绵羊肺炎支原体pcDNA3.1-TBP30-Hsp70融合表达质粒的构建及对小鼠细胞免疫应答的影响

doi:10.16431/j.cnki.1671-7236.2019.11.029

Abstract

Abstract: To investigate the effect of the fusion expression plasmid of Mycoplasma ovipneumoniae (Mo) pcDNA3.1-TBP30-Hsp70 on the cellular immune response in mice,a fusion expression plasmid of Mycoplasma ovipneumoniae pcDNA3.1-TBP30-Hsp70 was constructed.Using the constructed plasmids pMD19T-P30 and pMD19-Hsp70 as templates,primers were designed based on the principle of gene site-directed mutagenesis (SDM).The target gene fragments were amplified by SOE-PCR and directionally cloned into the expression vector pcDNA3.1(+) to construct pcDNA3.1(+)-TBP30 and the fusion plasmid pcDNA3.1(+)-TBP30-Hsp70.Mice were immunized with pcDNA3.1-TBP30,pcDNA3.1-TBP30-Hsp70,pcDNA3.1(+) and Elution Buffer.Serum levels of cytokines interleukin-2 (IL-2),IL-4 and interferon-γ (INF-γ) were detected by ELISA kit.The results showed that after digestion of pcDNA3.1-TBP30-Hsp70,the target gene fragments with the size of 1 413 bp and the vector bands with the size of 5 400 bp were visible.Compared with the blank control and the pcDNA3.1(+) groups,the immune recombinant plasmid groups could increase the secretion levels of cytokines INF-γ,IL-2 and IL-4 in the serum of mice.Compared with the blank control and the pcDNA3.1(+) groups,the difference was significant or extremely significant (P<0.05;P<0.01),but there was no significant difference between the blank control and blank plasmid groups (P>0.05).The final secretion of IL-2,INF-γ and IL-4 in serum of mice immunized with pcDNA3.1-TBP30 and pcDNA3.1-TBP30-Hsp70 increased,which indicated that the recombinant plasmids could stimulate the changes of IL-2,INF-γ and IL-4 in serum of mice,and showed the regularity of increasing and decreasing in time.The results showed that after immunizing mice with recombinant plasmid pcDNA3.1(+)-TBP30-Hsp70,the secretion levels of IL-2 and INF-γ increased,which enhanced the cellular immune function of the organism,and then regulated the cellular immunity of the organism to affect the secretion of T cells and macrophages,thereby improving the cellular immunity of the organism.Th2 differentiated into Th1,maintains the dominant state of Th1 and enhanced the cellular immune function of the body.The results provided a reference for the development of genetic engineering vaccine against Mycoplasma ovipneumoniae.

Key words: Mycoplasma ovipneumoniae; SOE-PCR; fusion plasmid; cytokine

CLC Number:

S858.26

ZHOU Yi, WANG Bolin, YANG Mei, HE Ling, QIN Lan, YUE Jun, ZHANG Shuangxiang, ZHOU Bijun, WANG Kaigong, CHENG Zhentao. Construction of Mycoplasma ovipneumoniae pcDNA3.1-TBP30-Hsp70 Fusion Plasmid and Its Effect on Cellular Immune Response in Mice[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(11): 3387-3395.

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Construction of Mycoplasma ovipneumoniae pcDNA3.1-TBP30-Hsp70 Fusion Plasmid and Its Effect on Cellular Immune Response in Mice

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