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

作　　者：赵超 宋春 单春兰 胡茜 王永璇 韩帅博 肖琨 文明[1,2] 朱二鹏 程振涛 ZHAO Chao;SONG Chun;SHAN Chunlan;HU Qian;WANG Yongxuan;HAN Shuaibo;XIAO Kun;WEN Ming;ZHU Erpeng;CHENG Zhentao(School of Animal Science,Guizhou University,Guiyang550025,China;Guizhou Province Key Laboratory of Animal Disease and Veterinary Public Health,Guiyang 550025,China;Liupanshui City Animal Disease Prevention and Control Center,Liupanshui,Guizhou 553001,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病与兽医公共卫生重点实验室,贵州贵阳550025 [3]六盘水市动物疫病预防控制中心,贵州六盘水553001

出　　处：《中国兽医学报》2024年第4期677-684,705,共9页Chinese Journal of Veterinary Science

基　　金：贵州省科技计划资助项目(黔科合支撑[2021]一般161号,黔科合支撑[2023]一般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)。

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pDCLP78,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pBHLP78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pBHLP78,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。To construct recombinant adenovirus vector vaccine expressing Mycoplasma synoviae(MS)LP78protein and evaluate its immune efficacy.After MS LP78protein was selected,LP78 was cloned into pMD19-T vector,and the recombinant pMD-LP78recombinant clone plasmid was connected to the shuttle vector pDC316-mCMV-EGFP using adenovirus Ad-Max system to construct the recombinant shuttle plasmid pDC-LP78.Recombinant adenovirus pBH-LP78was packaged into recombinant adenovirus PBH-LP78by transfecting HEK 293Acells with pBHGlox(delta)E1and 3cre.RT-PCR and Western blot were used to identify the recombinant adenovirus pBHLP78packaged in HEK 293Acell line.Virus titers were measured.Recombinant adenovirus pBHLP78was constructed by leg muscle immunization,and the secretion of specific antibodies and cytokines(IL-2,IFN-γ,IL-4and IL-6)in serum of immunized chickens was detected by ELISA method.PCR,double digestion and sequencing results showed that pMD-LP78and pDC-LP78were successfully constructed.RT-PCR and Western blot results showed that the target gene was stably expressed in HEK293Acells.ELISA results showed that serum levels of LP78specific antibodies and cytokines(IL-2,IFN-γ,IL-4and IL-6)were significantly increased(P<0.01),indicating that pBH-LP78recombinant adenovirus can stimulate humoral and cellular immune responses in chickens.This experiment successfully constructed MS recombinant adenovirus vector vaccine pBHLP78,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 

分 类 号：S852.62[农业科学—基础兽医学]