鸭寡腺苷酸合成酶样蛋白原核表达及多克隆抗体制备

doi:10.3864/j.issn.0578-1752.2019.23.024

中国农业科学 ›› 2019, Vol. 52 ›› Issue (23): 4429-4436.doi: 10.3864/j.issn.0578-1752.2019.23.024

• 专题：水禽疫病防控 • 上一篇

鸭寡腺苷酸合成酶样蛋白原核表达及多克隆抗体制备

毕可然^1,²,李银¹(),韩凯凯¹,赵冬敏¹,刘青涛¹,刘宇卓¹,黄欣梅¹,杨婧¹

¹ 江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室,南京210014
² 海洋生命与水产学院,江苏海洋大学,江苏连云港,222005

收稿日期:2019-04-23 接受日期:2019-07-22 出版日期:2019-12-01 发布日期:2019-12-01
通讯作者: 李银
作者简介:毕可然,Tel：15896101504;E-mail：bikeran@126.com
基金资助:
国家重点研发计划项目(2017YFD0500800);江苏省自然科学基金项目(BK20160064);国家博士后基金项目(2016M590430);江苏省农业科技自主创新资金项目(CX151008)

Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Oligoadenylate Synthase-Like Protein

BI KeRan^1,²,LI Yin¹(),HAN KaiKai¹,ZHAO DongMin¹,LIU QingTao¹,LIU YuZhuo¹,HUANG XinMei¹,YANG Jing¹

¹ Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Nanjing 210014
² College of Marine Life and Fisheries, Jiangsu Ocean University, Lianyungang 222005, Jiangsu

Received:2019-04-23 Accepted:2019-07-22 Online:2019-12-01 Published:2019-12-01
Contact: Yin LI

摘要/Abstract

摘要：

【目的】已有研究获得鸭寡腺苷酸合成酶样蛋白（oligoadenylate synthase-like protein,OASL）基因全长,并且证明其可以抑制鸭坦布苏病毒复制。因此在通过原核表达和多克隆抗体制备技术获得带有GST标签的鸭寡腺苷酸合成酶样融合蛋白和该融合蛋白的特异性抗体,为进一步明确鸭寡腺苷酸合成酶样蛋白抑制病毒复制分子机制提供物质基础。【方法】根据前期研究已经获得的鸭OASL基因开放阅读框（open reading frame,ORF）序列设计全长扩增引物pGEX-OAS-F和pGEX-OAS-R。利用动物组织总RNA提取试剂盒提取健康樱桃谷鸭幼鸭脾脏总RNA;通过RT-PCR,利用随机引物和M-MLV反转录酶扩增获得鸭脾脏cDNA,以cDNA为模板,利用设计合成的pGEX-OAS-F和pGEX-OAS-R引物进行PCR扩增,扩增后的产物经1.5%的琼脂糖凝胶电泳检测,检测后将片段大小与目的条带大小相同的PCR产物切胶纯化,纯化后的产物克隆到pEASY-T1载体,挑取单克隆菌落送基因公司测序,测序验证正确的PCR纯化产物通过限制性内切酶BamH I和Xho I酶切连接至带GST标签的原核表达载体pGEX-4t-1。重组质粒转化到大肠杆菌BL21（PLYSS） ^TM,经终浓度为0.5 mmol·L ^-1的IPTG诱导表达4 h后检测该融合蛋白的表达情况。离心收集诱导表达的大肠杆菌菌液,进行超声波破碎处理,收集上清及沉淀,采用SDS-PAGE和Western-blotting分析融合蛋白的表达。利用GST柱亲和纯化上清中的可溶性融合蛋白,经20 mmol·L ^-1Tris-HCL和0.10 mol·L ^-1NaCL溶液透析后获取纯化的融合蛋白,采用SDS-PAGE分析纯化的融合蛋白。采用皮下多点注射方法免疫新西兰白兔,经3次免疫后制备多克隆抗血清,采用SDS-PAGE和间接免疫荧光检测抗体的纯度和效价。【结果】克隆测序结果表明,设计合成的鸭OASL基因全长扩增引物pGEX-OAS-F 和 pGEX-OAS-R能够从樱桃谷鸭脾脏中获得鸭OASL基因ORF序列,并且序列组成与前期研究结果一致。被扩增的序列经酶切后,鸭OASL基因能够与pGEX-4t-1载体成功连接,IPTG诱导后可以在大肠杆菌BL21（PLYSS） ^TM中表达,表达产物经SDS-PAGE和Western-blotting分析显示,融合蛋白大小约为84 kD,主要以可溶性蛋白形式表达,在包涵体中也有少量表达。经GST亲和层析纯化上清中的可溶性鸭OASL融合蛋白,得到0.5 mmol·L ^-1纯度抗原蛋白。经3轮免疫新西兰大白兔制备多克隆抗体,纯化后ELISA结果显示鸭OASL蛋白抗体具有较好的灵敏度,最高效价为1:512 000,SDS-PAGE结果显示抗体大小为55 kD,与ORF理论值相同,且抗体纯度为90%以上。【结论】设计合成的引物能够成功地获得鸭脾脏OASL基因ORF序列,该ORF能够在原核细胞中成功表达,表达的融合蛋白通过免疫新西兰兔能够获得高纯度和高效价的鸭OASL多克隆抗体,研究成果为后续深入研究鸭OASL蛋白抑制病毒复制分子机制奠定坚实的物质基础。

