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.
Construction of recombinant adenovirus harboring mycoplasma synoviae LP78 protein and evaluation of its immune efficacy in chicken
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.
NETL
NSTL
万方数据
