基于倒毛鸡LSm14A分子序列分析的原核表达与免疫原性分析

doi:10.11843/j.issn.0366-6964.2016.07.027

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (7): 1511-1516.doi: 10.11843/j.issn.0366-6964.2016.07.027

基于倒毛鸡LSm14A分子序列分析的原核表达与免疫原性分析

伍昌华，陈绍品，温贵兰^*，李晨，李天珍，林汉卿，管国丹，王开功，文明，龚新勇

(贵州大学动物科学学院预防兽医学实验室，贵阳 550025)

收稿日期:2015-12-28 出版日期:2016-07-23 发布日期:2016-07-23
通讯作者: 温贵兰，E-mail：glwen@gzu.edu.cn
作者简介:伍昌华（1989-），男，仡佬族，贵州石阡人，硕士生，主要从事兽医微生物与免疫学研究，E-mail：1006492290@qq.com
基金资助:
贵州大学博士基金项目［贵大人基合字(2013)12］；国家自然科学基金(31460668)；贵州大学研究生创新基金项目(研农2015024)；贵州大学“本科教学工程”项目（JKSP2013004）；贵州省科技创新人才团队项目［黔科合人才团队（2015）4016］

Prokaryotic Expression of the LSm14A Gene in the Frizzle Chicken Based on the Sequence and Immunogenicity Analyses of the Expressed LSm14A

WU Chang-hua，CHEN Shao-pin，WEN Gui-lan^*，LI Chen，LI Tian-zhen，LIN Han-qing，GUAN Guo-dan，WANG Kai-gong，WEN Ming，GONG Xin-yong

(Preventive Veterinary Laboratory，College of Animal Science Guizhou University，Guiyang 550025，China)

Received:2015-12-28 Online:2016-07-23 Published:2016-07-23

摘要/Abstract

摘要：

旨在探明倒毛鸡LSm14A分子序列特征与免疫原性。通过提取倒毛鸡肝总RNA，经RT-PCR分别扩增倒毛鸡LSm14A全长CDS区与原核表达片段；采用分子生物学技术构建携带倒毛鸡LSm14分子的原核表达质粒；经IPTG诱导表达、His-tag镍柱纯化表达的重组蛋白质；纯化重组蛋白质rHis-cLSm14A分别免疫小鼠与兔制备相应的多克隆抗体；采用间接ELISA与Western blotting方法分析表达蛋白质的免疫原性。结果显示：倒毛鸡LSm14A全长CDS为1 386 bp，与原鸡源LSm14A相似性达100%，与鸭源的LSm14A相似性为95.1%；与鼠、爪蟾、猴、人、猪、鹅源的LSm14A相似性相对较低；鼠源、兔源抗cLSm14A多克隆抗体ELISA效价均高于1∶3 200；Western blotting结果显示，重组表达蛋白质分别被His抗体、鼠源与兔源抗cLSm14A多克隆抗体所识别，蛋白质条带大小约为19.6 ku。以上结果说明倒毛鸡源LSm14A分子具有较高的保守性与良好的免疫原性，研究结果为研究倒毛鸡的生物学特性与宿主的抗病毒天然免疫机制奠定了基础。

Abstract:

The aim of this study was to explore the sequence and the immunogenicity of LSm14A gene of the Frizzle chicken.Total cellular RNA was isolated from the livers of frizzle chickens hepatic tissue，and full length CDS region and prokaryotic expression fragment of LSm14A gene were amplified by reverse transcription polymerase chain reaction RT-PCR.The recombinant plasimd was transfected into BL21 competent cells，then His-tag Ni-NTA spin columns were applied to purify the recombinant protein which were produced by IPTG induced BL21.To generate the corresponding hyperimmune serum，rabbits and mice were immunized with the purified recombinant protein rHis-cLSm14A.Sequence analysis revealed that the full length of LSm14A CDS region transcripts from the frizzle chicken was amplified by RT-PCR.The amplified LSm14A CDS region had 1 368 bp and was 100% identical at the base sequence level to the sequence in GenBank.It has a close similarity to the duck source which reaches to 95.1%，on the contrary，the similarity to LSm14A from the mouse source，Xenopus，monkey source，human source，pig source，anser source were distant.The titers of serum mouse-anti-cLSm14A and rabbit-anti-cLSm14A antibodies were both higher than 1∶3 200.Western blotting assay showed that the recombinant protein displayed an immune response with the His-antibody，mouse-anti-cLSm14A hyperimmune serum and rabbit-anti-cLSm14A hyperimmune serum，The band weighted about 19.6 kDa in the western blotting.All the results indicated that the LSm14A molecule of frizzle chicken had a high conservative property and good immunogenicity，which laid a solid foundation for the further study of biological characteristics of frizzle chickens and antiviral natural immune mechanism of poultry.

中图分类号:

S852.4

伍昌华，陈绍品，温贵兰，李晨，李天珍，林汉卿，管国丹，王开功，文明，龚新勇. 基于倒毛鸡LSm14A分子序列分析的原核表达与免疫原性分析[J]. 畜牧兽医学报, 2016, 47(7): 1511-1516.

WU Chang-hua，CHEN Shao-pin，WEN Gui-lan，LI Chen，LI Tian-zhen，LIN Han-qing，GUAN Guo-dan，WANG Kai-gong，WEN Ming，GONG Xin-yong. Prokaryotic Expression of the LSm14A Gene in the Frizzle Chicken Based on the Sequence and Immunogenicity Analyses of the Expressed LSm14A[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(7): 1511-1516.

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基于倒毛鸡LSm14A分子序列分析的原核表达与免疫原性分析

Prokaryotic Expression of the LSm14A Gene in the Frizzle Chicken Based on the Sequence and Immunogenicity Analyses of the Expressed LSm14A

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