同周龄有显著或极显著变化,但在法氏囊组织中,各周龄间表达变化差异不显著;鸭感染大肠杆菌或者沙门氏菌后,第1天及第2天感染组表达量显著高于对照组,但在第3天时,感染组和对照组表达量差异不显著。研究分析dTLR2编码基因,预测胞外区段并设计引物,构建pET28a-TLR2重组表达质粒,重组表达dTLR2-His融合蛋白。经SDS-PAGE检测表明,重组蛋白成功高效表达,分子质量约29 ku。重组蛋白经镍柱纯化并透析后免疫家兔制备抗血清;所获家兔免疫抗血清能特异性的识别重组TLR2蛋白和组织中内源性TLR2蛋白。鸭dTLR2 mRNA组织中表达分析及多克隆抗体的成功制备将为进一步研究dTLR2的生物学功能提供生物素材。

关键词: 鸭, dTLR2 mRNA, 免疫组织, 重组表达, 多克隆抗体

Abstract:

In order to analyze the possible role of dTLR2 in duck innate immunity,we study the expression of dTLR2 mRNA in immune organs during 1 to 10 weeks,as well as the expression in blood after challenging with Escherichia coli and Salmonella enteritis by Fluorescence Quantitative PCR.We also prepared polyclonal antibodies of the extracellular domain of dTLR2 by recombinant protein expression for further studying the biological function of dTLR2.The results showed that dTLR2 mRNA was expressed in spleen,thymus and bursa at different weeks.The expression of dTLR2 mRNA was significantly different among different weeks in the spleen and thymus tissues,and there was no significant difference in bursa.After infection with E.coli and S.enteritis,the expression of dTLR2 mRNA was significantly higher than that of the control group in the first and second days,but there was no significant difference among groups in the third day.Analyzing the sequence of dTLR2 gene,we predicted the extracellular domain,designed primers for PCR assay,and built pET28a-TLR2 recombinant plasmid.The recombinant plasmid was translated into E.coli BL21 (DE3) for expression of dTLR2-His protein.The SDS-PAGE results showed that the fusion recombinant protein efficiently expressed in the supernatant,molecular mass was around 29 ku.The results of Western Blot showed that the antibody which harvested from immunizing rabbits could detect recombinant dTLR2 and endogenous dTLR2.The analysis of the expression of dTLR2 and successful preparation of polyclonal antibody provide favorable support for further studying of dTLR2 biology function.

Key words: Duck, dTLR2 mRNA, Immune organs, Recombinant expression, Polyclonal antibody

朱春红, 王志成, 刘宏祥, 陶志云, 宋卫涛, 王逸飞, 徐文娟, 章双杰, 李慧芳. 鸭TLR2在免疫组织和血液中的表达分析及多克隆抗体制备[J]. 华北农学报, 2024, 39(5): 212-218. doi: 10.7668/hbnxb.20194892.

ZHU Chunhong, WANG Zhicheng, LIU Hongxiang, TAO Zhiyun, SONG Weitao, WANG Yifei, XU Wenjuan, ZHANG Shuangjie, LI Huifang. Expression of Duck dTLR2 in Immune Organs and Blood and Preparation of Polyclonal Antibodies[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(5): 212-218. doi: 10.7668/hbnxb.20194892.

http://www.hbnxb.net/CN/Y2024/V39/I5/212

图/表 7

图1 不同周龄鸭免疫器官中dTLR2 mRNA表达不同大写字母表示在0.01水平上差异显著,不同小写字母表示在0.05水平上差异显著。图2同。

Fig.1 Expression of dTLR2 mRNA in duck immune organs in different weeks Different capital letters mean significantly different at 0.01 level,and different lowercase letters mean significantly different at 0.05 level. The same as Fig.2.

图2 鸭感染大肠杆菌、沙门氏菌后血液中dTLR2 mRNA表达

Fig.2 Expression of dTLR2 mRNA in the blood of ducks infected with E.coli and Salmonella

图3 dTLR2氨基酸结构域预测

Fig.3 Prediction of dTLR2 amino acid domain

图4 SDS-PAGE电泳分析dTLR2胞外区重组蛋白 M.标准蛋白;L.纯化抗原。

Fig.4 SDS-PAGE analysis of the purified recombinant protein of dTLR2 extracellular region M.Standard protein;L.Purified protein.

图5 dTLR2胞外区重组蛋白免疫血清Western Blot检测分析 1.重组蛋白,免前血孵育(兔1);2.重组蛋白,免前血孵育(兔2);3.重组蛋白10 ng,(兔1)抗血清孵育;4.重组蛋白25 ng,(兔1)抗血清孵育;5.重组蛋白10 ng,(兔2)抗血清孵育;6.重组蛋白25 ng,(兔2)抗血清孵育;二抗.羊抗兔 IgG(H+L)-HRP(1∶10 000);M.标准蛋白。

Fig.5 Western Blot analysis of the immune serum of dTLR2 recombinant extracellular region 1.Recombinant protein,pre-immuned blood (Rabbit 1);2.Recombinant protein,pre-immuned blood (Rabbit 2);3.Recombinant protein 10 ng,incubated with rabbit 1 antiserum;4.Recombinant protein 25 ng,incubated with rabbit 1 antiserum;5.Recombinant protein 10 ng,incubated with rabbit 2 antiserum;6.Recombinant protein 25 ng,incubated with rabbit 2 antiserum;Secondary antibody.Sheep anti-rabbit IgG (H+L)-HRP (1∶10 000);M.Standard protein.

图6 兔多抗血清效价结果

Fig.6 Results of serum titer of rabbit polyantiserum

图7 重组蛋白免疫血清检测鸭肝脏和脾脏组织内源性dTLR2蛋白 1.肝裂解液,免疫前兔血清孵育;2.脾裂解液,免疫前兔血清孵育;3.肝裂解液,(兔1)抗血清孵育;4.脾裂解液,(兔1)抗血清孵育;5.肝裂解液,(兔2)抗血清孵育;6.脾裂解液,(兔2)抗血清孵育;二抗.羊抗兔 IgG(H+L)-HRP(1∶10 000);M.标准蛋白。

Fig.7 Detection endogenous dTLR2 in duck liver and spleen by recombinant protein immune serum 1.Liver lysate,incubated with pre-immuned rabbit serum;2.Spleen lysate,incubated with pre-immuned rabbit serum;3.Liver lysate,incubated with rabbit 1 antiserum;4.Spleen lysate,incubated with rabbit 1 antiserum;5.Liver lysate,incubated with rabbit 2 antiserum;6.Spleen lysate,incubated with rabbit 2 antiserum;Secondary antibody.Sheep anti-rabbit IgG (H+L)-HRP (1∶10 000);M.Standard protein.

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鸭TLR2在免疫组织和血液中的表达分析及多克隆抗体制备

Expression of Duck dTLR2 in Immune Organs and Blood and Preparation of Polyclonal Antibodies

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鸭TLR2在免疫组织和血液中的表达分析及多克隆抗体制备

Expression of Duck dTLR2 in Immune Organs and Blood and Preparation of Polyclonal Antibodies

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