鸡滑液囊支原体LP78蛋白重组腺病毒载体的构建及雏鸡免疫原性评价
Construction of recombinant adenovirus harboring mycoplasma synoviae LP78 protein and evaluation of its immune efficacy in chicken
赵超 1宋春 1单春兰 1胡茜 1王永璇 1韩帅博 1肖琨 2文明 3朱二鹏 3程振涛3
作者信息
- 1. 贵州大学动物科学学院,贵州贵阳 550025
- 2. 六盘水市动物疫病预防控制中心,贵州六盘水 553001
- 3. 贵州大学动物科学学院,贵州贵阳 550025;贵州省动物疫病与兽医公共卫生重点实验室,贵州贵阳 550025
- 折叠
摘要
选取鸡滑液囊支原体(Mycoplasma synoviae,MS)LP78蛋白,将LP78基因克隆至pMD19-T载体上后,采用腺病毒Ad-Max系统将pMD-LP78重组克隆质粒连接到穿梭载体pDC316-mCMV-EGFP上构建重组穿梭质粒pDC-LP78,将pDC-LP78重组穿梭质粒与腺病毒大骨架pBHGlox(delta)E1、3cre共转染HEK 293A细胞包装成重组腺病毒pBH-LP78,RT-PCR及 Western blot鉴定HEK 293A细胞系包装的重组腺病毒pBH-LP78.测定病毒滴度;再通过腿部肌肉免疫接种构建的重组腺病毒pBH-LP78,并用ELISA方法检测免疫鸡血清中的特异性抗体和细胞因子(IL-2、IFN-γ、IL-4和IL-6)的分泌情况.PCR、双酶切鉴定和测序结果显示,成功构建了重组克隆质粒pMD-LP78和重组穿梭质粒pDC-LP78;RT-PCR及 Western blot结果显示,目的基因在HEK 293A细胞中稳定表达;ELISA结果显示,血清中LP78特异性抗体和细胞因子(IL-2、IFN-γ、IL-4和IL-6)的分泌量极显著升高(P＜0.01),表明pBH-LP78重组腺病毒能刺激鸡机体产生体液和细胞免疫应答.结果表明,成功构建了 MS重组腺病毒载体疫苗pBH-LP78,其可有效引起鸡机体产生体液和细胞免疫应答,为MS新型重组腺病毒载体疫苗的研发奠定了基础.
Abstract
To construct recombinant adenovirus vector vaccine expressing Mycoplasma synoviae(MS)LP78 protein and evaluate its immune efficacy.After MS LP78 protein was selected,LP78 was cloned into pMD19-T vector,and the recombinant pMD-LP78 recombinant clone plasmid was connected to the shuttle vector pDC316-mCMV-EGFP using adenovirus Ad-Max system to con-struct the recombinant shuttle plasmid pDC-LP78.Recombinant adenovirus pBH-LP78 was pack-aged into recombinant adenovirus PBH-LP78 by transfecting HEK 293A cells with pBHGlox(del-ta)E1 and 3cre.RT-PCR and Western blot were used to identify the recombinant adenovirus pBH-LP78 packaged in HEK 293A cell line.Virus titers were measured.Recombinant adenovirus pBH-LP78 was constructed by leg muscle immunization,and the secretion of specific antibodies and cy-tokines(IL-2,IFN-γ,IL-4 and IL-6)in serum of immunized chickens was detected by ELISA method.PCR,double digestion and sequencing results showed that pMD-LP78 and pDC-LP78 were successfully constructed.RT-PCR and Western blot results showed that the target gene was stably expressed in HEK293A cells.ELISA results showed that serum levels of LP78 specific antibodies and cytokines(IL-2,IFN-γ,IL-4 and IL-6)were significantly increased(P＜0.01),indicating that pBH-LP78 recombinant adenovirus can stimulate humoral and cellular immune responses in chick-ens.This experiment successfully constructed MS recombinant adenovirus vector vaccine pBH-LP78,which can effectively induce humoral and cellular immune responses in chickens,laying a foundation for the research and development of MS recombinant adenovirus vector vaccine.
关键词
鸡滑液囊支原体/LP78蛋白/重组腺病毒Key words
Mycoplasma synoviae/LP78 protein/recombinant adenovirus引用本文复制引用
基金项目
贵州省科技计划(黔科合支撑[2021]—般161号)
贵州省科技计划(黔科合支撑[2023]—般077号)
贵州省优秀青年科技人才资助项目(黔科合平台人才[2021]5646号)
出版年
2024
NETL
NSTL
万方数据