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

Abstract

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.

Key words: the frizzle chicken, cLSm14A, immunogenicity

CLC Number:

S852.4

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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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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