猪肺炎支原体p97 C末端基因重组腺病毒的构建及其免疫效果

Construction of the recombinant adenovirus expressing the C-Terminal of the Mycoplasma hyopneumoniae p97 gene and its immune response

【目的】构建携猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)黏附因子p97C末端基因的重组腺病毒并研究其诱导小鼠产生的免疫反应,为研制新型Mhp疫苗奠定基础。【方法】从Mhp基因组中扩增p97C末端基因,并将其克隆到穿梭载体pShuttle-CMV中,该重组穿梭质粒经PmeI线性化后电转化到BJ5183-AD-1细胞中进行同源重组获得重组腺病毒DNA,纯化后的重组腺病毒质粒经PacⅠ酶切线性化后转染AD293细胞以获得重组腺病毒。对该重组腺病毒进行RT-PCR、间接免疫荧光和Western blot鉴定,用氯化铯密度梯度离心纯化试剂盒对该重组腺病毒进行纯化并确定其滴度。重组病毒经肌注、滴鼻两种途径免疫小鼠并从体液免疫、粘膜免疫和细胞免疫3个方面对其免疫结果进行分析。【结果】PacⅠ酶切证实同源重组成功,RT-PCR、间接免疫荧光和Western blot鉴定证明了该重组腺病毒成功转录并表达了p97C末端蛋白,将该重组病毒进行纯化后滴度可达到5×1011TCID50/mL,重组病毒可诱导小鼠产生血清、肺匀浆液特异性IgG,通过滴鼻免疫方式还可诱导肺匀浆液SIgA的产生,但不能诱导特异的淋巴细胞增值。【结论】成功构建了表达p97末端基因的重组腺病毒,该病毒可诱发小鼠产生特异的的体液免疫和粘膜免疫,但不产生细胞免疫应答。

[ Objective] To construct recombinant adenovirus carrying C-Terminal of the adhesion factor gene p97 of Mycoplasma hyopneumoniae （Mhp） so as to provide basis for further studying new type Mhp vaccine. [Methods] We amplified p97 gene from the genome of Mhp and cloned into pShuttle-CMV plasmid. The correctly identified recombinant plasmid was linearized with Pme Ⅰ and transformed into E. coil BJS183-AD-1 competent cells containing adenovirus backbone vector to produce recombinant adenovirus DNA by homologous recombination. Purified recombinant adenovirus plasmid was linearized with Pac I , and transfected into AD293 cells to obtain recombinant adenovirus. The recombinant adenovirus was identified by RT-PCR, indirect hnmunofluorescence assay and Western blot, and purified by cesium chloride density centrifugation kit, then its titer was determined. Balb/c mice were immunized with recombinant adenovirus via intramuscular and intranasal routes and analysis the immunity results through humoral immunity, mucosal immunity and cell-mediated immunity aspect. [ Results] Digestion by Pac Ⅰ proved successful homologous recombination. RT-PCR, Indirect immunofluorescence assay and Western Blot showed that the recombinant adenovirus transcribed and expressed P97 C-terminal protein successfully, the titer could achieve to 5 × 10^11 TCID50/ mL after purification. Inoculation with the recombinant adenovirus by each route elicited P97 C-terminal protein specific serum and lung homogenate IgG and SIgA was induced by intranasal route, but the special lymphocyte proliferation was not induced by each route. [ Conclusion] The recombinant adenovirus expressing p97-Terminal gene was successfully constructed and it induced special humoral and mucosal immunity but no cell-mediated immunity.