关键词: 鸭寡腺苷酸合成酶, 原核表达, 多克隆抗体

Abstract:

【Objective】In previous study, we have obtained the full-length gene of duck oligoadenylate synthase-like protein (duOASL) and confirmed that duOASL could inhibit DTMUV replication. To further investigate the inhibitory effect of duOASL on DTMUV replication, the recombinant protein (pGEX-OASL) with GST tag and polyclonal antibody of duOASL protein was obtained by prokaryotic expression and polyclonal antibody preparation technology. These results would lay a foundation for performing further studies to determine mechanism(s) underlying the antiviral activity duOASL at the molecular level. 【Method】Primers were designed based on the ORF sequence of duOASL gene from healthy Cherry Valley ducks. Total RNA was extracted from the spleen tissues of duckling by using HP Total RNA Kits. The cDNA was synthesized using random primers and M-MLV reverse transcriptase. Full-length of duOASL gene was obtained by synthesising pGEX-OAS-F and pGEX-OAS-R primers by using the cDNA. The PCR results were detected by 1.5% agarose electrophoresis. The sequences of the same size as the target band were purified and cloned to pEASY-T1 vector. The cloned gene ORF was inserted into the expression vector pGEX-4t-1 with GST tag and by BamH I and Xho I digestion. The recombinant plasmid was transformed into E. coli BL21 and expressed under the induction with 0.5 mmol·L ^-1 IPTG for 4 h. The expression of the fusion protein was detected by SDS-PAGE and Western-blotting. Then the polyclonal antibody was obtained from rabbits which had immunized by the purified duOASL recombinant protein. The purity and titer of the polyclonal antibody were determined by SDS-PAGE and ELISA.【Result】The recombinant protein was a soluble protein and the molecular weight was 84 kD. The purity was 0.5 mg·mL ^-1. The antibody titer of polyclonal antibodies was 1:512 000.【Conclusion】Above results would lay a foundation for further studies of duOASL inhibiting DTMUV replication.

Key words: OASL, prokaryotic expression, polyclonal antibody

毕可然,李银,韩凯凯,赵冬敏,刘青涛,刘宇卓,黄欣梅,杨婧. 鸭寡腺苷酸合成酶样蛋白原核表达及多克隆抗体制备[J]. 中国农业科学, 2019, 52(23): 4429-4436.

BI KeRan,LI Yin,HAN KaiKai,ZHAO DongMin,LIU QingTao,LIU YuZhuo,HUANG XinMei,YANG Jing. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Oligoadenylate Synthase-Like Protein[J]. Scientia Agricultura Sinica, 2019, 52(23): 4429-4436.

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鸭寡腺苷酸合成酶样蛋白原核表达及多克隆抗体制备

Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Oligoadenylate Synthase-Like Protein

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